An Anisole Derivative in the Essential oil of Wurfbainia schmidtii with Virucidal Activity Against SARS-CoV-2 and Anti-Inflammatory Properties

Abstract

Objectives: Essential oils from many plants have been reported to have anti-inflammatory activity. However, essential oil of Wurfbainia schmidtii leaves has never been reported for anti-inflammatory activity. Moreover, previous works on the essential oil compositions of W. schmidtii (synonyms Amomum biflorum and Amomum schmidtii) reported varied compositions in its oils. This work aims at investigating the essential oil compositions of W. schmidtii and its anti-inflammatory activity, as well as virucidal activity against SARS-CoV-2 virus. Methods: The composition of W. schmidtii essential oil was analyzed by gas chromatography-mass spectrometry (GC-MS) technique. A crude hexane extract of W. schmidtii was separated by chromatographic method, giving a major phenylbutanoid, trans-p-(l-butenyl)-anisole (1), whose structure was elucidated by analysis of 1D and 2D ¹H nuclear magnetic resonance (NMR) spectra. Anti-inflammatory activity of the essential oil and the major compound 1 was evaluated, and virucidal activity of against SARS-CoV-2 virus was assessed for compound 1. Results: A major component in W. schmidtii essential oil was found to be trans-p-(l-butenyl)-anisole (1), accounting for 88.69%, and it exhibited virucidal activity against SARS-CoV-2 with EC₅₀ value of 119.60 μM. W. schmidtii essential oil and the major compound 1 exhibited anti-inflammatory activity with the IC₅₀ values of 0.0282 mg/mL and 12.74 μM, respectively. Conclusion: trans-p-(l-Butenyl)-anisole (1) was the major compound in the essential oil of W. schmidtii, and it displayed anti-inflammatory and virucidal activities. The essential oil of W. schmidtii showed anti-inflammatory activity.

Graphical Abstract

This is a visual representation of the abstract.

Keywords

Antiviral compounds Massage oils GC-MS Phenylbutanoid Aromatic plant

Introduction

Wurfbainia schmidtii (K.Schum.) Škorničk. & A.D.Poulsen commonly known in Thai as “Wan-Sao-Long”, belongs to the Zingiberaceae family, and it is a Thai medicinal herb that relieves flatulence and stomachache in northern and eastern Thailand.¹ This herbal plant, W. schmidtii (K.Schum.) Škorničk. & A.D.Poulsen, is an aromatic plant, which was previously known as Amomum schmidtii before Boer's group discovered that the genus Amomum could be separated into several genera.² W. schmidtii was named as synonyms Amomum biflorum and Amomum schmidtii, and previous studies referred to it by these names.^3–6 However, its name was recently revised to W. schmidtii (K.Schum.) Škorničk. & A.D.Poulsen.^2,7 Previous works showed that essential oils of Amomum species possess pharmacological activities such as antimicrobial, anti-inflammatory, antioxidant, antibacterial, and sedative activities.^3,8,9 The chemical constituents of essential oils that contribute to these biological activities are monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and non-terpenes.¹⁰ trans-p-(l-Butenyl)anisole (1) (Figure 1) was found to be a main compound in the essential oils from leaf, stem and root of A. schmidtti grown in Vietnam,¹¹ while camphor, α-bisabolol, camphene, and α-humulene were reported to be major compounds in an oil of A. biflorum, collected in Thailand.³ Interestingly, a recent study found that 1,8-cineole, camphene, and endo-borneol were major compounds in the essential oil of W. schmidtii.¹² The present work revealed that trans-p-(l-butenyl)-anisole (1) was a major compound in the essential oil of plant W. schmidtii (K.Schum.) Škorničk. & A.D.Poulsen (Figure 1).

Figure 1.

Structure of trans-p-(1-butenyl)-anisole (1) isolated from a crude hexane extract of W. schmidtii.

