**3. Results**

#### *3.1. Extraction Yield and Extracts Characteristics*

Yield of extractable compounds obtained from using different solvents evaluated and presented in Table 1. In general, the ranking of extraction yields obtained from different solvents are as follows in decreasing order: methanol > ethanol > water > hexane. There was a significant difference in yield between solvents used in extraction except for methanolic and ethanolic extracts of Tasmanian pepper leaf and ethanolic and water extracts of anise myrtle.

**Table 1.** Yields, total phenolic content, DPPH free radical scavenging and reducing power of Tasmanian pepper leaf, lemon myrtle and anise myrtle extracts.


DPPH: 2,2-diphenyl-1-picrylhydrazyl; LM: lemon myrtle; TPL: Tasmanian pepper leaf; AM: anise myrtle; mg GAE/gDW: milligram gallic acid equivalents/g dry weight. Reducing power results expressed from testing concentrations of 0.01 and 0.1 mg/mL of extracts. Means with different letters within the same row are significantly different at *p* < 0.05.

#### *3.2. Total Phenolic Content*

The TPC of all herbs from different solvent extracts shown in Table 1. Results of the total extractable phenols expressed as mg gallic acid equivalent (GAE) per g of sample dry weight. Lemon myrtle possessed the highest TPC followed by anise myrtle and Tasmanian pepper leaf. The extraction of phenolic compounds varied among solvents used in this experiment, methanol extracts had the highest phenols followed by ethanol, water and hexane.

#### *3.3. DPPH Radical Scavenging Activity*

The free radical scavenging capacity of herb extracts from different solvents given in Table 1. The results expressed in IC50 (half-maximal inhibitory concentration) values are interpreted as the concentration of antioxidants required to reduce the free radical DPPH by half in the solution; lower IC50 values represent strong free radical scavenging activity. The strongest scavenging capacity are shown in the extracts of lemon myrtle, followed by anise myrtle and Tasmanian pepper leaf. The radical scavenging activities of extracts significantly varied with different solvents and ranked in decreasing order as methanol = ethanol > water > hexane.

## *3.4. Reducing Power*

The reducing potential of a substance reflects its antioxidant capacity measured by utilizing the reducing power assay. The presence of antioxidant compounds (reductants) in the tested herb extracts, will react with the potassium ferricyanide (Fe3+) reducing it into potassium ferrocyanide (Fe2+) causing color transformation of the assay solution from yellow into different color spectrums of green and yellow which can be measured at 700 nm. Table 1 shows the reducing power of herbs extracted from different solvents. The reducing power ranking of herb extracts follows the same pattern (lemon myrtle > anise myrtle > Tasmanian pepper leaf) as the previous two assays, DPPH and TPC. The extracts reducing power potential is concentration dependent; it increases as extract concentration increases. Methanol and ethanol extracts had the highest reducing power, followed by water extracts having a moderate reducing potential while hexane extracts showed the least reducing capacity.

#### *3.5. Relationship between Total Phenolic Content and Antioxidant Capacities*

The antioxidant capacities of all tested herb extracts measured by the two assays DPPH and reducing power were found to have a significant linear correlation with the TPC. A regression analysis, correlation coefficient, performed to correlate TPC with reducing power (Figure 1a) and TPC with DPPH (Figure 1b). The overall correlation coefficient (R) between TPC and DPPH was −0.870 and between TPC and reducing power was 0.958. The correlation coefficient (R) values between TPC and DPPH were −0.988, −0.966 and −0.939 and between TPC and reducing power were 0.907, 0.979 and 0.983 for anise myrtle, lemon myrtle and Tasmanian pepper leaf, respectively. The strong relationship between TPC and antioxidant results is a clear indication that the phenolic compounds of the herb extracts contributed to the antioxidant capacity.

**Figure 1.** Correlation between total phenolic content and antioxidant capacities measured by DPPH and reducing power in Australian native herbs. (**a**) Correlation between reducing power and total phenolic content; (**b**) Correlation between DPPH and total phenolic content.

### *3.6. Antimicrobial Activities*

The mean values and standard deviations for inhibition zones of herb extracts from various solvents and standard antimicrobial drugs against yeasts and bacteria, using agar disc diffusion method, are given in Table 2. The herb extracts from different solvents showed varied antimicrobial activities against the tested microorganisms. Hexane extracts produced the largest inhibition zones showing strong antifungal activity (inhibition zone > 15 mm) in most weak-acid resistant yeasts, followed by methanol and ethanol extracts, while water extracts showed no activity against all tested microorganisms. Extracts from hexane, methanol and ethanol showed broad-spectrum antimicrobial activity against tested microorganisms showing moderate activity (inhibition zone 10 to 15 mm) and strong activity, except for the methanolic and ethanolic extracts of lemon myrtle and anise myrtle, which did not show inhibition against *E. coli*. In addition, the hexane extract of anise myrtle showed no inhibition against *E. coli.* The standard fluconazole had broad-spectrum activity against all tested yeasts showing moderate and strong activity, while amphotericin B had a narrow-spectrum that only inhibited *D. anomala*, *S. pombe* and *R. mucilaginosa*, with inhibition zones between medium and strong activity*.* All hexane extracts had significantly higher zones of inhibition in comparison to fluconazole

