Antioxidant, Antibacterial, Enzyme Inhibitory, and Anticancer Activities and Chemical Composition of Alpinia galanga Flower Essential Oil

Alpinia galanga is widely cultivated for its essential oil (EO), which has been used in cosmetics and perfumes. Previous studies of A. galanga focussed mostly on the rhizome but seldom on the flower. Therefore, this study was designed to identify the chemical composition of A. galanga flower EO and firstly estimate its antioxidant, antibacterial, enzyme inhibitory, and anticancer activities. According to the results of the gas chromatography with flame ionization or mass selective detection (GC-FID/MS) analysis, the most abundant component of the EO was farnesene (64.3%), followed by farnesyl acetate (3.6%), aceteugenol (3.2%), eugenol (3.1%), E-nerolidol (2.9%), decyl acetate (2.4%), octyl acetate (2.0%), sesquirosefuran (1.9%), (E)-β-farnesene (1.7%), and germacrene D (1.5%). For the bioactivities, the EO exhibited moderate DPPH and ABTS radical scavenging effects with IC50 values of 138.62 ± 3.07 μg/mL and 40.48 ± 0.49 μg/mL, respectively. Moreover, the EO showed strong-to-moderate antibacterial activities with various diameter of inhibition zone (DIZ) (8.79–14.32 mm), minimal inhibitory concentration (MIC) (3.13–6.25 mg/mL), and minimal bactericidal concentration (MBC) (6.25–12.50 mg/mL) values against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, and Proteus vulgaris. Interestingly, the EO possessed remarkable α-glucosidase inhibition (IC50 = 0.16 ± 0.03 mg/mL), which was equivalent to that of the positive control acarbose (IC50 = 0.15 ± 0.01 mg/mL) (p > 0.05). It showed moderate tyrosinase inhibition (IC50 = 0.62 ± 0.09 mg/mL) and weak inhibitory activity on acetylcholinesterase (AChE) (IC50 = 2.49 ± 0.24 mg/mL) and butyrylcholinesterase (BChE) (IC50 = 10.14 ± 0.59 mg/mL). Furthermore, the EO exhibited considerable selective cytotoxicity to K562 cells (IC50 = 41.55 ± 2.28 μg/mL) and lower cytotoxicity to non-cancerous L929 cells (IC50 = 120.54 ± 8.37 μg/mL), and it induced K562 cell apoptosis in a dose-dependent manner. Hence, A. galanga flower EO could be regarded as a bioactive natural product with great application potential in the pharmaceutical field.


Introduction
Essential oils are secondary plant metabolites with strong odors and are composed of a variety of volatile compounds [1,2]. Approximately 3000 essential oils are generated from more than 2000 plants, among which 300 are valuable from a commercial standpoint [3]. Essential oils have been employed extensively in pharmaceutical, agriculture, food, perfume, cosmetic, and sanitary industries due to their various pharmacological and biological effects, including antibacterial, antioxidant, anticancer, antidiabetic, virucidal, fungicidal, anti-inflammatory, analgesic, antimutagenic, and antiprotozoal activities [3][4][5][6]. As synthetic

Antioxidant Activity
DPPH and ABTS radical scavenging tests were used to assess the antioxidant activity of A. galanga flower EO, and the findings are presented in Table 2. Compared with BHT and ascorbic acid, the EO exhibited moderate DPPH and ABTS radical scavenging effects, with IC 50 values of 138.62 ± 3.07 µg/mL and 40.48 ± 0.49 µg/mL, respectively. Eugenol, which is a phenolic compound used as a spice, has been well established as possessing potent antioxidant and free-radical-scavenging activities in both in vivo and in vitro studies [40,41]. Aceteugenol, which is an acetylated derivative of eugenol, displayed significant activity in scavenging DPPH free radicals, with an IC 50 of 0.12 ± 0.03 µmol/L, and it showed a synergistic effect with eugenol regarding inhibiting the oxidation of sunflower oil [42]. Furthermore, isoeugenol is an isomer of eugenol, and methyleugenol is a methylated derivative of eugenol. These eugenol-related compounds may play a key role in the antioxidant activity of A. galanga flower EO. According to past reports, farnesyl acetate, E-nerolidol, octyl acetate, and germacrene D exhibit moderate-to-weak antioxidant effects [43][44][45]. Hence,

