Volatile constituents of Amomum argyrophyllum Ridl. and Amomum dealbatum Roxb. and their antioxidant, tyrosinase inhibitory and cytotoxic activities

2.1 Plant material

Fresh leaves and rhizomes of A. dealbatum (N: 20.1932°, E: 99.4856°) and A. argyrophyllum (N: 20.1927°, E: 99.4855°) were collected from the Doi Tung Development Project, Chiang Rai Province, Northern Thailand in May 2016. The plant was authenticated by Mr. Martin Van de Bult, a botanist at Doi Tung Development Project, Chiang Rai, Thailand. The voucher specimens (MFU-NPR0201 and MFU-NPR0202) were deposited at the Natural Products Research Laboratory of Mae Fah Luang University.

2.2 Chemicals

Gallic acid, l-ascorbic acid, kojic acid, C₈ - C₂₀ n-alkanes standard solution, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), tyrosinase from mushroom, 3,4-dihydroxy-l-phenylalanine (l-DOPA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mueller-Hinton broth was obtained from HiMedia Laboratories (Mumbai, India). Vancomycin hydrochloride was obtained from the EDQM Council of Europe (Strasbourg, France). Gentamycin sulfate and ampicillin sodium salt were obtained from Bio Basic Canada (Markham, ON, Canada). All chemicals and solvents used in this study were of analytical grade.

2.3 Headspace solid-phase microextraction (HS-SPME)

The volatile components from leaves and rhizomes of A. argyrophyllum and A. dealbatum were performed using a headspace solid-phase microextraction (HS-SPME). The SPME fiber was coated with 50/30 μm divinylbenzene/carboxen/polydimethyl-siloxane (DVB/CAR/PDMS) (Supelco, Bellefonte, PA, USA). Fresh samples (50 g) were transferred to a 250 mL glass septum bottle, then incubated in a water bath at 45 °C for 30 min. Volatile components were extracted by exposing the SPME fiber to the headspace for 30 min. For each extraction, the SPME fiber was preconditioned for 30 min at 220 °C by inserting into the injection port of GC–MS under helium atmosphere. The inlet temperature for volatile desorption was carried out at 250 °C for 5 min (Pintatum et al., 2020a).

2.4 Gas chromatography–mass spectrometry (GC–MS) analysis

An Agilent Technologies, Hewlett Packard model HP6890 gas chromatography with an HP model 5973 mass-selective detector (Agilent Technologies, Santa Clara, CA, USA) was used for GC–MS analysis. HP-5 ms (5 % phenylpolymethylsiloxane) capillary column (30 m length × 0.25 mm id × 0.25 μm film thickness, Agilent Technologies, CA, USA) and helium carrier gas (99.99 % purity) with a flow rate of 1 mL/min in split mode 1:70 was used. The oven temperature was set at 60 °C and increased at a rate of 3 °C/min to 220 °C. The temperatures of injector and detector were set at 250 °C and 280 °C, respectively. The detections were as follows, mass spectra with an ionization energy of 70 eV, scan a mass of m/z 29–300, and electron multiplier voltage of 1150 V, respectively. The temperatures of ion source and quadrupole were set at 230 °C and 150 °C, respectively (Pintatum et al., 2020a). All identified components were quantified using Kovát retention indices relative to the C₈–C₂₀ n-alkanes standard and the mass spectra of individual components with the reference mass spectra via Wiley and National Institute of Standards and Technology (NIST) database. The volatile constituents were summarized as a percent relative peak area, as shown in Table 1.

2.5 Rhizome extraction

One kilogram of each sample was macerated in ethyl acetate (EtOAc), (3 × 10 L, for 3 days) at room temperature (30 °C). Removal of the solvent at 40 °C under reduced pressure to provide the EtOAc extracts of A. argyrophyllum (18.53 g) and A. dealbatum (19.05 g), respectively. The extracts were stored at 4 °C for further studies.

2.6 Total phenolic content assay

The total phenolic concentration of the EtOAc extracts was determined according to the Folin-Ciocalteu method (Dudonné et al., 2009; Berker et al., 2013). The Folin–Ciocalteu reagent was diluted 10-fold with Milli-Q water. Gallic acid was used as the standard. One mg/mL of extract in ethanol was prepared. An aliquot of 100 µL of extract was pipetted into a test tube, then added 750 µL of Folin–Ciocalteu reagent, mixed and allowed to stand for 5 min at room temperature. Then, 750 µL of 6 % (w/v) sodium carbonate was added to the reaction mixture. The solution stood at room temperature for 1 hr. The absorbance at 750 nm wavelength was measured using a UV–vis Genesys 30 Visible spectrophotometer (Thermo Fisher Scientific, Fitchburg, WI, USA). Gallic acid with a serial dilution of 5, 10, 25, 50, and 100 µg/mL was used to generate a standard calibration curve. Total phenolic content in the samples was calculated and expressed as milligram gallic acid equivalents.

