Methylated Flavonols from Amomum koenigii J.F.Gmel. and Their Antimicrobial and Antioxidant Activities

Methylated flavonols form a special group with modulating biological activities in comparison with kaempferol and quercetin. The present study isolated ten compounds including two kaempferol methyl ethers: 5-hydroxy-3,7,4′-trimethoxyflavone (1), 3-hydroxy-5,7,4′-trimethoxyflavone (6); four quercetin methyl ethers: retusin (5-hydroxy-3,7,3′,4′-tetramethoxyflavone) (4), 3,5-dihydroxy-7,3′,4′-trimethoxyflavone (5), 3,4′-dihydroxy-5,7,3′-trimethoxyflavone (7), and 3,5,7,3′,4′-pentamethoxyflavone (9); β-sitosterol (2); 5-hydroxy-1-(4′-hydroxyphenyl)eicosan-3-one (3); p-hydroquinone (8); and vanillic acid (10) from the rhizomes and fruit of Amomum koenigii J.F.Gmel. (Zingiberaceae). Their structures were determined by MS, NMR, and X-ray spectroscopic techniques. Among the methylated flavonols, 1, 4–7, and 9 were isolated for the first time from the rhizomes, while 1, 4, and 5 were isolated from the fruit. Compounds 2, 3, 7, 8, and 10 were reported for the first time from the species. Three main methylated flavonols 1, 4, and 5 were quantitatively analyzed in the rhizomes of A. koenigii by RP-HPLC-DAD; their contents were determined to be 1.81% (1), 1.38% (4), and 1.76% (5). The antimicrobial assay against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Aspergillus niger, Fusarium oxysporum, Candida albicans, and Saccharomyces cerevisiae and antioxidant DPPH scavenging test were performed for the isolated methylated flavonols.


Introduction
Amomum Roxb. is a large genus with about 250 species and belongs to Zingiberaceae. In recent years, 21 Amomum species have been recorded in Vietnam [1]. e chemistry of a few Amomum species from Vietnam has been studied [2][3][4]. e fruit of A. koenigii has been used as an aromatic stomachic in China, India, and ailand [5,6]. A phytochemistry report on A. koenigii in China describes the isolation of eicosenones and methylated flavonols from the fruit of A. koenigii [5]. We investigated the occurrence of methylated flavonols in the fruit and rhizomes of A. koenigii from Vietnam (Figures S1 and S2) and isolated six methylated flavonols from the rhizomes for the first time. eir structures were analyzed by MS and 1D NMR spectroscopic techniques; the positions of methyl groups were determined by 2D NOESY and X-ray techniques. e contents of the main flavonoids in the rhizomes were analyzed by using RP-HPLC-DAD, and their antimicrobial and antioxidant activities were evaluated.

General Experimental
Procedure. ESI-MS spectra were measured on a ermo Fisher Scientific LTQ Orbitrap XL mass spectrometer in CH 3 OH solution. 1 H-NMR, 13 C-NMR, and DEPT spectra were recorded on a Bruker Avance 500 NMR spectrometer at 500 MHz for proton and 125 MHz for carbon-13. Tetramethyl silane (TMS) was used as the NMR internal standard. Diaion HP-20 (Mitsubishi, Japan) and silica gel (Merck, Germany) of 40-63 and 15-40 μm were used for column chromatography (CC).