Antibacterial Activity
The antibacterial properties of A. galanga flower EO were tested using diameter of inhibition zone (DIZ), minimum inhibition concentration (MIC), and minimum bactericidal concentration (MBC) values ( Table 3). The EO exhibited a broad-spectrum antibacterial property against the tested bacterial strains, with DIZ values ranging from 8.79 to 14.32 mm. Based on previous research, MIC values under 5 mg/mL are thought to have strong antibacterial activity [46]. Hence, the EO displayed a strong antibacterial effect against Staphylococcus aureus (MIC = 3.13 mg/mL, MBC = 6.25 mg/mL), Bacillus subtilis (MIC = 3.13 mg/mL, MBC = 6.25 mg/mL), Pseudomonas aeruginosa (MIC = 3.13 mg/mL, MBC = 12.50 mg/mL), and Proteus vulgaris (MIC = 3.13 mg/mL, MBC = 6.25 mg/mL), and showed moderate antibacterial property against Enterococcus faecalis (MIC = 6.25 mg/mL, MBC = 12.50 mg/mL) and Escherichia coli (MIC = 6.25 mg/mL, MBC = 12.50 mg/mL). Several studies attributed the antibacterial activity of essential oils to the active ingredient farnesene [47,48]. In a previous study, farnesyl acetate showed significant antibacterial activity against Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis, with MIC values ranging from 20 µg/mL to 28 µg/mL [49]. Additionally, other major constituents, including aceteugenol, eugenol, E-nerolidol, and germacrene D, were shown to possess antibacterial properties [45,[50][51][52]. Therefore, the significant antibacterial activity of the EO can be explained by the presence of these main components. According to these findings, A. galanga flower EO could be employed in the pharmaceutical field as a novel natural antibacterial agent.

Enzyme Inhibitory Activity
The A. galanga flower EO was tested for its ability to inhibit the following enzymes: α-glucosidase, tyrosinase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). All results are summarized in Table 4.  50 : the dose of the sample that inhibits 50% of enzyme activities. a,b Significant differences (p < 0.05) are indicated by different letters in the same column. * Galanthamine: IC 50 (µg/mL).
The A. galanga flower EO exhibited remarkable α-glucosidase inhibitory ability (IC 50 = 0.16 ± 0.03 mg/mL), and its inhibitory effect was equivalent to that of the positive control acarbose (IC 50 = 0.15 ± 0.01 mg/mL) (p > 0.05). Acarbose, which is an α-glucosidase inhibitor, is widely used to treat type 2 diabetes, as it can lower postprandial insulin and blood glucose levels by delaying carbohydrate absorption [53]. Concerns about acarbose's side effects, such as flatulence, diarrhea, and abdominal distension, have prompted the search for natural products that inhibit α-glucosidase's activity as complementary/alternative treatments for type 2 diabetes [54]. An earlier study demonstrated that farnesene, which is the most predominant component of the A. galanga flower EO, exerted significant α-glycosidase inhibitory activity [55]. Farnesyl acetate reduced postprandial blood glucose levels and was a potential α-glucosidase inhibitor [56]. In addition, past studies demonstrated that other major components, including aceteugenol, eugenol, and E-nerolidol, had α-glycosidase inhibitory effects [57][58][59]. Thus, the remarkable αglucosidase inhibitory activity of A. galanga flower EO could be attributed to these main constituents, and it could be used in the pharmaceutical industry as a new source of natural α-glucosidase inhibitors.
As depicted in Table 4, compared with the positive control arbutin (IC 50 = 0.19 ± 0.06 mg/mL), A. galanga flower EO showed a moderate tyrosinase inhibitory effect (IC 50 = 0.62 ± 0.09 mg/mL). Tyrosinase is a crucial enzyme in the enzymatic browning of fruits and mammalian melanogenesis [5]. Eugenol had a significant inhibitory effect on tyrosinase and could be used as a tyrosinase inhibitor [60]. Moreover, according to the research of Arung et al., eugenol and eugenol acetate significantly inhibit melanin formation in B16 melanoma cells [61]. Hence, the moderate tyrosinase inhibition of A. galanga flower EO may have been due to these main components.
As shown in Table 4, compared with the positive reference galanthamine, A. galanga flower EO showed weak inhibitory activity against AChE and BChE, with IC 50 values of 2.49 ± 0.24 mg/mL and 10.14 ± 0.59 mg/mL, respectively. Cholinesterase inhibitors enhance cholinergic neurotransmission by inhibiting the decomposition of acetylcholine, which has become an effective strategy for the treatment of Alzheimer's disease [62]. Eugenol, which is a major component of EO, was reported to display significant inhibition of AChE (IC 50 : 42.44 ± 1.21 µg/mL) and BChE (IC 50 : 63.51 ± 1.88 µg/mL) [63]. E-Nerolidol significantly reduces the AChE activity and oxidative/nitrosative stress, improves locomotor activity, and reverses motor incoordination and cognitive impairment after weight-drop-induced traumatic brain injury (TBI) in rats [64]. Thus, these major compounds could be responsible for the anti-cholinesterase activity of A. galanga flower EO.

