Effect of Extraction Method on Antimicrobial Activity Against Staphylococcus aureus of Tapak Liman (Elephantopus scaber L.) Leaves

Introduction

Tapak Liman (Elephantopus scaber L.) is a traditional medicinal plant widely distributed in tropical and subtropical regions, including Southeast Asia. The plant belongs to the Asteraceae family and has been used in traditional medicine for treating various ailments including wounds, fever, cough, and infectious diseases. The leaves of Elephantopus scaber have been reported to contain various bioactive compounds with potential medicinal properties.

Staphylococcus aureus is a gram-positive bacterium that causes a wide range of infections from mild skin infections to life-threatening conditions such as pneumonia, meningitis, and sepsis. The emergence of antibiotic-resistant strains of S. aureus, including methicillin-resistant S. aureus (MRSA), has become a global health concern, necessitating the search for new antimicrobial agents from natural sources.

The extraction method plays a crucial role in determining the yield and composition of bioactive compounds from plant materials. Different extraction techniques can selectively extract different compounds and affect the overall antimicrobial activity of the extract. This study examines the effect of various extraction methods on the antimicrobial activity of Tapak Liman leaves against Staphylococcus aureus.

Materials and Methods

Plant Material Collection

Fresh leaves of Elephantopus scaber L. were collected during flowering season from natural habitats. The leaves were identified and authenticated by a botanist. The collected leaves were washed thoroughly with distilled water, air-dried at room temperature in the shade, and then ground into fine powder using a mechanical grinder.

Extraction Methods

Four different extraction methods were employed in this study:

• Maceration: Powdered leaves were soaked in ethanol (96%) for 72 hours with occasional shaking, followed by filtration and solvent evaporation under reduced pressure.
• Soxhlet Extraction: The plant material was subjected to continuous extraction using ethanol in a Soxhlet apparatus for 6 hours.
• Ultrasonic-Assisted Extraction: Powdered leaves were extracted using ethanol in an ultrasonic bath for 30 minutes at 40C.
• Maceration with Water: Powdered leaves were soaked in distilled water for 72 hours at room temperature, followed by filtration and freeze-drying.

Phytochemical Screening

All extracts were subjected to qualitative phytochemical analysis to identify the presence of bioactive compounds such as alkaloids, flavonoids, tannins, saponins, terpenoids, and phenolic compounds using standard procedures.

Antimicrobial Assay

The antimicrobial activity of the extracts was evaluated against Staphylococcus aureus using the disc diffusion method. Bacterial strains were cultured on Mueller-Hinton agar plates. Sterile filter paper discs (6 mm diameter) were impregnated with different concentrations of each extract (50, 100, 150, and 200 mg/mL) and placed on the inoculated plates. Tetracycline (30 g/disc) was used as a positive control, while the respective solvent served as a negative control. After incubation at 37C for 24 hours, the zones of inhibition were measured.

Determination of Minimum Inhibitory Concentration (MIC)

The MIC values of the most active extracts were determined using the broth dilution method. Two-fold serial dilutions of the extracts were prepared in Mueller-Hinton broth, inoculated with bacterial suspension, and incubated at 37C for 24 hours. The lowest concentration that prevented visible growth was recorded as the MIC.

Results

Phytochemical Composition

All extraction methods yielded extracts containing various bioactive compounds, but there were notable differences in the phytochemical profiles:

• Ethanol extracts (maceration, Soxhlet, and ultrasonic-assisted) showed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, and phenolic compounds.
• The aqueous extract contained primarily tannins, saponins, and phenolic compounds, with only traces of flavonoids and no detectable alkaloids or terpenoids.
• The Soxhlet extraction yielded the highest amount of total phenolic content, followed by ultrasonic-assisted extraction and maceration.
• Flavonoid content was highest in the ultrasonic-assisted extract, followed by maceration and Soxhlet extraction.

Extraction Yields

The extraction yields varied significantly among methods:

• Soxhlet extraction: 12.8% w/w
• Maceration with ethanol: 10.5% w/w
• Ultrasonic-assisted extraction: 9.7% w/w
• Maceration with water: 8.3% w/w

Antimicrobial Activity

All extracts demonstrated varying degrees of antimicrobial activity against Staphylococcus aureus, with the ethanol-based extracts showing superior activity compared to the aqueous extract.

Table 1: Zones of inhibition (mm) of different Elephantopus scaber extracts against Staphylococcus aureus

Extraction Method	50 mg/mL	100 mg/mL	150 mg/mL	200 mg/mL
Maceration (Ethanol)	8.2 0.3	10.5 0.4	12.8 0.5	14.3 0.6
Soxhlet (Ethanol)	7.8 0.3	9.9 0.3	12.2 0.4	13.9 0.5
Ultrasonic-assisted (Ethanol)	9.5 0.4	12.3 0.5	15.1 0.6	17.2 0.7
Maceration (Water)	4.2 0.2	5.6 0.3	6.8 0.3	7.9 0.4
Tetracycline (Control)	-	-	-	22.5 0.9

The ultrasonic-assisted extract exhibited the highest antimicrobial activity at all concentrations tested, with the aqueous extract showing the weakest activity. The antimicrobial activity was concentration-dependent, with larger inhibition zones observed at higher concentrations.