Essential oils with anti-inflammatory properties have been used in various applications, for example, modulating colon pathophysiology,¹³ and management of rheumatoid arthritis.¹⁴ Recently, a number of works reported anti-inflammatory activity of essential oils.¹⁵ For example, essential oils of Homalomena pendula and H. cochinchinensis rhizomes display moderate anti-inflammatory activity,¹⁶ while those from Piper caldense, P. mosenii, and P. mikanianum showed anti-inflammatory activity in adult zebrafish models.¹⁷ Mint essential oils from Mentha arvensis, M. citrata, and M. spicata exert anti-inflammatory activity by inhibition of 5-lipoxygenase enzyme.¹⁸ Essential oils of Euodia lepta showed anti-inflammatory activity in lipopolysaccharides (LPS)-stimulated macrophages,¹⁹ whereas that of Artemisia vulgaris exhibited anti-inflammatory, antioxidant, and anti-bacterial activities.²⁰ Upon the above reports, we hypothesize that the essential oil of W. schmidtii may exhibit anti-inflammatory activity. Therefore, in this study, we evaluate anti-inflammatory activity of this plant, and report in this work. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent for COVID-19, whose pandemic has caused a global health problem. Infection with SARS-CoV-2 virus leads to considerable inflammation in different organs, causing excessive inflammatory responses and multi-organ damage.²¹ We also report virucidal activity of the essential oil from W. schmidtii against SARS-CoV-2 virus in this work.

Materials and Methods

Plant Materials, Extraction, and Isolation Procedure

Leaves of Wurfbainia schmidtii were obtained from Amnatcharoen Province, Thailand, and W. schmidtii was authenticated by Dr Wittaya Kaewsri, Mahidol University, and and a specimen (no. Kaewsri_58) was deposited at Bangkok Herbarium and Forest Herbarium, Thailand. The dried chopped leaves of W. schmidtii (457.57 g) were ground using a blender, and then extracted by maceration in 2L hexane (AR grade) overnight at room temperature. The extraction was repeated thrice, and filtrates were combined. The extracts were filtered using Whatman filter paper, and the filtrates were combined and dried using a rotary evaporator under reduced pressure, at 37 °C, giving 17.77 g of crude extract. A crude extract was fractionated by Sephadex LH-20 (3 × 75 cm) column chromatography and eluted with EtOH (AR grade), to obtain eleven fractions (F1-F11). Fraction 5 contained a major compound, trans-p-(1-butenyl)-anisole (1), with some impurities, therefore this fraction was further purified by Sephadex LH-20 (3 × 75 cm) column chromatography, eluted with EtOH, yielding nine fractions (F5.1-F5.9). Fraction F5.7 contained trans-p-(1-butenyl)-anisole (1), whose structure was elucidated by analysis of nuclear magnetic resonance (NMR) data (1D and 2D NMR techniques) and mass spectrometry (MS) data. ¹H, ¹³C and 2D NMR spectra of compound 1 are shown in the Supplemental Material.

The essential oils of W. schmidtii leaves were obtained by hydrodistillation using a Clevenger steam distillation apparatus. Forty grams of dried chopped leaves were placed in a round bottom flask, filled with 250 mL of distilled water. Hydrodistillation was performed for 2–4 h for each round to collect essential oil, and the hydrodistillation was repeated thrice. The oils were combined, and dried over anhydrous sodium sulfate, giving 495.93 mg of essential oils. Essential oils of W. schmidtii were stored in the freezer at −20 °C until further analyses or use for evaluation of biological activities.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

The chemical composition of essential oil obtained from W. schmidtii leaves was examined through gas chromatography-mass spectrometry (GC-MS). The analysis was carried out using an Agilent 6890N gas chromatograph equipped with an electron impact ionization and mass-selective detector (Agilent 5973). A fused-silica capillary DB5-MS column (30 m × 0.25 mm i.d., 0.25 μm) from J&W Scientific, USA, served as the chromatographic column, with helium serving as the carrier gas at a flow rate of 1.0 mL/min. The injection volume was set at 1.0 μL, and the injector temperature was maintained at 250 °C. The electron impact ionization was performed at an ionization energy of 70 eV, with both ion source and interface temperatures set to 250 °C. The temperature program of the oven was initiated at 60 °C and ramped up to 200 °C at a rate of 3 °C/min. The acquisition mode operated in scan mode, covering mass-to-charge ratios (m/z) from 30 to 300. Identification of volatile components relied on computer matching and comparison of mass spectra with the NIST20 libraries. Kovats retention indices were determined using linear interpolation of the retention times of n-alkanes, and literature values reported by Adams (2017)²² were cross-referenced. This comprehensive GC-MS analysis furnishes detailed insights into the sample's chemical composition, contributing valuable information for scholarly research and publications.