Anticancer Activity
The cytotoxic properties of A. galanga flower EO were investigated against human tumor cell lines (lung adenocarcinoma A549 cells, prostatic carcinoma PC-3 cells, leukemic K562 cells, and non-small-cell lung cancer NCI-H1299 cells) and a non-cancerous cell line (murine fibroblast L929 cells) using MTT assays. Cisplatin was used as a positive control. As shown in Table 5, A. galanga flower EO showed significant cytotoxic activities against the four human tumor cells, with IC 50 values ranging from 41.55 ± 2.28 µg/mL to 127.37 ± 4.15 µg/mL. In particular, EO exhibited considerable selective cytotoxicity against K562 cells (IC 50 = 41.55 ± 2.28 µg/mL), and its toxicity was almost 3 times that of non-cancerous L929 cells (IC 50 = 120.54 ± 8.37 µg/mL). Hence, the K562 cells were selected for subsequent studies.  Cancer cells are capable of evading apoptosis; therefore, triggering apoptosis is a key strategy for cancer therapies [65]. As shown in Figure 2A, the results of phasecontrast microscopy revealed typical apoptotic morphological alterations in EO-treated K562 cells, including cell shrinkage and fragmentation. Moreover, AO/EB staining and Hoechst 33,258 staining were used to observe the changes in the nuclear morphology of the K562 cells. According to the AO/EB staining results, after the EO treatment, the percentage of apoptotic cells with orange-red fluorescent nuclei increased, while the percentage of living cells with bright green fluorescent nuclei reduced ( Figure 2B). In the Hoechst 33,258 staining analysis, the nuclei of K562 cells treated with EO displayed brighter blue fluorescence, indicating that the nuclear chromatin of K562 cells was condensed with apoptotic features ( Figure 2C). In addition, the Annexin V-PE/7-AAD staining analysis was performed using a flow cytometer to quantitatively estimate the EO-induced apoptosis. As shown in Figure 3A,B, the percentage of apoptotic cells (Q2 + Q3) increased dose-dependently from 6.88 ± 0.07% in the control to 9.38 ± 1.12%, 18.36 ± 0.20%, 26.47 ± 0.30%, 32.13 ± 0.50%, and 55.04 ± 0.54% at the doses of 20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL, and 160 µg/mL of EO, respectively. These results suggested that EO significantly induced K562 cell apoptosis in a concentration-dependent manner.
Numerous farnesene-rich essential oils, such as those from Zornia brasiliensis, Malus domestica, and Streblus asper, were shown to have anticancer properties [66][67][68][69]. Farnesyl acetate had higher cytotoxicity against the tumor cells malignant melanoma MEWO (IC 50 = 734 µM) and promyelocytic leukemia HL-60 (IC 50 = 121 µM) compared with noncancerous cells (fibroblasts HFIG, keratinocytes HaCaT, and epithelium of the small intestine IEC6) (IC 50 > 1000 µM) [49]. The anticancer effects of eugenol have been well recognized, and it can induce apoptosis in different cancer cells [70]. Furthermore, the anticancer activities of other main components in EO, such as E-nerolidol, (E)-β-farnesene, and germacrene D, were demonstrated in previous research [71][72][73]. Thus, A. galanga flower EO's significant anticancer properties could be explained by these main components. Based on these findings, A. galanga flower EO may serve as a new source of natural anticancer agents in the pharmaceutical field. : the concentration of sample that reduces the cell growth by 50%. Cisplatin was used as a positive control. Cell lines: A549 (human lung adenocarcinoma cells), PC-3 (human prostatic carcinoma cells), K562 (human leukemic cells), NCI-H1299 (human non-small cell lung cancer cells), and L929 (murine fibroblast cells). a-e Significant differences (p < 0.05) are indicated by different letters in the same row.

