Abstract
Background
Artemisia vulgaris L. (AV) is a beneficial herb with therapeutic properties. This work aims to evaluate the compositions and bioactivity of essential oil from AV grown in Tiengiang, Vietnam.
Methods
The essential oils (AVEO) were extracted by hydrodistillation (HD), and the headspace volatiles (HS) were collected using the static headspace technique. Gas chromatography/mass spectrometry (GC/MS) was used to examine the compositions of oils in detail. The agar well-diffusion technique was used to conduct the antibacterial test. Multi-concentration dilution method was used for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination. The cell viability was determined by the MTT method. Nitric oxide and TNF-α in RAW 264.7 were analyzed by Griess reagent or ELISA as described by the manufacturer. In addition, DPPH, ABTS, and reducing power assay were used to determine the antioxidant activity.
Results
The HD method yielded 1,8-cineole, α-pinene, β -caryophyllene, borneol, camphor, and δ-elemene were the most abundant components. In contrast, the HS approach produced the primary constituents being camphor, 2-methylbutanal, 1,8-cineole, and camphene. The HD showed antibacterial activities against E.coli, S.typhimurium, S.aureus, P.aeruginosa, and S.mutans, and inhibited the production of NO, TNF-α in lipopolysaccharide-induced RAW264.7. Furthermore, the HD indicated moderate antioxidant activity. A hierarchical cluster analysis of essential oils from 22 regions shows that mugwort oils in Tiengiang, Vietnam, have a high level of δ-elemene (more than 5%), which is not common in oils from other species. The study's findings enhance our comprehension of the chemical components and chemical variety of mugwort oil across different sites and to help in the identification of the predominant species suitable for extracting essential oils for different purposes within the Artemisia vulgaris L. species.
Conclusion
These findings collectively suggest that essential oils derived from A. vulgaris have a range of potential therapeutic uses.
Introduction
One of the important medicinal plant species in the genus Artemisia, which is best known for its volatile oil, is Artemisia vulgaris (mugwort). 1 It is frequently used as a sedative, to improve circulation, to treat fevers, diarrhea, rheumatic aches, and dyspepsia. 2 Essential oils are intricate blends of naturally occurring materials with fragrant scents. They are often taken from plants that have aromatic secondary metabolites. Essential oils have long been employed as flavoring agents, perfumers, and in the preparation and preservation of food. 3 Many bioactive substances found in essential oils have a variety of biological effects, such as antibacterial, antiviral, anti-inflammatory, analgesic, antiparasitic, antidiarrheal, and anxiolytic effects.4,5 In Vietnam, A. vulgaris has historically been valued for its pain relief, anti-inflammatory, insecticidal, and antibacterial activities. Previous studies showed that essential oils of A. vulgaris (AVEO) contain myrcene, 1,8–cineole, borneol, sabinene, camphor, α-thujone, terpinene-4-ol, and its chemical compositions were dependent on climatic and soil conditions.6–8 The studies have documented the chemical constituents of oil extracted from the aerial parts of AV using hydrodistillation in countries including Egypt, 9 Cuba, 10 Vietnam, 11 France, 12 Italy, 13 and the Republic of Bashkortostan. 14
Moreover, Prior research has demonstrated that the application of mugwort oil in excessive doses can result in certain adverse effects. The European Food Safety Authority (EFSA) notes that when combined with food or dietary supplements, the components of AVEO, including α-thujone, β-thujone, camphor, and 1,8-cineole, may negatively impact human health. On the other hand, the EFSA highlights that the majority of studies have fobacused on a more concentrated oil, not a less concentrated extract. 15
Variable amounts of thujone are present in Artemisia species. European Medicines Agency (EMA) 16 highlighted the substance's toxicological potential. Although the concentration of α- and β-thujone in AV is typically small, it can differ. 17 The essential oil extracted from the stem and flowers of AV contains an estimated 56.3% α-thujone and 7.5% β-thujone. 15 However, the precise amount extracted via traditional infusion is challenging to determine due to the low solubility of these compounds in water. Neurotoxic effects can be attributed to both α-thujone and β-thujone, with α-thujone exhibiting a potency that is three to four times greater than β-thujone. 18 The rapid modulation of GABA-gated chloride channels by thujone is responsible for the neurotoxicity and epileptiform convulsions that are commonly observed in cases of acute poisoning. 19
The above studies show that the pharmacological effects of mugwort oil will depend on its constituents and content. Variations in these parameters can occur simultaneously based on different geographical locations. Therefore, to ensure the safe and effective use of mugwort oil, it is crucial to research to clarify the ingredients and their biological effects, especially studying the mugwort oil's ingredient originating in the location without having previously studied it.