Minimum Inhibitory Concentration (MIC)

The MIC values for ethanol extracts against Staphylococcus aureus were as follows:

• Ultrasonic-assisted extract: 6.25 mg/mL
• Maceration extract: 12.5 mg/mL
• Soxhlet extract: 12.5 mg/mL

The aqueous extract had an MIC value of 50 mg/mL, indicating significantly lower antimicrobial potency.

Discussion

The results of this study demonstrate that the extraction method significantly influences the antimicrobial activity of Elephantopus scaber leaves against Staphylococcus aureus. Among the methods evaluated, ultrasonic-assisted extraction produced the most potent antimicrobial extract, as evidenced by the largest inhibition zones and lowest MIC value. This can be attributed to several factors.

Firstly, the ultrasonic-assisted extraction likely achieved a more efficient extraction of bioactive compounds due to acoustic cavitation, which disrupts plant cell walls and enhances solvent penetration. This is supported by the higher flavonoid content observed in the ultrasonic-assisted extract, as flavonoids are known to contribute significantly to antimicrobial activity through multiple mechanisms including disruption of bacterial cell membranes and inhibition of enzymatic functions.

Secondly, the shorter extraction time (30 minutes for ultrasonic-assisted versus 72 hours for maceration) may have minimized the degradation of thermolabile or labile compounds, preserving their antimicrobial properties. This is particularly important for compounds such as certain terpenoids and sesquiterpene lactones which have been reported to possess antimicrobial activity but may degrade with prolonged extraction times.

The Soxhlet extraction, despite producing a higher yield than maceration and ultrasonic-assisted methods, did not result in proportionally higher antimicrobial activity. This suggests that prolonged heating and repeated extraction cycles may extract additional compounds that do not contribute to antimicrobial activity or may even dilute or interfere with the active compounds.

The aqueous extract demonstrated the weakest antimicrobial activity despite using a much higher extraction volume and duration compared to other methods. This can be explained by the limited solubility of many antimicrobial compounds in water, particularly non-polar compounds such as terpenoids and certain alkaloids. Water primarily extracted polar compounds like tannins and some saponins, which have weaker antimicrobial effects against Staphylococcus aureus compared to the broader spectrum of compounds extracted by ethanol.

The difference in phytochemical profiles among the extraction methods correlates with their antimicrobial activities. Ethanol, being a semi-polar solvent, extracted a wider range of compounds including both polar and non-polar constituents, resulting in a more comprehensive phytochemical profile and enhanced antimicrobial activity.

When comparing the activities of the extracts to the standard antibiotic (Tetracycline), even the most potent extract (ultrasonic-assisted at 200 mg/mL) showed smaller inhibition zones. However, it is important to consider that these are crude extracts containing multiple compounds at relatively low concentrations, whereas the antibiotic is a pure compound at a lower concentration. Further isolation and purification of active compounds from the extracts would likely yield compounds with higher specific activity.

The concentration-dependent antimicrobial activity observed for all extracts is consistent with other studies on plant antimicrobials and suggests that higher concentrations result in increased bacterial membrane disruption and interference with cellular processes.

These findings have significant implications for the development of Elephantopus scaber-based antimicrobial agents. Ultrasonic-assisted extraction offers several advantages including reduced extraction time, improved yields of active compounds, and enhanced antimicrobial potency. This method also uses less solvent compared to Soxhlet extraction, making it more environmentally friendly and cost-effective for large-scale applications.

Conclusion

This study demonstrates that the extraction method significantly influences the antimicrobial activity of Elephantopus scaber leaves against Staphylococcus aureus. Among the methods evaluated, ultrasonic-assisted extraction using ethanol as solvent produced the most active extract, followed by maceration and Soxhlet extraction. The aqueous extract exhibited the weakest antimicrobial activity.

The enhanced antimicrobial activity observed with ultrasonic-assisted extraction can be attributed to more efficient extraction of bioactive compounds, particularly flavonoids, while minimizing compound degradation through shorter extraction times. These findings highlight the importance of optimizing extraction methods to maximize the therapeutic potential of medicinal plants.

The results also suggest that Elephantopus scaber leaves contain promising antimicrobial compounds that could be further investigated for development as alternative or complementary treatments against Staphylococcus aureus, including antibiotic-resistant strains. Future research should focus on isolating and identifying the specific compounds responsible for the observed antimicrobial activity, as well as conducting comprehensive toxicity studies to evaluate safety for potential therapeutic applications.

Furthermore, the use of more advanced extraction techniques such as microwave-assisted extraction, supercritical fluid extraction, or pressurized liquid extraction could be explored to potentially improve the yield and activity of antimicrobial compounds from Elephantopus scaber leaves.

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