and amphotericin B at the tested concentrations, except against *D. anomala* for Tasmanian pepper leaf and anise myrtle. Furthermore, methanol and ethanol extracts of lemon myrtle and anise myrtle exhibited comparable inhibition zones to fluconazole and amphotericin B. In the case of bacteria, chloramphenicol showed significantly higher zones of inhibition than herb extracts. The negative controls, ethanol and water, produced no zones of inhibition.

**Table 2.** Inhibition zone (mm) of Australian native herb extracts from hexane, methanol and ethanol against yeasts and bacteria.


TPL: Tasmanian pepper leaf; LM: lemon myrtle; AM: anise myrtle; M: methanol; E: ethanol; H: hexane; NT: not tested. Extract concentration of 2 mg/6 mm disc. Positive controls: 30 μg/6 mm disc chloramphenicol against bacteria, 20 μg/6 mm disc fluconazole and amphotericin B against yeasts. Columns sharing different letters within the same herb extract treatment are significantly different at *p* < 0.05. a Tasmanian pepper leaf; b lemon myrtle and c anise myrtle. Water extracts showed no inhibition. Criteria for antimicrobial activity: <10 mm, weak; 10–15 mm, moderate; >15 mm, strong.

#### *3.7. Mode of Antifungal Action*

Lemon myrtle hexane extract showed the most effective anti-yeast activity and selected to investigate the antifungal mechanism of action against *S. cerevisiae. S. cerevisiae* is a widely studied eukaryotic model yeas<sup>t</sup> with a relatively large cell size for better observation and investigation for morphological changes [36]. The mechanism of antifungal action of hydrophobic bioactive compounds is reported as penetrating and damaging cellular cytoplasmic membrane [37,38]. Images of *S. cerevisiae* untreated, 30 min and 60 min treated cells are given in Figure 2. The untreated cells appear to have a normal elongated-oval shape with no signs of deformation or damage to cell structure (Figure 2a). Cells exposed to treatment for a period of 30 min have undergone structural modification where cells became swollen and changed from being oval into a circular shape; in addition, in some cells the membrane collapsed and reduced in size (Figure 2b). When treatment increased to 60 min, cell damage became more profound showing cells with ruptured membrane (Figure 2c). The DAPI staining observed to be brighter on damaged cells compared to non-damaged cells this could be due to the stain accumulating on the rough layer of damaged cell membrane. It was also noticeable when cells were exposed to the fluorescence microscope light source for a longer period, the stain started to fade away at a faster rate only in membrane ruptured cells. This phenomenon indicates that the stain is contained within the boundary of non-ruptured cells and is protected inside the cell and requires a longer light exposure time to fade away compared to the stain that has leaked out of the damaged cells.

**Figure 2.** Illustration of morphological changes to yeas<sup>t</sup> cells of *S. cerevisiae* at different treatment stages. (**a**) Untreated cells having a normal oval-shape; (**b**) Lemon myrtle extract (4% *v*/*v*) cell treated for 30 min showing swollen round-shaped cells next to a membrane damaged cell and (**c**) treatment for 60 min exhibits clear cell membrane rupture.

#### *3.8. UHPLC-MS Analysis of Herb Extracts*

UHPLC-MS analysis showed hexane extracts contained the highest concentration of major compounds, polygodial, citral and anethole, whereas no detectable readings were found in water extracts due to the hydrophobic characteristic of these compounds. Methanol and ethanol extracts indicate similar concentration of major compounds, which were significantly lower (*p* < 0.05) than the hexane extracts (Figure 3). The major compounds identified in herb extracts and their extracted quantities are listed as follows: polygodial found in Tasmanian pepper leaf at 41.70%, 4.43% and 3.91%; citral found in lemon myrtle at 45.7%, 9.62% and 9.29%; and anethole found in anise myrtle at 37.1%, 1.80% and 4.16%; from hexane, ethanol and methanol, respectively.

**Figure 3.** *Cont*.

**Figure 3.** Chromatogram of major compounds extracted from Australian native herbs in different solvents. Citral in lemon myrtle extracts (**a**); polygodial in Tasmanian pepper leaf extracts (**b**) and anethole in anise myrtle extracts (**c**).