Moreover, mugwort is widely grown wild or as a cultivated plant in Vietnam. Studies have been conducted on the essential oils derived from the mugwort plant in the Northern regions of Vietnam, specifically Ha Noi and Vinh Phu. To the best of our knowledge, no research has been conducted on the composition of oils obtained by hydrodistillation of the AV aerial parts from Tiengiang – a southern province of Vietnam. Thus, it is essential to thoroughly look into the chemical composition and biological activities of mugwort essential oil to increase the value as well as a reasonable use in medicine of AV planted in Tiengiang, Vietnam. Therefore, the primary goals of this study were to determine the biological characteristics of AVEO obtained by hydrodistillation extraction from the aerial part of A. vulgaris plants cultivated in Tiengiang, Vietnam, and to evaluate their chemical compositions and headspace analysis. Furthermore, our study employed the hierarchical cluster analysis method to evaluate the variances between our essential oil composition and prior publications, specifically the two mugwort oils in Hanoi and Vinh Phu.
Results and Discussion
Chemical Composition of Hydrodistillation Essential oil (HD) and Headspace (HS) Analysis of A. vulgaris by GC–MS
The yield of AVEO by hydrodistillation was 0.26%. From the oils and headspace specimens of A. vulgaris, 94 compounds were identified, 60 for HD (Table 1 and Figure S1) amounting to 100% of the total amount of essential oil, and more components for the HS (Table 2 and Figure S2) measurements of AVEO with 68 identified compounds, corresponding to 100%. Among 94 constituents, 34 were in both HD and HS analysis. The HD yielded 1,8-cineole (24.25%), α-pinene (10.57%), β - caryophyllene (7.10), borneol (8.89%), camphor (6.87%), and δ - elemene (6.05%), while the major volatile compounds in headspace method were camphor (27.16%), 2 - methylbutanal, (20.94%), 1,8 - cineole (14.88%), and camphene (7.38%). Tables 1 and 2 demonstrated that the HD method detected numerous components with higher boiling points in the mugwort species in Tien Giang, including oxygenated monoterpenes and sesquiterpenes. Conversely, the HS method primarily identified volatile substances, such as aldehydes, alcohols, and ketone compounds, with a low carbon number and low boiling point. It is important to emphasize that the HD method contains a higher concentration of hydrocarbon sesquiterpene compounds, such as δ - elemene and β - caryophyllene, than the HS method. Upon examining the chemical compositions of the two procedures, HD and HS, we made an exciting discovery: the levels of oxygenated monoterpene and oxygenated sesquiterpene were rather constant in both approaches (Tables 1 and 2). In the HS approach, there was a substantial increase (27.70%) in the number of non-terpene compounds compared to the HD method (1.22%). However, the HS approach resulted in a considerable decrease in the hydrocarbon sesquiterpene group by 8.50%, in contrast to the corresponding group in the HD method which showed a level of 22.26%. The HS method only used a heating temperature of 90 0C, thereby inhibiting the evaporation of elevated boiling point compounds, such as hydrocarbon sesquiterpenes, from the sample and impeding their identification.
Chemical Composition of Hydrodistillation Essential Oils of Artemisia vulgaris L. Using GC-MS.
RT: Retention time. RI: Retention index; MS: Mass spectrometry; a: Calculated retention index; b: References retention index.
Chemical Composition of Headspace Essential Oils of Artemisia vulgaris L. Using GC-MS.