Plant Material
Alpinia galanga flower was harvested from Yulin City, Guangxi Province, China, in June 2020. It was identified by Prof. Guoxiong Hu from the College of Life Sciences,

Preparation of Essential Oil
The fresh, finely chopped flower of A. galanga (2.5 kg) was hydrodistilled for 4 h in an all-glass Clevenger-type apparatus. After dehydration with anhydrous Na 2 SO 4 , the essential oil (2.76 g, 0.11% w/w) was kept in a sealed vial at 4 • C for further analysis.

Analysis of Essential Oil
Quantitative analysis of the EO was carried out using a gas chromatograph (GC) equipped with a flame ionization detector (FID) (model 6890, Agilent Technologies, Santa Clara, CA, USA). Column: HP-5MS capillary column (60 m × 0.25 mm, 0.25 µm film thickness). The GC settings were as follows: injection volume (1 µL), split ratio (1:20), carrier gas helium (1 mL/min), and oven temperature program (kept at 70 • C for 2 min, increased to 180 • C at 2 • C/min, raised to 310 • C at 10 • C/min, and finally kept at 310 • C for 14 min). The gas chromatograph-mass spectrometer (GC-MS) (model 6890/5975C, Agilent Technologies, Santa Clara, CA, USA) was used for qualitative analysis of the EO. GC parameters and the column were the same as in the GC-FID analysis. The MS was operated as follows: ion source temperature at 230 • C, interface temperature at 280 • C, ionization voltage at 70 eV, and a scan range of m/z 29 to 500 amu. The relative abundance (%) of the chemical constituents was determined using the peak area. Standard n-alkanes (C 9 -C 30 ) were used for the calculation of the retention index (RI). Each component of EO was determined by comparing the RI and mass spectra in the Wiley 275 (Wiley, New York, NY, USA) and NIST 2020 (National Institute of Standards and Technology, Gaithersburg, MD, USA) databases.

DPPH Assay
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging effect was assayed by utilizing Tian et al.'s method [74]. Briefly, the DPPH solution (2 mL, 0.08 mM) was blended with the sample solution (2 mL) and then kept at room temperature for 30 min in the dark. The optical density at 517 nm was recorded. Ascorbic acid and butylated hydroxytoluene (BHT) served as positive controls. Data were presented as IC 50 values.

ABTS Assay
The 2,2 -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging ability was determined by utilizing the method reported by Tian et al. [74]. ABTS• + solution was generated by mixing ABTS solution (50 mL, 0.7 mM) with K 2 S 2 O 8 solution (50 mL, 2.45 mM) and incubating at room temperature for 12 h in the dark. Subsequently, methanol was used to dilute the ABTS• + solution, yielding an absorbance of 0.70 ± 0.02 at 734 nm. The sample solution (0.4 mL) and diluted ABTS• + solution (4 mL) were blended and incubated at room temperature for 10 min in the dark, and then the optical density at 734 nm was measured. BHT and ascorbic acid were used as positive controls. The results were expressed as IC 50 values.

