MATERIALS AND METHODS

Sample collection and identification: Complete organisms of Artemisia vulgaris, collected and donated from a private garden in southern Laredo, Texas, were taken to Mr. Willis E. Gentry, a United States Department of Agriculture Identifier, to confirm the correct species was gathered. The study was conducted from February 2014-May 2014 as part of an undergraduate Medicinal Chemistry class at Texas A and M International University, Laredo, Texas.

Table 1:	Summary of procedure

Extraction process: The extraction method from Ynalvez et al. (2012) was adopted with few modifications. The plants were sundried for five days over aluminum foil and then introduced in small pieces into a laboratory oven at 60°C for a day. Once dried, the leaves, stems and roots were partially crushed utilizing a mortar and pestle. The pieces were enveloped in cheesecloth (in place of a thimble) and introduced into a Soxhlet apparatus. The solvents chosen were the following: petroleum ether, acetone and 90% ethanol in water. Each solvent was used for continuous extraction until the solvent in the arm of the extractor was clear (Table 1).

The solvents of the acetone and ethanol/water extracts (petroleum ether extract was discarded) were evaporated using a Heidolph rotary evaporator at a temperature no higher than 60°C. A Branston sonicator was employed to remove the residues from the flasks by adding about 35 mL of distilled water. The remaining liquid from the evaporation procedure, were displaced to two 50 mL VWR centrifuge tubes and frozen in a -80°C freezer. Then the tubes were uncapped and covered with kimwipes secured with rubber bands to then be lyophilized in a LABCONCO FreeZone 2.5 freeze dryer until all the water was removed and each tube had powder contents. Concentrations of 5, 10, 25, 50 and 100 mg mL^–1 were prepared for each organic sample.

Microorganism used: Antimicrobial studies were carried out using gram positive bacteria Staphylococcus aureus (Presque Isle Cultures 4651), Methicillin Resistant Staphylococcus aureus (Presque Isle Cultures 4656) and Bacillus subtilis (Presque Isle Cultures 420). These organisms were chosen based on their availability.

Inoculation: Antimicrobial analysis was carried out using cultured Methicillin Resistant Staphylococcus aureus, Staphylococcus aureus and Bacillus subtilis. One colony was transferred to a test tube containing 2 mL of LB broth; the process was repeated eight more times in addition to two negative controls. The tubes were left in a water bath shaker set to 110 rpm for 16-18 h at 37°C. Turbidity of the broth was adjusted to the value of absorbance 0.132±0.005 at 625 nm on a spectrophotometer (Bausch and Lomb Spectronic 20). Hundrad microliter of the newly adjusted inoculum was added to 10 previously prepared Mueller-Hinton agar plates, distributing the inoculum evenly utilizing sterile L-shaped rods.

Biological assay: The disc diffusion method was employed to determine antibacterial inhibition of the plant extracts (Bonev et al., 2008; Ynalvez et al., 2012). Each 6 mm filter paper disc was infused with 20 μL of each plant extract (ethanol and acetone) and extract concentrations along with DMSO (negative control) and ready bought penicillin discs (positive control). After incubation for 18-20 h at 38°C, the zones of inhibition were measured in mm. The tests were repeated five times and were set up in sextuplicate.

RESULTS

Preliminary screening of the antimicrobial properties for the ethanol and the acetone extracts of A. vulgaris were tested using DMSO as negative control and Penicillin as positive controls. The negative control showed no inhibition, which indicates that any inhibition observed in the bioassay can be attributed to the penicillin (positive control) or the antibacterial properties exhibited by active component of the extracts. Antimicrobial activity of petroleum extract was not done because the sample size was not enough for analysis. Result of the bioassays is presented in the Fig. 1(a-b).

Acetone extract
Bacillus subtilis: Artemisia vulgaris extracts prepared from acetone showed most activity against B. subtilis. Result of antimicrobial activity of acetone extract is represented in the graph below. The result shows acetone extract had a positive correlation (R² = 0.998) against B. subtilis as evidenced by an increase in the Zone Of Inhibition (ZOI) as the concentration of acetone extract was increased (Fig. 2). The acetone extracts had well-defined zones of inhibition.

It took 5 mg mL^–1 of acetone extract just like B. subtilis to inhibit the growth of S. aureus. The inhibition zones however, were not as large when compared to inhibition zones of B. subtilis and it had a much weaker correlation coefficient (R² = 0.840).