2.7 DPPH free radical scavenging assay

The antioxidant activity was determined using a DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay (Liyanaarachchi et al., 2018; Pintatum et al., 2020a, 2020b). The extract was tested at serially diluted concentrations of 5, 10, 25, 50, and 100 µg/mL in methanol. DPPH methanolic solution (6 × 10^-5 M, 100 µL) was incubated with 100 µL of the extract in the dark at room temperature for 30 min. The absorbance of the reaction solution was recorded against a blank at wavelength of 517 nm using the microplate reader (Biochrom Asys UVM 340 Microplate Reader, Biochrom, Cambridge, UK). Ascorbic acid at serially diluted concentrations (0.5, 1, 2, 4, and 8 µg/mL in methanol) was used as the positive control. The DPPH radical scavenging activity was expressed as the inhibitory concentration at 50 % (IC₅₀), which was calculated in comparison with the standard ascorbic acid (Li et al., 2016).

2.8 ABTS radical cation scavenging assay

The ABTS radical scavenging activity of extract was determined based on the method described previously (Dudonné et al., 2009; Pintatum et al., 2020a, 2020b) with some modifications. The working solution of ABTS radical cation (ABTS^•+) was prepared from the reaction of equal volumes of 7 mM of ABTS with 2.45 mM of potassium persulfate in the dark at room temperature for 16 h before use. The working solution of ABTS^•+ was adjusted to the absorbance of 0.70 ± 0.02 at 734 nm with ethanol. The extract was tested at serially diluted concentrations of 50, 100, 150, 200, and 300 µg/mL in ethanol. An aliquot of 20 µL of extract was mixed with 180 µL of ABTS^•+ solution and allowed to stand in the dark at room temperature for 5 min, then the absorbance of the reaction solution was measured at 734 nm using the microplate reader (Biochrom Asys UVM 340 Microplate Reader, Biochrom, Cambridge, UK). Serially diluted concentrations of ascorbic acid (1.5, 3, 6, 12, and 25 µg/mL) were used as the positive controls. The ABTS radical cation scavenging activity of extract was expressed as the inhibitory concentration at 50 % (IC₅₀), which was calculated in comparison with the standard ascorbic acid.

2.9 Ferric reducing antioxidant power (FRAP) assay

The ferric reducing power of the extract was determined based on the method modified version of the FRAP assay (Dudonné et al., 2009). The working FRAP reagent was prepared daily by mixing 1 vol of 10 mM TPTZ (solution in 40 mM HCl), with 1 vol of 20 mM ferric chloride solution, and 10 volumes of 300 mM acetate buffer, (pH 3.6). The FRAP reagent was warmed up to 37 °C in a water bath. Fifty microliters of extract and 150 µL of deionized water were added to 1.5 mL of FRAP reagent, and then incubated at 37 °C in a water bath for 30 min. The absorbance of the reaction solution was measured at 593 nm using a microplate reader (Biochrom Asys UVM 340 Microplate Reader, Biochrom, Cambridge, UK). Acetate buffer was used as blank. Ascorbic acid with a serial dilution of 50, 100, 200, 300, and 400 µM was used to generate a standard calibration curve. The results were expressed as µM ascorbic acid equivalents (Pintatum et al., 2020a).

2.10 Inhibition of tyrosinase assay

The mushroom tyrosinase inhibition activity of the extract was determined using the method described previously (Gomółka et al., 2021; Li et al., 2019; Pintatum et al., 2020a). The extract was dissolved in DMSO at a concentration of 10 mg/mL. An aliquot of 40 µL of extract was mixed with 80 µL of 0.1 M phosphate buffer (pH 6.8), and 40 µL of tyrosinase from mushroom, enzyme commission number 1.14.18.1 (48 units/mL). Following the addition of 40 µL of l-DOPA (2.5 mM), and then allowed to stand at room temperature for 30 min. the absorbance of the reaction solution was measured at 490 nm using the microplate reader (Biochrom Asys UVM 340 Microplate Reader, Biochrom, Cambridge, UK). Each sample was accompanied by a blank sample containing all of the components without l-DOPA. Kojic acid was used as a positive control.