Cell Line and Virus

The human epithelial lung carcinoma cells, which stably express human angiotensin I-converting enzyme-2 (A549-hACE2), were obtained from InvivoGen (San Diego, CA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM)/High glucose (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA) and antibiotics.²³ The SARS-CoV-2 Delta variant (B.1.617.2) was provided by Dr Anan Jongkaewwattana (National Center for Genetic Engineering and Biotechnology or BIOTEC, Thailand).²³

Cytotoxicity Test

Cytotoxicity test was performed according to a previous report.²⁴ Suspension of RAW 264.7 cells (mouse macrophage cell line, ATCC TIB-71, Rockville, MD, USA) in phenol red-free medium was seeded in 96-well plate at 30,000 cells/100 µL/well and cultured at 37 °C for 24 h to allow cell attachment. RAW 264.7 cell line (ATCC number: TIB-71) was obtained from the American Type Culture Collection (ATCC). Then, 25 µL of the medium containing various concentrations of test sample was added to each well, and the cells were further incubated for 24 h. Since RAW 264.7 cells attached to the plate weakly, a modified MTT assay for non-adherent cells was used to determine cell viability.²⁵ At the end of treatment, an MTT solution (25 µL) was added to each well and further incubated for 4 h. Subsequently, lysis solution (20% SDS in 10 mM of HCl, 100 µL/well) was added to lyse cells and solubilize formazan crystals formed by mitochondrial activity of viable cells. The plate was left in the dark at room temperature for another 2 days. The absorbance at wavelength 550 nm was measured and subtracted with absorbance at the reference wavelength 650 nm. The absorbance of untreated control cells was assigned as 100% cell viability. Three replicates were investigated for each sample.

Anti-inflammatory Assay

Anti-inflammatory effects of the essential oil and trans-p-(1-butenyl)-anisole (1) were determined according to previously described,²⁴ by measuring ability to suppress nitric oxide production in RAW 264.7 cells. A cell suspension (30,000 cells/100 µL) in a phenol red-free medium was seeded into 96-well plate and cultured at 37 °C for 24 h. After that, 25 µL of sample was added to the cells and incubated for 3 h, and then the cells were stimulated with 10 µg/mL LPS from Escherichia coli (Sigma Aldrich, Saint Louis, MO, USA) for 24 h. To determine nitric oxide level, 50 µL of culture medium from each well was mixed with 100 µL of Griess reagent (Promega, Madison, WI, USA) and incubated at room temperature in the dark for 20 min. A standard curve of sodium nitrite (0-100 µM) was prepared in parallel using the same protocol. The absorbance was measured at 540 nm, and the nitrite concentration of the culture medium was calculated from the standard curve. An anti-inflammatory compound, vermelhotin (at 10 μM), was used as a positive control. Vermelhotin, a natural product, was previously found to display potent anti-inflammatory activity through the inhibition of nitric oxide production in LPS-stimulated RAW 264.7 macrophage cells.²⁶ Therefore, vermelhotin was used as a positive control in this work. For IC₅₀ value calculation, nitric oxide production in LPS-stimulated untreated cells was assigned as 100%.

Virucidal and Antiviral Assays against SARS-CoV-2

Virucidal activity is the ability of agent to inactivate virus before infection to host cells. While antiviral activity is the ability of agent to suppress virus propagation inside host cells after infection. Evaluation of virucidal and antiviral activities was followed the method described previously.²⁷ The major compound, trans-p-(l-butenyl)-anisole (1), in the essential oil of W. schmidtii was assessed for its virucidal and antiviral activities against SARS-CoV-2 virus. All assays against SARS-CoV-2 were conducted in a biosafety level-3 (BSL-3) laboratory at BIOTEC, Thailand. For the virucidal assay, the host cells, A549-hACE2, were seeded overnight in a 96-well plate at a density of 40,000 cells/100 µL/well. Prior to infection, the virus (at a multiplicity of infection, MOI of 0.0014) was incubated with varying concentrations of compound 1 at 37 °C for 1 h. The virus treated with DMSO served as a control, representing 100% viral infectivity. Subsequently, the cells were exposed to the virus mixture at 37 °C for 1 h. The excess virus was washed out with phosphate-buffered saline (PBS), and the infected cells were cultured in the absence of compound 1 for 96 h. Viral infectivity was determined by assessing the cytopathic effects caused by the infection using the Viral ToxGlo Assay (Promega, Madison, WI, USA). Briefly, the culture supernatant was replaced with Viral ToxGlo (diluted with PBS at a ratio of 1:1) and the cells were incubated at room temperature for 10 min. The luminescence was quantified using the EnSight Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). The relative viral infectivity was calculated by normalizing the luminescence values to those obtained from the DMSO control. Due to the unavailability of a suitable chemical virucidal reagent that is non-toxic to host cells for use as a positive control in virucidal assays, we previously validated our assay protocol using an anti-SARS-CoV-2 spike antibody, which can neutralize viral particles and prevent host cells from the infection.²⁷ The half-maximal effective concentration (EC₅₀) was calculated by creating a dose-response curve using GraphPad Prism software (version 9.0).