RP-HPLC Analysis of Compounds 1, 4, and 5.
Qualitative and quantitative HPLC analysis was performed with an HPLC-DAD Shimadzu SIL-20AC autosampler using a 5 µm particle analytical RP-18 column (4.6 mm × 250 mm). e mobile phase was acetonitrile (HPLC grade)-deionized H 2 O gradient: 56% acetonitrile (0-5 min) and 100% acetonitrile (5.1-15.5 min). e flow rate was 1 mL/min. e injection volume was 20 μL. e column temperature was 30°C. e HPLC conditions were optimized for the baseline, precision, accuracy, repeatability, LOD, and LOQ at the corresponding signal-to-noise ratios 3 and 10 (Table S1). A wavelength of 306 nm was selected for the detection of methyl flavonols. An accurate weight of the methanol rhizome extract (34.7 mg) was dissolved in 1 mL MeOH of HPLC grade. e sample was passed through a silica gel solid-phase extraction (SPE) cartridge eluting with MeOH to remove impurities. e methanol solution was filtered through a 0.45 μm Millipore membrane filter then analyzed directly by HPLC. e measurements were performed in triplicate to calculate the average values. A calibration curve showing the linear relationship between amounts of the standards injected (μg, x-axis) and the peak area (y-axis) was constructed. e calibration standards were prepared by serial dilution of 1000 ppm concentration stock solutions of 1, 4, and 5 (HPLC purity ≥ 95%) ( Figure S3).

Antimicrobial Activity Assay.
e antimicrobial assay was performed in 96-well plates, and minimum inhibitory concentration (MIC) was determined as described by Vanden Berghe and Vlietinck [7]. e bacterial test organisms were cultured on tryptone soya agar (TSB, Sigma-Aldrich) and the test fungi on Sabouraud's dextrose agar (SDB, Sigma-Aldrich). e bacterial inocula were adjusted to yield a density of 1.5 × 10 8 colony forming units (CFU/mL) (0.5 McFarland standard). e samples were dissolved in dimethylsulfoxide (DMSO) by vortexing and filtered through a 0.02 μm microfilter to produce stock solutions. e wells were filled with 50 μL stock solutions in DMSO and 50 μL organism suspension. e positive controls were prepared in DMSO: ampicillin (50 mM), tetracycline (10 mM), and nystatin (0.04 mM). MIC values of the positive controls were as follows: ampicillin 21.83 μg/mL, tetracycline 6.87 μg/mL, and nystatin 1.44 μg/mL (for yeasts) and 2.89 μg/ml (for fungi). e negative control was DMSO. e wells were allowed to diffuse for 24-hour incubation at 37°C and 48-hour incubation at 30°C for bacteria and fungi, respectively. e results were positive when no growth was observed (CFU < 5). MIC was determined by using serial dilution of the stock solutions. MIC is defined as the lowest concentration of the compounds that showed no growth compared with growth in control (DMSO) wells. All determinations were performed in triplicate.

X-Ray Crystallographic Analysis of Methylated Flavonols 1 and 4.
Single crystals of 1 (crystal size 0.43 × 0.23 × 0.12 mm) and 4 (crystal size 0.37 × 0.07 × 0.04 mm) were prepared by recrystallization. We confirmed the locations of the methyl substituents (A-ring 5,7-substitution and B-ring 1,4-and 1,3,4-substitution) in 1 and 4 by X-ray crystallographic analysis. e unit cell of 1 was determined to be the triclinic crystal system, P-1 space group with Z � 2 and molecular formula C 18 H 16 O 6 ( Figure S4; Table S2). e molecule is nonplanar with the dihedral angle between the benzopyranone ring and the 4-substituted phenyl ring of 28.971(6)°. In the crystal, molecules of 1 are linked by weak intermolecular C-H. . .O hydrogen bonds forming ribbons lying parallel to (100) ( Figure S5; Table S3). e unit cell of 4 was determined to be orthorhombic crystal system, Pbca space group with Z � 47 and molecular formula C 19 H 18 O 7 ( Figure S6; Table S4). Excluding the methyl moieties of the substituted methoxy groups, the molecule of 4 is essentially planar with non-H atoms exhibiting mean and maximum deviations from coplanarity of 0.0808 and 0.0896Å, respectively. In the crystal, molecules are linked by weak intermolecular C-H· · ·O hydrogen bonds forming ribbons lying parallel to (100) ( Figure S7; Table S5). 1, 4, and 5. Previously, compounds 1, 4, 3,7-dihydroxy-5,4′-dimethoxyflavone, and 3,7-dihydroxy-5,3′,4′-trimethoxyflavone were analyzed in the seeds and pericarps of A. koenigii collected from Guangxi Province and Yunnan Province of China [6]. A typical analytical HPLC chromatogram of the rhizome methanol extract is shown in Figure S8. Compounds 1, 4, and 3,5dihydroxy-7,3′,4′-trimethoxyflavone (5) showed three main peaks in the HPLC chromatogram of the rhizome MeOH extract of A. koenigii from Vietnam. e retention times of 1, 4, and 5 were 12.6, 11.9, and 10.3 min, respectively. According to the HPLC quantification, the flavonoids were present in the rhizomes of A. koenigii in dry quantities of 1.81% (1), 1.38% (4), and 1.76% (5). e content of the major compound 4 (retusin) was almost 0.5% in the seeds [6] which is much lower than its content in the rhizomes in the present study.