2.11 Antibacterial assay

Four Gram-positive bacteria, Bacillus cereus TISTR 687, Staphylococcus epidermidis TISTR 2141, Bacillus subtilis TISTR 1248, and Staphylococcus aureus TISTR 746, and four Gram-negative bacteria, Salmonella typhimurium TISTR 1470, Pseudomonas aeruginosa TISTR 1287, Escherichia coli TISTR 527, and Serratia marcescens TISTR 1354, were obtained from the Microbiological Resources Centre of the Thailand Institute of Scientific and Technological Research. A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) (Pintatum et al., 2020a; Singtothong et al., 2013; Wikaningtyas and Sukandar, 2016; Yang et al., 2008). The extract was diluted with DMSO, and then loaded in Mueller-Hinton broth microdilution with serially dilution (twofold). One hundred microliter of microbial culture an approximate of 1.0 × 10⁶ CFU/mL was added into 96-well microtiter plates. The last row was containing only the extract without microorganisms, was used as a negative control. The broth cultures of each strain were incubated at 37 °C for 24 h. The MIC values were determined as the lowest concentration of the extract that completely inhibits the growth of microorganisms. Vancomycin, gentamycin, and ampicillin were used as the positive controls (Table 2).

2.12 MTT assay

The cytotoxicity against the human keratinocyte cells (HaCaT) was determined using the MTT assay. Cells were grown in the Dulbecco’s modified eagle’s medium, supplemented with 10 % fetal bovine serum, 2 % of sodium bicarbonate (7.5 % solution), 1 % of sodium pyruvate (100 mM) and 1 % of penicillin–streptomycin (10,000 Units/mL). The cells were incubated in a humidified 37 °C, 5 % CO₂ incubator, until reaching a subconfluent (approximately 80 %). The HaCaT cells were plated out in 96-well plates containing 100 µL of growth medium with a cell concentration of 2 × 10⁴ cells/well and incubated for 24 h in an incubator. The cells were treated with increasing concentrations of extracts (12.5–200 µg/mL) for 24 h at 37 °C in 5 % CO₂, after which 10 µL of MTT solution (5 mg/mL) was added and incubated the cells for another 4 h. An aliquot 90 µL of 10 % SDS–0.01 M HCl was added in order to solubilize the formazan product. The absorbance was measured at 595 nm after 24 h using a microplate reader (Envision Plate Reader, Perkin Elmer, USA). Withaferin A was used as positive control (Abe et al., 2018; Pintatum et al., 2020b; Septisetyani et al., 2014; Zanette et al., 2011).

2.13 Luciferase assay

The HaCaT cells stably expressing p(NFκB)₃50-luc was used for this assay. The cells were plated at a density of 10⁵ cells/well in 24-well plates for 24 h recovery period. To determine NFκB dependent transcription, the cells were preincubated for 2 h with a dose range of extract, followed by stimulation with TNFα (2 ng/mL) for 6 h at 37 °C. Then, the cells were lysed in 1 X lysis buffer (25 mM Tris‐phosphate (pH 7.8), 2 mM DTT, 2 mM CDTA, 10 % glycerol, and 1 % Triton X‐100). Luciferase activity was measured by the instructions of the “luciferase assay kit” (Promega, Madison, WI, USA), following 25 μL of lysates were placed in opaque 96 well plates, and then added 50 µL of luciferase substrate (1 mM luciferin or luciferin salt, 3 mM ATP, and 15 mM MgSO4 in 30 mM HEPES buffer, pH 7.8). Bioluminescence was measured by using the Envision multilabel reader (Perkin Elmer, Waltham, MA, USA). Withaferin A was used as positive control (Cavin et al., 2007; Bremner et al., 2009; Zanette et al., 2011).

2.14 Statistical analysis

All values given were performed in triplicate and expressed as the means ± standard deviation (SD) using Microsoft Excel. The statistical analyses included the Analysis of Variance (ANOVA), the hierarchical cluster analysis (HCA; Ward’s method), and the Principal Components Analysis (PCA) were performed with SPSS version 23.0 package (SPSS Inc., Chicago, USA). Significant differences are reported as p-value < 0.05.