To assess the cytotoxicity of compound 1 in the virucidal assay, A549-hACE2 cells were seeded in a 96-well plate at a density of 40,000 cells/100 µL/well overnight. The cells were treated with varying concentrations of compound 1 at 37 °C for 1 h, washed with PBS, and then cultured in medium without compound for 96 h. The cells treated with DMSO were used as a non-cytotoxic control, representing 100% cell viability. The cytotoxicity of the sample was assessed by measuring the viability of the treated cells using the Viral ToxGlo assay, as mentioned earlier. The relative cell viability was obtained by normalizing the data to the DMSO control. The half-maximal cytotoxic concentration (CC₅₀) was estimated by fitting the relative cell viability data to a dose-response curve using GraphPad Prism software. The selectivity index (SI) was determined by dividing the CC₅₀ value by the EC₅₀ value (CC₅₀/EC₅₀).

For the antiviral assay, the effect of trans-p-(1-butenyl)-anisole (1) at the post-infection stage was evaluated. A549-hACE2 cells (seeded overnight in a 96-well plate at 40,000 cells/100 µL/well) were exposed to SARS-CoV-2 (MOI 0.0007) at 37 °C for 1 h. The excess virus was washed out, and the infected cells were treated with compound 1 at varying concentrations for 96 h. The infected cells treated with DMSO were used as 100% viral infectivity control. The relative viral infectivity and EC₅₀ value were obtained as described earlier. The cytotoxicity of compound 1 in the antiviral assay was evaluated by treating uninfected A549-hACE2 cells with compound 1 for 96 h. The relative cell viability, CC₅₀, and SI values were derived as mentioned above.

Statistical Analysis

Statistical analysis was performed by using GraphPad Prism version 10.1.2 for anti-inflammatory assay. The results are presented as mean ± SD, and analyzed by Student's t-test. A p value less than 0.05 was considered statistically significant. The EC₅₀ and CC₅₀ values of antiviral and virucidal activities were determined by fitting data to a sigmoidal dose-response curve using the log(inhibition) versus normalized response with a variable slope equation in GraphPad Prism software (version 9.0).

Results and Discussion

Chemical Constituents of Wurfbainia schmidtii

Leaves of W. schmidtii were extracted with hexane to give a crude hexane extract, and they were hydrodistilled to obtain essential oil. A crude hexane extract of W. schmidtii was separated by chromatographic technique to give trans-p-(l-butenyl)-anisole (1) (Figure 1). The structure of compound 1 was elucidated through the analysis of NMR data, and the ¹H, ¹³C and 2D NMR spectra of 1 are provided in the Supplemental Material. When comparing ¹H NMR of trans-p-(l-butenyl)-anisole (1) (Figure 2A) with those of essential oil (Figure 2B), and a crude hexane extract (Figure 2C), we found that the essential oil and a crude extract had compound 1 as a major compound. ¹H NMR spectra also indicated that the essential oil (Figure 2B) contained trans-p-(l-butenyl)-anisole (1) as a major compound, but a crude hexane extract (Figure 2C) had both trans-p-(l-butenyl)-anisole (1) and fatty acids as major compounds. Signals of fatty acids in the ¹H NMR spectrum of a crude hexane extract were at 5.10, 2.05, 1.60, 1.25, and 0.85 ppm (Figure 2C). We then performed GC-MS analysis of W. schmidtii essential oil, revealing the presence of 41 identified constituents, accounting for 99.65% of the total relative area (Table 1). As shown in Table 1, trans-p-(l-butenyl)-anisole (1) was found to be a major component in the essential oil, accounting for 88.69%. Other compounds in the essential oil were β-caryophyllene (1.84%), sabinene (1.64%), β-elemene (0.65%), borneol (0.54%), E,E-α-farnesene (0.43%), and myrtenyl acetate (0.41%) (Table 1).