Antimicrobial Activity of Compounds 1 and 4-7.
Five methylated flavonols 1 and 4-7 were tested for their antibacterial and antifungal activity as described by Vanden Berghe and Vlietinck [7]. e microorganisms used included Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25923), Bacillus subtilis (ATCC 27212), Staphylococcus aureus (ATCC 12222), Aspergillus niger (439), Fusarium oxysporum (M42), Candida albicans (ATCC 7754), and Saccharomyces cerevisiae (SH 20). e samples were tested for their antimicrobial activity, and the active samples underwent serial 2-fold dilutions to determine MIC values. e MIC is defined as the lowest concentration of compounds that inhibits any visible growth of microorganisms after incubation. Compounds were tested at the concentrations of 100 μg/mL (1, 4, and 5) and 50 μg/mL (6 and 7); only 4 showed potent activity against A. niger with an MIC value of 100 μg/mL (Table 1). Quercetin is a broadrange antibacterial compound; it is active against S. aureus (MIC 100 μg/mL), P. aeruginosa (MIC 100 μg/mL) [18], and E. coli (MIC 50 35.76 μg/mL) [19]. In accordance with our results, high levels of methylation in 4, 5, and 7 decreased the antibacterial activity, and the presence of the hydroxyl groups at C-3′ and C-4′ in the catechol moiety of the flavonoid B ring was important for the activity, while the free hydroxyl groups at C-3, C-5, and C-4′ were not critical. Kaempferol is much less active than quercetin, showing MIC values against S. aureus and B. cereus of 500 μg/mL, but it is strongly active against E. coli (MIC 50 25 μg/mL) [20]. Çitoglu et al. noted that the presence of free hydroxyl groups at C-5 and C-4′ was not significant for strong antibacterial activity of kaempferol methyl ethers [21]. No activity was observed for 6 showing that the free hydroxyl group at C-3 was not critical for strong antibacterial activity.

Antioxidant Activity of Compounds 4, 5, and 7.
e antioxidant activity of three methylated quercetins 4, 5, and 7 was evaluated based on the scavenging activity test against DPPH. Compounds 4, 5, and 7 were selected because quercetin with its catechol (3,4-dihydroxyphenyl) structure in the flavonoid B ring is a much stronger antioxidant than kaempferol [22]. e samples were dissolved in DMSO, and DPPH was added to ethanol 96%. e absorbance of DPPH was read at λ � 515 nm. e tests were triplicated, and the results were averaged (p < 0.05). As a result, compounds 4 (30.03%, 100 μg/mL) and 5 (14.20%, 100 μg/mL) showed a lower scavenging activity in comparison with that of quercetin (72.78%, 44 μg/mL) ( Table 2). e results provided evidence for the importance of the 3,4-dihydroxyphenyl moiety of the flavonoid B ring in the scavenging activity of quercetin derivatives [22]. No activity was observed for 9 having no free hydroxyl group implying that the C-ring α,  β-unsaturated carbonyl alone is not a requisite for the scavenging activity without the free hydroxyl group at C-3. A slight increase in the scavenging activity of 7 (42.61%, 50 μg/ mL) in comparison with those of 4 and 5 was related to the presence of a free hydroxyl group at C-4′ since the 5hydroxyl group was already blocked and the SC value of 5 with a free hydroxyl group at C-3 was relatively low.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.