A hierarchical cluster analysis (HCA) was conducted on the percentages of the main compounds in mugwort oils from various locations. The analysis, based on the Euclidean distance between groups, resulted in the formation of three distinct clusters, as shown in Figure 1 and Table S1. Our HCA, which incorporated 43 essential oil compositions of AV from 26 reports9–14,20–33 and our samples, yielded some unexpected results. Contrary to our initial expectations, the chemical profile of mugwort oil from different locations in Vietnam was found to be scattered across various classes (Figure 1). The study conducted a similarity analysis for mugwort species collected in Vietnam. The similarity between the oil of Tiengiang and Hanoi was 93.25%. At the level of the dendrogram, the oils extracted from AV in Tiengiang were grouped within the same class of AV in Hanoi, confirming intra-class homogeneity between these species. However, the level of similarity between Tiengiang and Vinhphu decreased to 46.35%. In the case of comparison with the mugwort species grown outside of Vietnam, the high homogeneity was observed with three samples from Wuhan (China), Nis (Serbia), and Siauliai (North Lithuania), with a striking resemblance of 78.49%. These oils contained the most predominant amount of 1,8-cineole, borneol, β-caryophyllene, β-thujone, and camphor. The oils extracted from AV cultivated in Wuhan, Ha Noi, North Lithuania, Germany, Serbia, France, and England showed the 1,8-cineole chemotype similar to the Tiengiang sample. Camphor was the common chemotype in our sample compared to mugwort oils cultivated in Giza (Egypt), Montpellier (France), Lucknow (India), Italy, Japan, and Morocco. It is worth noting that the monoterpene hydrocarbon α-pinene is exclusively found in the essential oils of Tiengiang, Hanoi, London, and the Republic of Bashkortostan. Meanwhile, the isomer β-pinene is exclusively present in samples collected from Tiengiang, Hanoi, and the Republic of Bashkortostan. Our research has revealed a noteworthy discovery: our oils, unlike other A. vulgaris oils, contain a significant quantity of δ-elemene (6.05%). To our knowledge, mugwort essential oils containing δ-elemene, which is higher than 5%, are rare. Actually, the elemene chemotype previously recognized in most A. vulgaris essential oils was β-form. The δ-elemene chemotype was also not found in AV cultivated in Hanoi and Vinhphu (Vietnam). The occurrence of δ-elemene in mugwort oils is uncommon, and its presence can be regarded as a reference for oils derived from Tiengiang (Vietnam). This is a fascinating fact to consider when studying natural compounds and essential oils. The chemical and biological variety of aromatic and medicinal plants is known to be influenced by climate factors, vegetation techniques, and genetic parameters. 34 These elements have an impact on the biosynthetic pathways of the plant, which causes variations in the amount and properties of various phytoconstituents. This leads to a variety of chemical structures in secondary natural products, as well as opportunities for the discovery of new biological activity based on the diversity of chemical structures.
Dendrogram represents the similarity relation of the main compounds in mugwort oils growing in different locations.
Among the identified composition, 1,8-cineole is a monoterpene cyclic ether and possesses many bioactivities such as antimicrobial, antioxidant, mucolytic, broncholytic, and anti-inflammatory properties. 35 Borneol is a bicyclic monoterpene found in several Artemisia species. In vitro experiments demonstrated the antibacterial, anti-inflammatory, analgesic, and antithrombotic properties of this compound. 36 Moreover, camphor, a monoterpene ketone, has several biological characteristics, including antibacterial, antiviral, and antitussive activities. 37 The results on the toxicity of different isomers of thujones ranged from relatively weak to extremely strong. At higher concentrations, this ketone has significant toxicity against brain, liver, and kidney cells in both humans and animals.
Our findings indicate that mugwort oil originating from Tiengiang, Vietnam, contains small amount of thujone isomers. This implies that the utilization of AVEO will be acceptable for customers.
Antibacterial Activity of AVEO
Using the inhibitory zone method, the antibacterial effect of AVEO extracted from A. vulgaris was assessed against the following bacteria: Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 27853), Streptococcus mutans (ATCC 25175), and Escherichia coli (ATCC 8739). By measuring the dimension of the inhibitory zone of the selected bacteria, the antibacterial activity was determined.
As shown in Figure 2, AVEO exhibited notably inhibited bacteria, especially Staphylococcus aureus and Streptococcus mutans in a concentration-dependent manner. Results revealed that the zone of inhibition (mm) were 16.0, 14.5, 13.5, and 12.0 for Staphylococcus aureus, whereas those values for Streptococcus mutans were 14.5, 12.0, 12.0, and 11.0, at concentrations of 50, 25, 10, and 5 μl/well, respectively. At the examined doses, AVEO demonstrated weak and moderate inhibition for the other bacterial strains. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the essential oil against five bacterial strains ranged from 25 to 150 μl/mL (Table 3). As we knowed bacteriostatic activity, which inhibited the development of bacteria without necessarily killing them, and bactericidal activity, which killed bacteria, were the two categories of anti-bacterial effects. We are able to differentiate between these two categories based on the ratio MBC/MIC (R): if R < 4, it falls into the bactericidal group, and if R > 4, it falls into the bacteriostatic group.