Figure 2.

¹H NMR spectra of A, trans-p-(1-butenyl)-anisole (1); B, essential oil of W. schmidtii; and C, a crude hexane extract of W. schmidtii.

Table 1.

Chemical Constituents of W. schmidtii Essential oil as Revealed by GC-MS Analysis.

Peak	Time (min)	Compound	Adam RI	% Relative area
1	6.59	Tricyclene	920	0.18
2	7.87	Sabinene	969	1.64
3	9.67	1,8-Cineole	1026	0.23
4	12.21	Linalool	1095	0.08
5	15.01	Borneol	1165	0.54
6	15.48	Terpinen-4-ol	1174	0.06
7	16.06	α-Terpineol	1195	0.15
8	18.75	p-Anisaldehyde	1247	0.39
9	21.07	Anisyl formate	1326	0.06
10	21.65	Myrtenyl acetate	1330	0.41
11	22.33	butenyl anisole isomer	1350	0.27
12	24.58	Trans-p-(l-butenyl)-anisole	1380	88.69
13	24.78	β-Elemene	1389	0.65
14	25.55	α-cis-Bergamotene	1411	0.11
15	25.77	β-Caryophyllene	1417	1.84
16	26.86	epi-β-Santalene	1445	0.18
17	27.13	α-Humulene	1452	0.08
18	27.21	E-β-Farnesene	1454	0.11
19	27.38	β-Santalene	1457	0.07
20	28.09	Butylated hydroxy anisole	1488	0.26
21	28.37	Germacrene D	1480	0.22
22	28.44	Widdra-2,4(14)-diene	1482	0.24
23	28.77	β-Selinene	1489	0.27
24	29.3	E,E-α-Farnesene	1505	0.43
25	29.57	α-Bulnesene	1509	0.13
26	29.71	Anisyl propanoate	1510	0.09
27	30.21	E-γ-Bisabolene	1529	0.08
28	30.45	4'-Methoxybutyrophenone	1533	0.07
29	31.99	Germacrene D-4-ol	1574	0.23
30	32.07	Trans-sesquisabinene hydrate	1577	0.22
31	32.21	Caryophyllene oxide	1582	0.05
32	32.37	Gleenol	1586	0.12
33	32.46	Allo-hedycaryol	1589	0.11
34	33.95	10-epi-γ-Eudesmol	1622	0.15
35	34.83	Pogostol	1651	0.23
36	36.06	Ishwarone	1680	0.11
37	36.19	5-neo-Cedranol	1684	0.09
38	38.03	Isobicyclogermacrenal	1733	0.13
39	38.85	β-Z-Curcumen-12-ol	1754	0.25
40	39.05	Aristolone	1763	0.18
41	39.99	γ-Eudesmol acetate	1784	0.25
Total percentage of relative area				99.65

As noted in the introduction, W. schmidtii was formerly classified within the Amomum genus and commonly referred to as either Amomum schmidtii or Amomum biflorum. Previous studies often used the nomenclature A. schmidtii or A. biflorum.^3–6 However, the chemical compositions of A. schmidtii or A. biflorum varied across these reports, and the variations in oil composition might be from: i) plants grown in different geographical areas with difference in climate and soil properties may have different chemical compositions, and ii) an error in the taxonomic classification because plants in the Amomum genus closely resemble the Wurfbainia genus. For instance, trans-p-(1-butenyl)-anisole (1) was identified as the major compound in A. schmidtii,¹¹ whereas camphor and α-bisabolol were identified as major compounds in the essential oil of A. biflorum.³ While the present work revealed trans-p-(l-butenyl)-anisole (1) as the major compound in the essential oil of W. schmidtii, recent findings by Vietnamese chemists highlighted 1,8-cineole, camphene, and endo-borneol as the major compounds in the essential oil of W. schmidtii.¹² Previously, the essential oil extracted from A. schmidtti (synonym for W. schmidtii) collected in Vietnam was reported to contain trans-p-(l-butenyl)-anisole (1) as the main compound.¹¹ This suggests a potential taxonomic discrepancy regarding W. schmidtii, warranting further investigation into its classification. Fortunately, recent taxonomic revisions have clarified the taxonomy of plants previously known as A. biflorum and A. schmidtii, now recognized as W. schmidtii.^2,7 We propose that the major compound, trans-p-(l-butenyl)-anisole (1), in the essential oil of W. schmidtii may be used as a chemical marker for this plant. Trans-p-(l-butenyl)-anisole (1) is a phenylbutanoid, and plants in the family Zingiberaceae normally have phenylbutanoids as secondary metabolites, for example, Zingiber cassumunar.²⁸ But trans-p-(l-butenyl)-anisole (1) is a major phenylbutanoid found only in W. schmidtii, not in other Zingiberaceae plants; therefore, it may serve as a chemotaxonomic marker for W. schmidtii.