3.1 Volatile oils composition

HS-SPME is simple and useful to analyse the volatiles in fragrant plants (Yang et al., 2009; Huang et al., 2012). HS-SPME-GC/MS on HP-5MS column allowed the identification of 49, 47, 49, and 34 components, comprising 99.5 %, 96.8 %, 99.6 %, and 98.8 % of the total peak areas from rhizomes and leaves of A. argyrophyllum and A. dealbatum (Fig. 1), respectively. The chemical compositions of leaves and rhizomes volatiles are shown in (Table 1). The volatiles were dominated by 75.8 %, 52.1 %, 87.7 %, and 96.7 % of monoterpenes, followed by 13.2 %, 2.1 %, 0.8 %, and 0.2 % of oxygenated monoterpenes, 10.3 %, 39.0 %, 11.3 %, and 2.8 % of sesquiterpenes, and 0.2 %, 1.1 %, 0.07 %, and 0.05 % of oxygenated sesquiterpenes, respectively. The major components of leaves and rhizomes of A. argyrophyllum were identified as camphene (0.4 % ± 0.03 %, 10.8 % ± 0.5 %), β-pinene (16.7 % ± 1.4 %, 16.7 % ± 2.2 %), o-cymene (20.7 % ± 1.9 %, 1.1 % ± 0.1 %), limonene (1.9 % ± 0.2 %, 9.7 % ± 0.8 %), (Z)-β-ocimene (2.0 % ± 0.1 %, 10.6 % ± 0.5 %), and (E)-β-ocimene (2.4 % ± 0.4 %, 8.7 % ± 0.5 %). Whereas, the main constituents of A. dealbatum were β-pinene (45.2 % ± 2.1 %, 55.1 % ± 2.0 %), α-pinene (23.0 % ± 1.4 %, 24.7 % ± 1.4 %), limonene (3.5 % ± 0.1 %, 5.6 % ± 0.5 %), (Z)-β-ocimene (4.6 % ± 0.3 %, 0.8 % ± 0.1 %), and (E)-β-ocimene (4.4 % ± 0.3 %, 1.2 % ± 0.2 %), respectively. The other constituents identified in the volatile are compiled in Table 1.

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Fig. 1

HS-SPME chromatogram of fresh rhizomes and leaves from Amomum argyrophyllum and Amomum dealbatum.

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Most of these compounds have already been reported by previous studies in different Amomum species (Ao et al., 2019; Edris, 2007; Kurup et al., 2018; Yang et al., 2008). In comparison with the previous studies, 1,8-cineole (61.3 %), α-terpineol (7.9 %), α-pinene (3.8 %), β-pinene (8.9 %), and allo-aromadendrene (3.2 %) were reported as the main volatile components in A. subulatum (Gurudutt, 1996). In addition, allo-aromadendrene (16.2 %), β-pinene (8.7 %), and (E)-caryophyllene (8.5 %) were reported as major component in the rhizome oil of A. agastyamalayanum, and santolina triene (42.2 %), and α-pinene (17.1 %) were the major constituents in rhizome oil of A. newmanii, respectively (Kurup et al., 2018). Moreover, the main constituents of the essential oil from leaves and root barks of A. villosum were as β-pinene (56.6 %, 34.7 %) and α-pinene (22.0 %, 11.6 %), respectively (Dai et al., 2016). Finally, the major constituents in the leaves of A. maximum were β-pinene (40.8 %), α-pinene (9.7 %), β-elemene (10.9 %) and β-caryophyllene (8.3 %), whereas β-pinene (28.0 %), α-pinene (15.0 %) and β-phellandrene (11.6 %) were the main constituents of the root (Huong et al., 2019). The leaves oil of A. muricarpum presented major constituents as α-pinene (48.4 %), β-pinene (25.9 %) and limonene (7.4 %), while α-pinene (54.7 %), β-pinene (14.3 %) and β-phellandrene (8.3 %) were the major in the roots, respectively (Huong et al., 2019). The results of volatile components in this study had partial agreement with the previous reports. The present results represent the first identification of the volatile constituents of rhizomes and leaves of A. argyrophyllum and A. dealbatum.