Disc Diffusion Assay
The disc diffusion method was employed to determine the diameter of inhibition zone (DIZ) [5]. In brief, bacterial suspension (100 µL, 1 × 10 6 CFU/mL) was evenly spread on Mueller-Hinton agar plates. Afterward, the filter paper discs (diameter 6 mm) with 20 µL of pure EO or streptomycin (100 µg/mL) were added and incubated for 24 h at 37 • C. Finally, the DIZ was measured and recorded.

MIC and MBC Assays
The microdilution broth assay was utilized to detect the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values [5]. The EO solution (100 mg/mL, w/v in 0.1% DMSO) was two-fold serially diluted with the medium to concentrations of 50.00, 25.00, 12.50, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, and 0.10 mg/mL. Streptomycin solution (100 µg/mL, w/v in distilled water) was also diluted with the medium to the dose range of 0.10-50 µg/mL. The bacterial suspensions (100 µL) were seeded into each well of 96-well plates at a density of 5 × 10 4 CFU/well. Subsequently, 100 µL of the diluted EO or streptomycin solution was added and cultured at 37 • C for 24 h. Resazurin aqueous solution (10 µL, 0.01%) was utilized as a microbial growth indicator, added to each well, and incubated in the dark for 2 h at 37 • C. The minimum sample concentration that did not cause a color change was its MIC value. Furthermore, 10 µL of culture from the wells with no change in color was spread on the Mueller-Hinton agar plate and inoculated at 37 • C for 24 h. The MBC values were determined as the lowest concentration of EO that induced no visible growth of the tested bacteria.
3.6. Enzyme Inhibitory Activities 3.6.1. α-Glucosidase Inhibitory Activity The α-glucosidase inhibitory effect was performed following the protocol reported by Hong et al. [5]. A total of 90 µL of EO solution or acarbose solution (positive control) was mixed with α-glucosidase solution (10 µL, 0.8 U/mL) and added to each well of the 96-well plates. After incubation at 37 • C for 15 min, the reaction was started by adding 10 µL of p-nitrophenyl-α-D-glucopyranoside (p-NPG) substrate (1 mM) and maintained at 37 • C for 15 min, then halted by the addition of 80 µL Na 2 CO 3 solution (0.2 M). Subsequently, the optical density at 734 nm was recorded using a microplate reader (Varioskan Lux Multimode, Thermo Fisher Scientific, Waltham, MA, USA). The results of the α-glucosidase inhibitory activity were presented using IC 50 values.

Tyrosinase Inhibitory Activity
The tyrosinase inhibition was performed based on the protocol reported by Tian et al. and utilized arbutin as a positive control [74]. A total of 70 µL of EO solution and 100 µL of tyrosinase solution (100 U/mL) was mixed and incubated at 37 • C for 5 min in a 96-well plate. After that, the L-tyrosine substrate (80 L, 5.5 mM) was added to start the reaction and incubated at 37 • C for 30 min. Then, the optical density at 492 nm was recorded, and the tyrosinase inhibitory activity was presented using IC 50 values.

Cytotoxic Activity
The cytotoxic activities against human cancerous cells (A549 lung adenocarcinoma cells, PC-3 prostatic carcinoma cells, K562 leukemic cells, and NCI-H1299 non-small-cell lung cancer cells) and non-cancerous cells (L929 murine fibroblast cells) were evaluated using MTT assays with a slight modification [76]. The EO solution (160 mg/mL, w/v in DMSO) or cisplatin solution (40 mg/mL, w/v in DMSO) was diluted with RPMI-1640 medium, where the final concentration of DMSO was lower than 0.1%. The cell suspensions (80 µL) were seeded into a 96-well plate (5 × 10 3 cells/well). After 24 h of incubation, cells were treated with EO (0, 10, 20, 40, 80, and 160 µg/mL) or cisplatin (0, 1.88, 3.75, 7.5, 15, and 30 µg/mL) for 48 h. Subsequently, MTT solutions (10 µL, 5 mg/mL in PBS) were added and incubated for 4 h. Finally, the formazan crystals in each well were dissolved in 150 µL of DMSO, and a microplate reader was used to detect the absorbance at 490 nm. The results of the cytotoxicity were presented as IC 50 values.