Antibaterial activity of AVEO. EC: Escherichia coli, PA: Pseudomonas aeruginosa, SA: Staphylococcus aureus, SM: Streptococcus mutans, ST: Salmonella typhimurium. Pos: Chloramphenicol 20 μg/well. 50, 25, 10, 5: volume of AVEO/well (μl). Data are expressed as mean ± SD of three independent experiments.
The MIC and MBC of AVEO.
AVEO: Artemisia vulgaris essential oil. Standard: Chloramphenicol. MIC: Minimum inhibitory concentration, MBC: minimum bactericidal concentration, BC: bactericidal activity. R = MBC/MIC ratio.
From Table 3, the essential oil of A. vulgaris (AVEO) showed bactericidal activity for five bacteria. The A. vulgaris essential oil had very little antibacterial activity against gram-negative bacteria and our findings were similar to the previous document. 38 The volatile components that had been discovered above and found in the essential oil may be responsible for the results of our present investigation of the AVEO's antibacterial capacity against the strains investigated. A little amount of research has been done on the essential oil of A. vulgaris from southern of Vietnam, even though there are many studies on the antibacterial activity of the essential oil of A. vulgaris. Essential oils are thought to be rich sources of antibacterial substances, particularly those that combat bacterial infections. 39 The bacterial cell membrane and mitochondria's lipids partition due to the hydrophobic nature of the essential oil, disrupting the cell's structural integrity and increasing permeability. 40 Additionally, reports have indicated that the chemical components of the essential oil broke down the bacterial cell wall by attaching themselves to the cell surface and entering the target areas via plasma membranes or enzymes attached to membranes. 41 The previously identified compounds in the essential oil could clarify our study's results against the tested strains. Despite many findings on the anti-bacterial properties of A. vulgaris extract, no study has been performed on A. vulgaris essential oil from Southern of Vietnam. These herbal antibiotics can replace synthetic drugs and be selective, effective, biodegradable, and environmentally beneficial. 42
Antioxidant Activity of AVEO
Free radicals, also known as radicals, are chemical molecules with unpaired electrons and highly reactive. They are often produced due to normal cellular processes in biological systems, such as reactive oxygen species (ROS).43,44 ROS refers to a comprehensive group of significant reactive oxygen species molecules generated during oxygen metabolism. 45 Antioxidants help balance the free radicals’ levels in cells, thereby preventing oxidative stress and its associated diseases. 46 The overproduction of ROS is caused by an imbalance in the antioxidant system. This excessive production leads to oxidative damage, which deteriorates cellular macromolecules such as enzymes, lipids, DNA, and proteins.47,48
Antioxidants provide a biological means of protection against oxidative stress in the human body. They relate to the ability to eliminate ROS by providing atoms of hydrogen/electrons, binding transitional metal ions, enhancing antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and preventing oxidation. However, these protective approaches may not always be sufficient due to the extensive diseased condition. Therefore, the addition of antioxidants plays an essential role in preventing oxidative damage in the body. 49
DPPH and ABTS radical scavenging and reducing power activities were used to determine the antioxidant properties of A. vulgaris essential oil. The AVEO presented moderate DPPH and ABTS free radical-scavenging activities with IC50 values of 74.66 ± 2.78 and 23.05 ± 1.74 μg/ml, respectively (Figure 3). However, the activity was lower than the positive control (IC50 of 5.52 ± 1.50, and 2.21 ± 0.32 μg/ml).
Biological activity (DPPH assay, ABTS assay and RP assay) of A) standard compounds (vitamin C, Trolox) and B) AVEO sample. Data are expressed as mean ± SD of three independent experiments.
