# Antimicrobial Air Filter Coating with Plant Extracts Against Airborne Microbes
## 3 Results and Discussion  
### Comparison of the Antimicrobial Activities of the Three Plant Extracts  
The antimicrobial activity of the tea-tree oil, rosemary, and garlic against two microbial strains was investigated and is presented in Figure 2. These plant extracts showed antimicrobial activities against both gram-positive and negative strains. All plant extracts showed a dose-dependent pattern of the antimicrobial function. Cell inactivation increased with the concentrations of plant extracts. Typically, the tea-tree oil showed a higher cell inactivation than the other two plant extracts. To quantitatively compare the extract's antimicrobial activities, the MIC50 (minimum inhibition concentration) values, where 50% of cell inactivation is found, were calculated. The MIC50 values against the _E. coli_ were 1.62, 224, and 407 mg/mL for tea-tree oil, rosemary, and garlic, respectively (Table 1). The MIC50 values against the _M. luteus_ were 2.06, 181, and 419 mg/mL for tea-tree oil, rosemary, and garlic, respectively. Tea-tree oil showed potent cell inactivation effects on both _E. coli_ and _M. luteus_ and caused cell inactivation even at low concentration. In contrast, adding rosemary and garlic extracts caused cell inactivation initially, but the cell growth recovered at longer cultivation times (Figures S1 and S2). This suggests that the microorganisms adapted to the antimicrobial mechanism of both rosemary and garlic extracts. However, the tea-tree oil maintained its antimicrobial activity for a long time. For example, a concentration of 0.9 mg/mL of tea-tree oil-induced an approximately 22% inactivation at 1 h after being added and maintained this level of antimicrobial activity for 6 h (Figures S1 and S2). A concentration of 9 mg/mL tea-tree oil inhibited the cell growth by approximately 53% after 1 h, and by 88 and 82% for _M. luteus_ and _E. coli_, respectively, at 6 h (Figures S1a and S2a). These results indicate that the antimicrobial activity of the tea-tree oil increased as the culturing time increased, implying that the microorganisms did not adapt to the tea-tree oil during cultivation time. Rosemary and garlic extracts showed cell inactivation at higher concentrations and a clear decrease of antimicrobial activity during cell culturing. This could be due to their weaker antimicrobial properties or their instability in aqueous solutions. The phytochemicals present in the plant extracts chosen are listed in Table 2. The plant extracts were composed of 30, 6, and 6 phytochemicals for tea-tree oil, rosemary, and garlic, respectively. Typically, tea-tree oil contains various phytochemicals. We found through a literature survey that twenty-two phytochemicals in tea-tree oil are related to antimicrobial activities. Among them, thirteen chemicals--\(\alpha\)-pinene, \(\beta\)-pinene, \(\alpha\)-phellandrene, \(\alpha\)-terpinene, cymene, dipentene (D-limonene), eucalypt (1,8-cineole), \(\gamma\)-terpinene, terpinolene, linalool, \(\alpha\)-terpineol, (-)-terpinen-4-ol, and (-)-globul--exhibited an antimicrobial activity in previous studies. Nine compounds--sabinene, \begin{table}
\begin{tabular}{c c c} \hline \hline  & _E. coli_ & _M. luteus_ \\ \hline Tea-tree oil & 1.62 & 2.06 \\ Rosemary & 224 & 181 \\ Garlic & 407 & 419 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Minimum inhibitory concentration 50 (MIC50) of the plant extracts against _E. coli_ and _M. luteus_ (unit: mg/mL). trans-\(\beta\)-ocimene, (-)-trans-pinocarveol, \(\beta\)-terpineol, isoborneol, (-)-\(\alpha\)-gurjunene, (+)-aromandrene, \(\beta\)-selinee, viridioflorol--were proven to feature antimicrobial activities in other studies (references are listed in Table 2). A further nine phytochemicals--sabinene, trans-\(\beta\)-ocimene, (-)-trans-pinocarveol, \(\beta\)-terpineol, isoborneol, (-)-\(\alpha\)-gurjunene, (+)-aromandrene, \(\beta\)-selinee, and viridioflorol--were found as components in antimicrobial plant extracts.