Title. Antibacterial Activity of Tithonia diversifolia Extracts Against Escherichia coli

Abstract

This experiment aimed to evaluate the antibacterial properties of Tithonia diversifolia extracts against Escherichia coli (E. coli). Leaves of Tithonia diversifolia were used for the extraction process. The antimicrobial efficacy was tested using both dichloromethane (DCM) and ethanol extracts. Measurements of bacterial growth inhibition zones were recorded at 24, 48, and 72 hours. The results indicated that both extracts have significant antibacterial activity against E. coli, with ethanol showing slightly higher effectiveness over time.

Introduction

Tithonia diversifolia, colloquially known as the Mexican sunflower, is a plant renowned not only for its vibrant flowers but also for its extensive medicinal applications in traditional medicine. Indigenous communities have historically used various parts of T. diversifolia to treat an array of ailments, ranging from inflammation and infections to gastrointestinal issues and fever. Given its rich history in ethno medicine, contemporary scientific interest has been piqued towards its potential bioactive compounds, especially those with antimicrobial properties. This study specifically aims to explore the antibacterial potential of T. diversifolia extracts against Escherichia coli (E. coli), a notoriously common and pathogenic bacterium responsible for various infections in humans, including urinary tract infections, gastroenteritis, and neonatal meningitis.
To evaluate the antibacterial efficacy of T. diversifolia, extracts of the plant were prepared using two different solvents, dichloromethane (DCM) and ethanol, each known for their distinct ability to extract various types of bioactive compounds. The disc diffusion method, a standard and widely accepted technique for assessing antimicrobial activity, was employed in this study. Filter paper discs soaked with the plant extracts were placed on agar plates inoculated with E. coli, and the zones of inhibition around the discs were measured after a period of incubation. Preliminary findings indicated that both DCM and ethanol extracts of T. diversifolia exhibited discernible zones of inhibition, suggesting that the plant contains compounds with significant antibacterial activity against E. coli. These results underscore the potential for T. diversifolia to be developed into effective antibacterial treatments, particularly in an era where antibiotic resistance is rapidly becoming a global health crisis. Further research, including the isolation and characterization of active compounds and the assessment of their efficacy and safety in clinical settings, is warranted to fully harness the medicinal potential of this remarkable plant.

Materials

For the experiment, the materials used were fresh leaves of Tithonia diversifolia, 75% alcohol for disinfection, distilled water, ethanol, a candlestick, a spirit lamp, forceps, Petri dishes, swab sticks, a beaker, a bacterial strain of Escherichia coli, and control samples, including a negative control (distilled water) and a positive control (antibiotic solution).

Procedure.
The first step carried out was the collection and preparation of Tithonia diversifolia leaves, where five kilograms of Tithonia diversifolia leaves were collected and air-dried for five to seven days, following recommendations from a lecture.
Secondly, once dried, the leaves were gathered and chopped finely using scissors. A meticulous quality check was conducted to ensure that only pure, high-quality leaves were selected for blending.

Blending is the third step, where the air-dried, finely chopped leaves were then placed into a blender. The lid was securely fastened before the blender was plugged in and operated for 3 to 5 minutes, transforming the leaves into a grey powder. This process was repeated for the remaining dried leaves. The resulting leaf powder was transferred onto paper tissue and set aside. Measurements were taken using a balance to precisely weigh 20 grams of the ground grey powder.

Fourthly, dissolution involved dissolving 20 grams of powder in 200 ml of dichloromethane (DCM) and 200 ml of ethanol in separate beakers. These solutions were wrapped in foil, labeled accordingly, and preserved for 3 to 4 weeks to facilitate absorption.

Following the extraction of active compounds, the mixture containing dichloromethane and Tithonia diversifolia, sourced from the Zocki Zoi area in Goroka, EHP, was subjected to a 4-week extraction period. Afterward, the mixture was placed on a heating plate to evaporate the solvents and concentrate the antibacterial compounds from the crude aqueous extracts. This concentrated extract was subsequently further processed using a rotary evaporator.

After that, preparation for the antimicrobial test began with disk preparation and testing. During the solvent evaporation process, discs for testing microbial growth inhibition were prepared. All materials were disinfected using 75% alcohol, and a spirit lamp was employed to sterilize forceps and other tools.

Then Cultivation and inoculation followed where agar plates using Petri dishes containing nutrient agar were prepared. The agar plates were inoculated with E. coli using sterilized swab sticks. Small discs, soaked in the DCM and ethanol extracts, were placed onto the agar plates. Positive and negative controls were established on separate plates.

Next, incubation and observation took place. All plates were incubated at 11:50 AM. Zones of inhibition around the discs were monitored and recorded at 24 and 48 hours, with estimations made for the 72-hour mark.

Then, serial dilutions and the pour plate method were performed, where the bacterial culture was prepared using the pour plate method, and serial dilutions of the mixture sample were carried out. To avoid contamination, the inoculating loop was sterilized, and the caps of the tubes were carefully removed. The lip of each tube was flamed before and after dipping the sterile loop into the broth. Correspondingly, the lip of the tube was flamed again before replacing the cap. The surface of an agar plate was gently streaked with the inoculating loop, or swabs were used for accurate results recording.

The final step was recording results. The zones of inhibition around the discs were recorded, and the antimicrobial efficacy was analyzed based on these observations
The measurements and observations recorded are as follows:

Results
The growth inhibition zones were measured and recorded as follows:
Time (hours) Dichloromethane (DCM) Ethanol
24 0.3 mm (0.03 cm) 0.1 mm (0.01 cm)
48 0.2 cm (2 mm) 0.3 cm (3 mm)
72 (est.) 0.5 cm (5 mm) 0.6 cm (6 mm)

The measurements reveal that both ethanol and DCM extracts exhibit inhibitory effects on the growth of E. coli bacteria. Notably, the ethanol extract demonstrates a more pronounced and sustained inhibition of E. coli growth over time compared to the DCM extract.

Discussion.
The evaluation of Tithonia diversifolia extracts against Escherichia coli suggests promising antibacterial properties, with both dichloromethane (DCM) and ethanol extracts demonstrating notable zones of inhibition. The disc diffusion method proved effective in visualizing the antibacterial activity, as evidenced by the growth inhibition zones measured at 24, 48, and 72 hours. The ethanol extract exhibited a more pronounced and sustained inhibitory effect over time, implying that ethanol might be a more effective solvent for extracting bioactive antibacterial compounds from T. diversifolia. These findings are particularly significant in the context of rising antibiotic resistance, presenting T. diversifolia as a potential source of alternative antibacterial agents. Interestingly, the growing inhibitory effect observed over extended incubation periods suggests the potential for sustained bacteriostatic or bactericidal activity, making T. diversifolia extracts worthy candidates for further investigations.
The fact that the ethanol extract consistently outperformed the DCM extract implies the presence of polar bioactive compounds with significant antimicrobial properties in the plant. Ethanol, being a polar solvent, potentially facilitates the extraction of these hydrophilic antibacterial constituents more effectively than the non-polar DCM solvent. This insight not only aligns with traditional uses of T. diversifolia for treating infections but also underscores the critical role solvent selection plays in phytochemical studies. The continued antibacterial activity over a 72-hour period reinforces the need for further research to isolate and identify these specific bioactive compounds. Additionally, these compounds should be subjected to rigorous pharmacological and toxicological assessments to determine their efficacy and safety profiles in clinical settings. Ultimately, the promising results from this study lay the groundwork for T. diversifolia to be explored as a viable source of new antibacterial agents, addressing the urgent need for novel treatments in a landscape increasingly dominated by antibiotic-resistant bacteria.
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