To verify the antioxidant properties further, the reducing power assay was carried out. The reducing power test is frequently used to assess an antioxidant's capacity to provide an electron. 50 This test measured the extracts’ capacity to convert Fe3+ to Fe2+. The ferric cyanide complex Fe3+ was reduced to the ferrous cyanide form Fe2+ as a result of the extracts’ antioxidant content. Depending on the reducing power of the phytocompounds, the antioxidants in the reducing power test caused the reduction of Fe3+ into Fe2+, which resulted in the solution shifting from green to blue in varying colors. 51 Nonetheless, Perl's Prussian blue color was created by strong reducing compounds, which were absorbed at 700 nm. As shown in Figure 3, it was discovered that the reference chemical, trolox (OD0.5 of 71.81 ± 1.43 μg/ml), had a greater reducing power than AVEO (OD0.5 of 138.16 ± 2.30 μg/ml). This indicated that AVEO contained a moderated amount of reductones and could reduce ferric ions. In order to transform free radicals into more stable substances and stop the free radical chain events, AVEO may therefore function as electron donors and our results are similar to Bhatt ‘s study. 52
Anti-Inflammatory Effect of AVEO
In order to identify the appropriate concentrations of essential oil (AVEO) for anti-inflammatory application, the toxicity of the AVEO on RAW 264.7 cells was assessed (Figure 4A). The dose of AVEO affected cell toxicity. Cell survival exceeded more than 80% when AVEO concentrations were less than 125 μg/ml, there was no statistically significant difference between the concentrations of AVEO at 125; 62.5, and 31,25 compared to untreated cells (P < 0.01). Notably, cell viability surpassed 100% at low doses, and cell growth was stimulated when compared to the untreated group (P > 0.01). As a defensive strategy against outside stimuli, essential oils increase cell growth. 53 Thus, in this experiment, the RAW 264.7 cells were treated with essential oils at a concentration of below 100 μg/ml to assess the anti-inflammatory effects of the AVEO.
Anti-inflammatory activity of AVEO. A) RAW 264.7 cell viability. B) NO production in LPS-induced RAW264.7 macrophages. C). The secretion of TNF-α in LPS-induced RAW 264.7 macrophages. Neg: untreated cell group; Dex: dexamethasone group; LPS: LPS-activated group. Data are expressed as mean ± SD of three independent experiments. @ P < 0.01 compared to DMSO. ns: non-significant. # P < 0.01 compared to Untreat; * P < 0.01 compared to LPS.
The levels of NO dissolved in the cell supernatants were evaluated in vitro to assess the macrophages’ phagocytosis-related activities. Figure 4B displayed the NO concentrations in the LPS-activated RAW 264.7 cells. Compared to the untreated cells (Neg, 11.92 ± 0.75 μM), the NO level of the LPS-treated cells (LPS, 22.05 ± 1.11 μM) was around two times greater (P < 0.01). The administration of 100 nM dexamethasone (Dex) significantly reduced the amount of NO in the positive control by 56.4% in contrast to the LPS group (P < 0.01). Meanwhile, in comparison to the LPS group, the AVEO treatment reduced NO generation by 9.48–58.43% in LPS-stimulated RAW 264.7 cells. It's important to note that AVEO reduced NO content more than the positive control did at a concentration of 100 μg/mL. The results showed that AVEO at a dose of 100 µg/ml had a good inhibitory effect on NO production, with a statistically significant difference compared to the untreated group (P < 0.01).
TNF-α is one of the cytokines that contribute to inflammatory reactions in macrophages. 54 Consequently, this cytokine is employed as a crucial indicator of anti-inflammatory action. In this instance, the AVEO markedly reduced the expression of TNF-α in LPS-activated RAW 264.7 cells (Figure 4C). When compared to the untreated cells, LPS-stimulated RAW 264.7 cells elevated TNF-α expression by about 23.8 times, and the above difference was statistically significant (P < 0.01). Conversely, dexamethasone 100 μM inhibited the increase in TNF-α expression about 91.4%. Interestingly, the AVEO exhibited an 11.6–55.6% reduction of the inflammatory response when compared to the LPS group (P < 0.01). Except for the 12.5 µg/ml concentration, AVEO at other concentrations all statistically significantly reduced the expression of the cytokine TNF-a (P < 0.01) compared to the LPS group.
According to the current findings, AVEO has good anti-inflammatory property, making it an appropriate anti-inflammatory source for the study and development of natural anti-inflammatory medications.
The Limitation of the Current Study
This study provided new information about mugwort essential oil cultivated in Tiengiang, a province in the Mekong delta region of southern Vietnam. Via GC-MS in combination with headspace techniques, 94 compounds were identified which belong to hydrocarbon monoterpene, oxygenated monoterpene, hydrocarbon sesquiterpene, oxygenated sesquiterpene, and other. This study emphasizes the potential application of mugwort essential oil in health care products due to its anti-oxidant and anti-inflammatory properties. Along with health benefits, the outstanding action of mugwort essential oil in anti-bacterial tests suggested the application of this oil in food packing or agricultural products. In addition, due to suitable conditions, mugwort thrives in Tiengiang; therefore, the result provided the foundation for developing the product in the industry.