Cytotoxicity and Anti-Inflammatory Activity

The anti-inflammatory activity of the essential oil from W. schmidtii leaves and its major compound, trans-p-(1-butenyl)-anisole (1), was evaluated. Raw 264.7, murine macrophage-like cells were treated with essential oil and trans-p-(1-butenyl)-anisole (1) at various concentrations for 24 h (Tables 2 and 3). The cytotoxicity of the samples was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and results showed that essential oil at concentrations ranged from 0.000977 mg/mL to 0.03125 mg/mL were not toxic to the cells, as the viability of the treated cells was greater than 85.66% (Table 2). Similarly, trans-p-(1-butenyl)-anisole (1) at concentrations between 0.48 µM and 125 µM were also not toxic to the cells, as cell viability was greater than 97% (Table 3). We then determined anti-inflammatory activity at these non-toxic concentrations of both essential oil and compound 1 by measuring the inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells.

Table 2.

Cytotoxicity and Inhibition of LPS-Induced Nitric Oxide Production by W. schmidtii Essential oil.

Sample	Sample concentration (mg/mL)	Cell viability(% of control)	Nitric oxide concentration(µM)
W. schmidtii essential Oil	0.000977	109.49 ± 5.06	21.13 ± 0.57
	0.001953	109.18 ± 4.01	21.82 ± 0.84
	0.003906	105.83 ± 3.01	21.44 ± 0.92
	0.007813	109.95 ± 2.16	19.86 ± 0.48
	0.015625	107.86 ± 2.53	17.11 ± 0.38
	0.03125	85.66 ± 13.00	14.31 ± 1.45
	0.0625	5.56 ± 2.94	Not determined
	0.125	4.20 ± 1.58	Not determined
	0.25	3.36 ± 0.03	Not determined
	0.5	3.75 ± 0.07	Not determined
	1.0	3.50 ± 0.28	Not determined
	2.0	3.09 ± 0.14	Not determined

Table 3.

Cytotoxicity and Inhibition of LPS-Stimulated Nitric Oxide Production by Trans-p-(1-Butenyl)-anisole (1).

Sample	Sample concentration(mg/mL)	Sample concentration(µM)	Cell viability(% of control)	Nitric oxide concentration(µM)
trans-p-(1-Butenyl)-anisole (1)	0.000077	0.48	109.18 ± 1.21	21.97 ± 0.30
	0.000157	0.97	109.49 ± 2.00	22.70 ± 0.09
	0.000316	1.95	108.68 ± 3.84	20.78 ± 0.77
	0.000632	3.90	111.87 ± 1.33	19.44 ± 0.33
	0.001267	7.81	117.41 ± 2.85	16.84 ± 0.32
	0.002534	15.62	108.32 ± 7.47	12.24 ± 0.21
	0.005069	31.25	107.10 ± 5.83	8.52 ± 0.23
	0.010139	62.5	107.10 ± 2.35	7.91 ± 0.23
	0.020278	125	97.06 ± 2.708	8.45 ± 0.09
	0.040557	250	63.41 ± 4.75	Not determined
	0.081114	500	5.99 ± 2.90	Not determined
	0.162228	1000	3.25 ± 0.17	Not determined