Morphology Assay
K562 cells were seeded into a 6-well plate (5 × 10 5 cells/well) and cultured for 24 h. Subsequently, the cells were subjected to the new medium with different doses of EO (0, 20, 40, 80, and 160 µg/mL) for 48 h. Finally, morphological alterations of K562 cells were recorded using a Leica DMi8 inverted microscope (Leica Microsystems, Germany).
Acridine orange/ethidium bromide (AO/EB) staining and Hoechst 33,258 staining assays were used to detect the nuclear morphology changes of the K562 cells. After the treatment with EO as mentioned above, the cell suspensions (1.5 mL) were collected, centrifuged at 1000× g for 5 min, fixed with 4% paraformaldehyde (0.5 mL) for 10 min, washed twice with PBS (1 mL), and resuspended in 50 µL PBS. Afterward, the cell suspension (10 µL) was dropped onto the glass slides. For AO/EB staining, cells were stained with AO/EB dye (10 µg/mL AO and 10 µg/mL EB) for 5 min. After washing twice with PBS (0.5 mL), the stained cells were viewed using a fluorescence microscope. For Hoechst 33,258 staining, K562 cells were stained with 500 µL of Hoechst 33,258 for 5 min, washed twice with PBS (0.5 mL), and recorded using a Leica DMi8 inverted fluorescence microscope (Leica Microsystems, Germany).

Flow Cytometry Assay
Quantitative analysis of the EO-induced apoptosis of K562 cells was carried out using the Annexin V-PE/7-AAD apoptosis kit (Multi Sciences (Lianke) Biotech, Co., Ltd., Hangzhou, China) based on the manufacturer's instructions. Briefly, cells were seeded into a 6-well plate (3 × 10 5 cells/well), incubated for 24 h, and then treated with a new medium supplemented with EO at different doses of 0, 20, 40, 60, 80, and 160 µg/mL. After 48 h of incubation, cells were collected, rinsed with pre-cold PBS, and resuspended in 1× binding buffer (500 µL). Then, the cells were labeled with Annexin V-PE (5 µL) and 7-AAD (10 µL) for 5 min in the dark at room temperature. The apoptosis rate of cells was analyzed using an ACEA NovoCyte TM flow cytometer (ACEA Biosciences, San Diego, CA, USA).

Statistical Analysis
Each experiment was repeated at least three times independently, and the results were presented as the mean ± SD (standard deviation). SPSS software (version 25.0) was used for the statistical analysis. The significant differences between groups were analyzed using one-way analysis of variance (ANOVA) and the least significant difference (LSD) test at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Conclusions
To our knowledge, this is the first study on the antioxidant, antibacterial, enzyme inhibitory, and anticancer activities of A. galanga flower essential oil. Fifty-seven chemical components were identified using GC-FID/MS. For the bioactivities, the A. galanga flower EO exhibited moderate DPPH and ABTS radical scavenging effects. Moreover, it showed strong-to-moderate antibacterial activities against S. aureus, B. subtilis, E. faecalis, P. aeruginosa, E. coli, and P. vulgaris. In addition to the weak inhibition against AChE and BChE, as well as moderate tyrosinase inhibition, an interesting finding was that it displayed remarkable α-glucosidase inhibition comparable to that of acarbose. Furthermore, it exhibited considerable selective cytotoxicity to K562 cells and lower cytotoxicity to non-cancerous L929 cells, and it induced K562 cell apoptosis in a concentration-dependent manner. Hence, A. galanga flower EO could be regarded as a bioactive natural product with great application potential in the pharmaceutical field.

Conflicts of Interest:
The authors declare no conflict of interest.