*3.2. Antibacterial Activity against Antibiotic-Resistant Bacteria*

The agar diffusion method was used to evaluate systematically the antibacterial activity of 67 spice extracts on antibiotic-resistant *S. aureus* SJTUF 20978 and *S. enteritidis* SJTUF 10987, with each standard strain used as the comparison. The results of DIZ were presented in Table 1. A significant variation in the antibacterial activity reflected by different DIZ values was observed, depending on the type of spice extracts and the subject bacteria.

For antibiotic-resistant *S. aureus* SJTUF 20978, a significant proportion of the spices (38, approximately accounting for 56.7% of the total tested samples) exhibited antibacterial activity, with DIZ in the range of 8.7–25.6 mm. Besides, 11 of these spice extracts (accounting for 16.4%) showed a relatively superior antibacterial activity, with DIZ values greater than 15 mm. Among there, galangal, the rhizome of *Alpinia galanga* (L.) Willd., showed exceptional antibacterial capacity, with the DIZ reaching 25.6 mm, followed by cinnamon, fructus galangae (ripe fruit from galangal), yellow mustard seed, rosemary, and marjoram, with DIZ values of 20.7, 20.2, 18.6, 18.3, and 17.2 mm, respectively. Moreover, the MIC and MBC results of these 11 samples were determined and were shown in Table 3. The MIC values ranged from 0.40–6.25 mg/mL, and MBC values ranged from 0.40–12.5 mg/mL, which were one or two times higher than MICs. Among them, clove fruit, sage, rosemary, and liquorice had the lowest MIC value (0.40 mg/mL), and fructus galangae, galangal, and yellow mustard seed showed a relatively high value (6.25 mg/mL). However, for drug-resistant *S. enteritidis* SJTUF 10987, the overall antibacterial effects of tested spice extracts were relatively low, and only four samples showed inhibitory activity, including cinnamon (DIZ = 16.0 mm), male clove (DIZ = 11.0 mm), thorn amomum villosum (DIZ = 8.6 mm), and female clove (DIZ = 8.5 mm).


**Table 3.** Minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) values of selected 11 spice extracts with good antibacterial activity.

In general, the antibacterial activity of spice extracts against antibiotic-resistant bacteria was somewhat less effective compared to corresponding standard strains *S. aureus* ATCC 25923 and *S. enteritidis* ATCC 13076 (data shown in Table 1). Besides, spice extracts showed much better antibacterial activity against Gram-positive *S. aureus* than Gram-negative *S. enteritidis*. Therefore, we further tested whether spice extracts had a broad spectrum antibacterial effect on drug-resistant *S. aureus*. 11 spice extracts with DIZ more than 15 mm on drug-resistant *S. aureus* SJTUF 20978 were selected to verify their antibacterial capacity against another 10 antibiotic-resistant strains of *S. aureus*. As shown in Figure 1, all selected spice extracts exhibited inhibitory effects against the validated antibiotic-resistant strains of *S. aureus*. Among them, galangal (DIZ = 25.6–31.1 mm), fructus galangae (DIZ = 21.2–27.8 mm), and cinnamon (DIZ = 18.3–25.1 mm) showed the strongest antibacterial effects, followed by yellow mustard seed (DIZ = 18.0–21.4 mm) and rosemary (DIZ = 16.2–19.9 mm). Overall, selected spice extracts possessed a broad spectrum antibacterial effect against antibiotic-resistant *S. aureus*.

**Figure 1.** The antibacterial activity of selected spice extracts on 10 antibiotic-resistant strains of *S. aureus*.

#### *3.3. Cytotoxicity of Spice Extracts*

Spices were generally considered to be non-toxic or less toxic because of their natural origin and long use as food additives and medicine for ailment treatments. Meanwhile, some studies on the efficacy and safety of plants pointed out that some phytochemicals displayed certain cytotoxicity, genotoxicity, and carcinogenic effects when used chronically [30]. Therefore, it was necessary to determine the cytotoxicity of the selected 11 spice extracts with good activity against multi-drug resistant *S. aureus*. The cytotoxicity of these chosen 11 spices were determined by using the in vitro assay with HFF cells, and LC50 values were calculated. Of 11 spices tested, eight spice extracts did not show any cytotoxicity against HFF cells after 24 h of treatment with the highest concentration tested (100 μg/mL), suggesting that their LC50 values higher than 100 μg/mL. However, other three spices, including galangal, rosemary, and sage, were able to inhibit the growth of HFF cells at the LC50 of 9.32 ± 0.83, 19.77 ± 2.17, and 50.54 ± 2.57 μg/mL, respectively, indicating their potential safety issue. Overall, the results found that most spice extracts with high antibacterial effect were low toxic, and could be used as potential antimicrobial agents in food industry.