3.2 Statistical analysis of volatile components

In order to study the variability of chemical components within and between the studied populations, the Hierarchical Cluster Analysis (HCA) and the Principal Components Analysis (PCA) were carried out. This analysis was employed to provide an overview of chemical components of volatile oil based on GC–MS data. With a dissimilarity 77, the HCA using Ward’s method was indicated in three groups (A, B, and D) according to similarity of their chemical components (Fig. 2). The A group was characterized by the presence of β-pinene and α-pinene as major components. Group B was further indicated into two sub-groups (B1 and B2). Sub-group B1 was characterized by o-cymene, β-copaene, β-cubebene, γ-patchoulene, and α-humulene. Sub-group B2 consisted of limonene, (E)-β-ocimenene, allo-ocimene, (Z)-β-ocimene, thymol methyl ether, and camphene. With a dissimilarity of 64 components in group C. In addition, the PCA was employed to all volatile constituents. Fig. 3 shows a PCA plot of the volatile constituents of rhizomes and leaves of A. argyrophyllum and A. dealbatum. The first principal components (PC1) explained 46.6 % of the variation across the samples, whereas the second principal components (PC2) explained 35.4 % of the variance. The samples exhibited similar major components, with different levels of adulteration. As shown in Fig. 3, the volatile distributions at negative axis were highly influenced by α-pinene, β-pinene, camphene, o-cymene, limonene, β-myrcene, (Z)-β-ocimene, (E)-β-ocimene, allo-ocimene, endo-Fenchyl acetate, caryophyllene, copaene, and aromadendrene, all of which were present in large amounts. The PCA results supported the differentiation of the samples obtained by the HCA analysis. The results indicated that the classification proposed by HCA and PCA is acceptable.

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Fig. 2

Dendrogram obtained by cluster analysis, representing chemical composition similarity relationships of 77 volatile components from the rhizomes and leaves of A. argyrophyllum and A. dealbatum.

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Fig. 3

Principal component analysis (PCA) loading plots revealing the compounds present in rhizomes and leaves of A. argyrophyllum and A. dealbatum.

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3.3 Total phenolic content and antioxidant activities

The total phenolic content in different extracts of A. argyrophyllum and A. dealbatum rhizomes are shown in Table 2. The total phenolic content of the extract was 2.9 ± 0.5 and 2.1 ± 0.6 mg gallic acid equivalence (mg GAE), respectively. From the results, total phenolic content was found to be lower than previous reports, A. chinense (8.3 mg GAE), A. tsao-ko (7.2 mg GAE), and A. villosum (9.3 mg GAE) (Gan et al., 2010; Butsat and Siriamornpun, 2016).

Antioxidant activities of the extracts were determined using DPPH, ABTS, and FRAP assays, respectively. As indicated in Table 2, the DPPH and ABTS radical scavenging activity of A. argyrophyllum and A. dealbatum rhizomes extracts were showed IC₅₀ value of (179.8 ± 3.9 µg/mL, 392.9 ± 2.6 µg/mL) and (120.3 ± 2.5 µg/mL, 328.6 ± 3.3 µg/mL). Ascorbic acid was used as a positive control, with an IC₅₀ value of 1.6 ± 0.8 µg/mL and 5.2 ± 0.8 µg/mL, respectively. For the FRAP value, the extracts showed the lowest reducing ability with FRAP value of 76.5 ± 7.8 µM ascorbic acid equivalence (mM AAE) and 84.9 ± 4.4 µM AAE, respectively. Similarly, some Amomum species exhibited lower antioxidant activity than synthetic antioxidant agents (Yang et al., 2010; Prakash et al., 2012). The A. kravanh and A. subulatum exhibited DPPH radical scavenging activity with IC₅₀ value of 13.8 µg/mL and 431.2 µg/mL, respectively (Shrestha, 2017; Zhang et al., 2020). In addition, A. subulatum also presented an IC₅₀ value of 8.3 µg/mL by DPPH assay (Prakash et al., 2012).

Phenolic compounds are secondary metabolites, which play important roles in neutralizing free radicals and preventing oxidative damage (Pintatum et al., 2020a, 2020b). In this study, the A. argyrophyllum and A. dealbatum rhizome extracts exhibited weak total phenolic content. This implies why the lowest antioxidant activity was indicated in the extracts. These results are in accordance with previous studies, reporting a correlation between phenolic contents and antioxidant properties (Owen et al., 2000; Yang et al., 2010; Minatel et al., 2016).

3.4 Tyrosinase inhibitory activity

The A. argyrophyllum and A. dealbatum rhizomes extracts showed weak tyrosinase inhibitory activity at 69.0 ± 3.6 mg kojic acid equivalence (mg KAE) and 53.7 ± 7.4 mg KAE (Table 2). Findings from this study, the lowest of total phenolic content and antioxidant properties would mean the lowest of tyrosinase inhibition ability, as well. The tyrosinase inhibitory effects may have depended on the phenolic compounds and antioxidant properties (Pintatum et al., 2020a, 2020b).