Cells cultured without treatment (untreated cells) were used as a control, while vermelhotin (10 µM) was used as a positive control.²⁶ There was an increase in nitric oxide production in LPS-stimulated untreated cells to a level of 26.34 µM, compared to the basal level of 11.21 µM in untreated cells without LPS stimulation. Both W. schmidtii essential oil and trans-p-(1-butenyl)-anisole (1) reduced nitric oxide production in LPS-stimulated cells in a dose-dependent manner (Figure 3). The essential oil of W. schmidtii exhibited anti-inflammatory activity with the IC₅₀ value of 0.0282 mg/mL (Figure 3A), while trans-p-(1-butenyl)-anisole (1) had the IC₅₀ value of 12.74 μM (Figure 3B). Trans-p-(l-butenyl)-anisole (1) is a phenylbutanoid, and its anti-inflammatory activity has not previously been reported. Previously, a phenylbutanoid-rich extract of Zingiber cassumunar was shown to exert anti-inflammatory activity through the inhibition of nitric oxide production in RAW264.7 cells.²⁹ W. schmidtii collected in Vietnam had the major compounds, 1,8-cineole, camphene, and endo-borneol in its essential oil.¹² Previously, 1,8-cineole (eucalyptol), a major compound of eucalyptus oil, was found to be an anti-inflammatory agent with potential application for suppressing airway inflammation,³⁰ While camphene inhibited inflammatory and neuropathic pain via Cav3.2 T-type calcium channels.³¹ A. biflorum (synonym W. schmidtii) collected in Thailand had camphor, α-bisabolol, camphene, and α-humulene as the major components in its essential oil.³ Camphor was found to exhibit anti-inflammatory activity,³² whereas α-bisabolol exerted anti-inflammatory activity by downregulating expression of iNOS and COX-2 genes through the inhibition of NF-κB and AP-1 signaling.³³ α-Humulene could reduce the eosinophil recruitment to the BALF, the IL-5, CCL11 and LTB₄ levels in BALF, and IL-5 production in mediastinal lymph nodes.³⁴

Figure 3.

Anti-inflammatory effects of essential oil and compound 1 on RAW 264.7 macrophages. LPS-stimulated cells were treated with W. schmidtii essential oil and trans-p-(1-butenyl)-anisole (1) at non-cytotoxic concentrations A) Nitric oxide produced in activated RAW 264.7 cells treated with W. schmidtii essential oil. B) Nitric oxide produced in activated RAW 264.7 cells treated with compound 1. Nitric oxide production in LPS-stimulated untreated cells was assigned as 100%.

Virucidal and Antiviral Activities of W. schmidtii Essential Oil

¹H NMR spectrum (Figure 2B) of the essential oil from W. schmidtii revealed that trans-p-(l-butenyl)-anisole (1) was a major component, and GC-MS analysis indicated that the essential oil contained 88.69% of compound 1 (Table 1). We then evaluated virucidal and antiviral activities of trans-p-(l-butenyl)-anisole (1) against SARS-CoV-2 using virucidal and antiviral assays, as previously described.²⁷ Prior to the evaluation of virucidal and antiviral activities, cytotoxicity of compound 1 was evaluated in the test system of an antiviral assay using A549-hACE2 cells, which are host cells of SARS-CoV-2 virus. The cytotoxicity of the compound is represented by the CC₅₀ value, which is the concentration that reduces cell viability by 50%. In contrast, virucidal and antiviral activities are expressed in the EC₅₀, representing the concentration required to inhibit viral infectivity by 50%. As shown in Figure 4, trans-p-(l-butenyl)-anisole (1) exhibited virucidal activity against SARS-CoV-2 with EC₅₀ value of 119.60 μM (Figure 4A) and CC₅₀ value more than 308.21 μM (Figure 4B). However, compound 1 did not display antiviral activity against COVID-19 virus, having EC₅₀ and CC₅₀ values of more than 308.21 μM (Figures 4C and 4D). Compound 1 is a phenylbutanoid, and to our knowledge, phenylbutanoids including compound 1 have never been reported to have virucidal activity against SARS-CoV-2 virus.

Figure 4.

Virucidal and antiviral activities of trans-p-(1-butenyl)-anisole (1) against SARS-CoV-2. (A) The virucidal effect was determined by treating SARS-CoV-2 with trans-p-(1-butenyl)-anisole (1) at 37 °C for 1 h. Subsequently, hACE2-A549 cells were infected with the treated virus and cultured for 96 h without the addition of compound. DMSO-treated virus served as a control, representing 100% viral infectivity. Viral infectivity was quantified by measuring the cytopathic effect from the infection using the Viral ToxGlo assay. (B) Cytotoxicity of compound 1 in the virucidal assay was accessed by treating hACE2-A549 cells as mentioned above, without virus infection. DMSO-treated cells served as a non-cytotoxic control, representing 100% cell viability. Cell viability was subsequently quantified using the Viral ToxGlo assay. (C) The antiviral activity of compound 1 was evaluated by infecting hACE2-A549 cells with SARS-CoV-2 at 37 °C for 1 h. The infected cells were subsequently treated with compound for 96 h. Viral infectivity was quantified by Viral ToxGlo assay. (D) The cytotoxicity of compound 1 in the antiviral assay was determined by treating hACE2-A549 cells with compound for 96 h, and cell viability was subsequently quantified using the Viral ToxGlo assay. The EC₅₀ and CC₅₀ values of trans-p-(1-butenyl)-anisole (1) were estimated by fitting the data to a dose-response curve using GraphPad Prism software. Data are presented as mean values ± SD from a minimum of two independent experiments (each performed in duplicate).