Methicillin resistant Staphylococcus aureus: The activity of A. vulgaris acetone extract against MRSA was observed at 25 mg mL^–1, whereas the activity of the ethanol extract was not observed in any of the concentrations.

Fig. 1(a-b):

Antibacterial activities of Artemisia vulgaris extracts against Bacillus subtilis, Staphylococcus aureus and MRSA, (a) AcBS: Acetone extracts against Bacillus subtilis, AcSa: Acetone extracts against Staphylococcus aureus, AcMRSA: Acetone extracts against MRSA and (b) EtBs: Ethanol extracts against Bacillus subtilis, EtSa: Ethanol extracts against Staphylococcus aureus and EtMRSA: Ethanol extracts against MRSA

Fig. 2:	Antibacterial activities of Artemisia vulgaris acetone extract against Bacillus subtilis and Staphylococcus aureus

Ethanol extract: Unlike acetone extract, the ethanol extract was not very active against all strains tested. Antimicrobial inhibition was observed at 25 mg L^–1 against B. subtilis and 50 mg mL^–1 against S. aureus and no inhibition against MRSA.

DISCUSSION

The results of both bioassays indicate that the acetone extracts have a greater activity against both strains tested bacteria than ethanol extract. Various reasons can explain why such behavior occurred between the two extracts. Sequential extraction with solvents of increasing polarity allows different solvents to extract different compounds in the plant material. In this case, the active component was extracted more efficiently using acetone than ethanol, thus the zones of inhibition of acetone were more well-defined because there is a higher concentration of the active component in the acetone extract. This indicates that acetone is able to extract more compounds that are active antibacterials than the ethanol extract. Previous studies on acetone extracts of plants revealed greater antibacterial activity of less polar extracts compared to more polar solvents (Doughari and Okafor, 2008; Eloff et al., 2008). This can explain the higher activity observed in this current study. Furthermore, different parts of the plant contain different active components (Cowan, 1999). In this study, the leaves stem and roots of the A. vulgaris plant were all crushed together and placed in the Soxhlet apparatus. This can result in either enhanced antimicrobial activity (as a result of active compounds from other parts) or reduced antimicrobial activity (as a result of dilution effects from other compounds from other parts). The slightly higher antimicrobial activity against S. aureus in this study (5 mg mL^–1) compared to previous studies (6.25, 10 mg mL^–1) can be inferred that other parts of A. vulgaris contain compounds that exhibit antibacterial activity.

Valsaraj et al. (1997) reported ethanolic extract of leaf of A. vulgaris having antibacterial activity against Staphylococcus aureus of 6.25 mg mL^–1 which was quite different from what was reported in this current study. This current study involved whole plant as opposed to leaf. Extra compounds extracted from the other parts of the plant (stem and root) enhanced the concentration of the active components and hence increased the potency of the extract. When the antibacterial activity of the ethanolic extract in this study against S. aureus (25 mg mL^–1) is compared to that reported in literature (~10 mg mL^–1), it can be inferred that the active antibacterial component was extracted during the extraction with acetone, hence the observed great antibacterial activity with acetone extract (Chen et al., 1989).

CONCLUSION

Overall, the suspected antibacterial activity of A. vulgaris was clearly determined to be attributed to secondary compounds in the acetone extract. The MIC for acetone extract against B. subtilis was 5 mg mL^–1 and against S. aureus was 5 mg mL^–1. While the ethanol extract was 25 mg mL^–1 against B. subtilis, 50 mg mL^–1 against S. aureus and no activity against MRSA. This justifies the use of A. vulgaris in traditional medicine for microbial infections. Because the plant’s extracts were not purified before the bioassay, it is not known which compound(s) in the plant and in which part of it, exhibit the shown antibacterial properties. As such, purification of the acetone and ethanol extracts may give better results and thus, offer better insight to the behavior of the active compound(s) of A. vulgaris. Further research may give the lead compound which can be modified later to optimize its activity against bacterial infections.

ACKNOWLEDGMENTS

The authors express their appreciation to Kassandra Compean, Elester Williams, Idalia Lim and Dr. Ruby Ynalvez for helping them with the bioassays. The Labconco freeze-dry system was acquired through NSF-MRI grant award (NSF DBI 0959395) to Ruby Ynalvez.