3.5 Antibacterial activity

The antimicrobial activity of the A. argyrophyllum and A. dealbatum rhizome extracts was investigated using the Mueller-Hinton broth microdilution method against eight types of resistant bacteria. As indicated in Table 3, A. dealbatum rhizomes extracts exhibited the smallest MIC value only to Gram-positive bacteria, Bacillus cereus and Bacillus subtilis in concentrations of 640 µg/mL. Whereas, the extracts showed less activity or were inactive toward the other bacteria strains in concentrations of 1280 µg/mL or more. In this study, vancomycin, gentamicin, and ampicillin were used as standard antibiotics.

According to some researchers, some Amomum species have a wide variety of secondary metabolites such as tannins, alkaloids and flavonoids (Gurudutt et al., 1996). They play important roles in preventing oxidative damage and antimicrobial properties (Gurudutt et al., 1996; Pintatum et al., 2020a, 2020b). The essential oil isolated from A. subulatum showed good antimicrobial activity against B. pumilus, S. aureus, S. epidermidis, P. aeruginosa, and S. cerevisiae (Agnihotri and Wakode, 2010). In addition, the essential oil of A. tsao-ko also showed strongest antimicrobial activity against S. aureus (Yang et al., 2008). It was clear for the results, because the extracts contained less phenolic content and antioxidant properties. It could be the cause of lowest antimicrobial activity.

3.6 In vitro cytotoxicity

The crude rhizome extracts of A. argyrophyllum and A. dealbatum were tested to assess cytotoxicity on HaCaT keratinocyte cells using MTT assay. The HaCaT keratinocyte cells were treated with increasing doses of extract, (12.5, 25, 50, 100, and 200 µg/mL) for 24 h. The cytotoxic effects of these extracts are presented in Fig. 4. Percentage of cell viability is reported as the mean ± SD of three independent experiments. MTT results showed that exposure to 200 µg/mL concentrations of A. argyrophyllum extract inhibited the growth of cells, with percentages of cell viability of 65.2 ± 4.4 %. Nevertheless, the A. argyrophyllum extract in a concentration range of 12.5 – 100 µg/mL and A. dealbatum extract in all concentrations used did not inhibit the growth of the cell lines. The percentages of viable cells remained above 80 %, it is concluded that A. argyrophyllum and A. dealbatum does not exert a cytotoxic effect on HaCaT cells.

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Fig. 4

Relative HaCaT viability (%) by increasing concentrations of A. argyrophyllum and A. dealbatum.

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3.7 Anti-inflammatory activity

Anti-inflammatory activity was represented by the inhibitory effects on nuclear factor-κB (NF-κB) reporter gene cells in TNF-α treated. The different treatments were applied to the cells. The luciferase reporter gene activity was measured in lysates in presence of ATP/luciferin reagent (Promega, WI, USA). The total emitted bioluminescence (relative light units, RLU) was measured during 30 s (Envision multiplate reader, Perkin Elmer). The result is shown in Fig. 5. The proinflammatory TNF-α increased luciferase gene expression, as compared to the control and extracts without TNF-α. The extracts displayed dose-dependent decreased luciferase gene expression. The extracts showed anti-inflammatory effects on NF-κB activity.

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Fig. 5

Anti‐inflammatory effects of A. argyrophyllum and A. dealbatum measured in HaCaT NF‐κB reporter gene cells.

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TNF-α is another inflammatory cytokine that plays important role in some pain models, inflammatory, and neuropathic hyperalgesia (Zhang and An, 2007). The extracts and chemical constituents of some Amomum plants have been reported as antioxidant properties, and anti-inflammatory activity. From the previous report, labdane and norlabdane diterpenoids were isolated from the rhizomes of A. villosum. The compounds were evaluated for anti-inflammatory activity using inhibitory effects on nitric oxide (NO) production. The compounds from the rhizomes of A. villosum showed significant inhibition of NO production (Yin et al., 2019). In addition, the compounds isolated from dried fruits of A. tsaoko were also investigated for inhibitory effect on NO production. The compounds exhibited anti-inflammatory activity in a dose-dependent manner (Zhang et al., 2016). The A. compactum extract was examined for potential anti-inflammatory effects on LPS-induced inflammatory models. The measurement of accumulation of nitrite in the culture media. The results revealed that A. compactum extract decreased the production of NO and PGE₂ (Lee et al., 2012). The present study A. argyrophyllum and A. dealbatum extracts showed moderate anti-inflammatory NF-κB effects. This could probably be due to the lower phenolic content and the radical scavenging activities.