Although this work shows anti-inflammatory activity of W. schmidtii essential oil and the major compound, trans-p-(1-butenyl)-anisole (1), as well as virucidal activity of compound 1 against the SAR-CoV-2 virus, there are a few limitations as follows: i) the evaluation of biological activities was performed only for the major compound 1, while other components were not tested for the biological activities; ii) the isolation of the hexane extract focused only on the major compound, while other components were not isolated from the extract; iii) the essential oil of W. schmidtii has strong distinctive odor, which may affect its applications.

Conclusions

The leaves of W. schmidtii were chemically explored, and trans-p-(1-butenyl)-anisole (1) was found as a major component in this plant. Compound 1 may be used as a chemotaxonomic marker for W. schmidtii. Both the essential oil of W. schmidtii and trans-p-(1-butenyl)-anisole (1) were found to exhibit anti-inflammatory property and virucidal activity against the SAR-CoV-2 virus. Given its anti-inflammatory effect, the essential oil of W. schmidtii may be used as an anti-inflammatory agent for further applications.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251323479 - Supplemental material for An Anisole Derivative in the Essential oil of Wurfbainia schmidtii with Virucidal Activity Against SARS-CoV-2 and Anti-Inflammatory Properties

Supplemental material, sj-docx-1-npx-10.1177_1934578X251323479 for An Anisole Derivative in the Essential oil of Wurfbainia schmidtii with Virucidal Activity Against SARS-CoV-2 and Anti-Inflammatory Properties by Charmaine Sagayap, Piyachat Chuysinuan, Nopporn Chutiwitoonchai, Patcharee Pripdeevech, Wittaya Kaewsri, Sanya Sureram, Narisara Chantratita, Kriengsak Lirdprapamongkol, Jisnuson Svasti, Supanna Techasakul, Chulabhorn Mahidol, Somsak Ruchirawat and Prasat Kittakoop in Natural Product Communications

Footnotes

Acknowledgements

C. Sagayap thanks Chulabhorn Graduate Scholarship supported by Thailand International Cooperation Agency (TICA) and Chulabhorn Graduate Scholarship Commemorating the 84^th Birthday Anniversary of His Majesty King Bhumibol Adulyadej the Great. We acknowledge the financial support and facilities of the Chulabhorn Research Institute (CRI) and Chulabhorn Graduate Institute (CGI), as well as the grant from the National Research Council of Thailand (NRCT) and Thailand Science Research and Innovation (TSRI). We are thankful to Dr Anan Jongkaewwattana for providing SARS-CoV-2 for our virucidal and antiviral assays.

Author Contributions

C.S.: Investigation, Formal analysis, Writing-original draft; P.C.: Investigation, Writing-original draft; N.C.: Investigation, Writing-original draft; P.P.: Investigation, Writing-original draft; W.K.: Formal analysis; S.S.: Formal analysis; N.C.: Formal analysis; K.L.: Formal analysis; J.S.: Formal analysis; S.T.: Formal analysis; C.M.: Supervision; S.R.: Supervision, Funding acquisition; P.K.: Conceptualization, Funding acquisition, Writing-original draft, Writing-review & editing.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Ethical Approval is not applicable for this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research project is supported by Chulabhorn Graduate Institute (Fundamental Fund by National Science Research and Innovation Fund (NSRF): fiscal year 2025) (FRB680079/0518 Project code 209039). This work is partially supported by Thailand Science Research and Innovation (TSRI, Chulabhorn Research Institute, Grant No. 36827/4274406).

ORCID iDs

Charmaine Sagayap

Prasat Kittakoop

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supporting Material

¹H, ¹³C NMR and 2D NMR spectra and mass spectrum of trans-p-(1-butenyl)-anisole (1) (PDF).

Supplemental Material

Supplemental material for this article is available online.

Trial Registration

Not applicable, because this article does not contain any clinical trials.

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