More details on the chemical composition of essential oils should be provided to develop this resource for industrial use. The compounds in the essential oil would vary depending on the time of harvest (daytime, season, etc), the time of extraction, etc Therefore, the main chemotype for each target biological function should be studied. Based on this result, the enrichment process should be performed. In this way, the standardization and generalization of results become easy. Although the anti-oxidative and anti-inflammatory capacities were proved in this study, the results were done in vitro; this information should be verified on animals. Likewise, the resistant capacity of bacteria against essential oils needs to be examined to conclude whether our essential could be used as an antibiotic. Finally, to provide enough scientific evidence about the application of essential oils in health products, acute and chronic toxicity needs to be supported in the future.
Conclusion
The above results show that mugwort essential oil in Vietnam accounted for 0.26% of the aerial parts of A. vulgaris. The main chemical components of essential oils are monoterpene (α-pinene, 1,8-cineol, borneol, camphor), and sesquiterpene (δ-elemene, β-caryophyllene). Essential oils possess interesting biological activities such as antioxidant, anti-inflammatory, and antibacterial effects, especially against gram-positive bacteria. Further, in vivo studies need to be conducted to be able to apply mugwort essential oils in cosmetics and pharmaceuticals.
Materials and Methods
Material
The aerial parts of A. vulgaris were collected from plants growing in Tien Giang province, Vietnam in October 2023. The plant was identified and authenticated by Dr Luu Hong Truong. A voucher specimen (2023/D-10AV) was kept at the Department of Biopharmaceutical Materials, Institute of Applied Materials Science, Ho Chi Minh, Vietnam.
AVEO Extraction
Freshly obtained A. vulgaris samples were cleaned, dried, cut, and manually ground to a fine powder. The fresh materials (500 g) in 2000 ml of water contained in a 5-liter flask were hydro-distilled for four hours using a Clevenger device. Anhydrous sodium sulfate was used to dry the essential oils, which were stored in a freezer until use.
Headspace Analysis
A 20 mL headspace vial containing 500 mg of aerial parts was filled with 3 mL of distilled water for the static headspace investigations. 500 µL of the vapor produced by the specimens was extracted from the vial using a gas-tight syringe (90 °C) and injected straight onto the chromatographic column after the samples had been heated to 90 °C for 30 min. The next mixing program involved shaking the samples for 5 s and pausing for 2 s.
GC-MS Analysis
A GC (Agilent 6890N, USA) equipped with an HP-5MS Capillary Column (30.0 m × 0.25 mm, film thickness 0.25 µm, USA) was used to examine the AVEO (25 µL). The temperature program was run at 5 °C per minute from 50 to 150 °C, 3 °C per minute from 150 to 200 °C, 10 °C per minute from 200 to 280 °C, and 20 min at 280 °C. Electron impact ionization (70 eV) at full scan mode (40 to 500 m/z) with an ion source operating at 200 °C was used to acquire the mass spectra. Based on a standard sample including a homologous series of n-alkanes (C8-C30, Sigma Co., USA), the retention index was calculated. Retention indexes (RI) and mass spectra were compared with information from the NIST 11.0 MS collection and published works to determine the identity of the essential oils. 55
Antibacterial Activity
The agar well-diffusion technique was used to carry out the antibacterial test. 56 The bacterial inoculums were made in sterile normal saline with turbidity equal to 0.5 McFarland standard, which is 1.5 × 108 CFU/mL. Utilizing sterile cotton swabs, the produced inoculums of various bacteria were evenly distributed in distinct Mueller-Hinton Agar (MHA, Sigma Co., USA) media. Before the experiment, mugwort essential oil was diluted in DMSO with a 100-fold dilution, and the diluted essential oil was the sample used in the study of antibacterial activity. The procedure was carried out three times, with the plate being rotated at a 60° angle in between each streaking. The 90 mm-diameter wells were bored into the MHA plates using a sterile metal cork borer with a 6 mm diameter. Next, 50 µL of diluted essential oil dissolved in DMSO (Sigma Co., USA) (v/v) at concentrations of 5%, 10%, 25%, and 50% were added to the wells. This resulted in final essential oil concentrations in the wells of 2.5, 5.0, 12.5, and 25.0 µL, respectively. The solvent, or negative control, was DMSO, while the positive control was chloramphenicol (1 mg/mL). After allowing the essential oil and antibiotic to diffuse for an hour in the fridge, the plates were incubated for twenty-four hours at 37 °C. Every experiment was carried out in three duplicates. Next, a scale was used to measure the zones of inhibition. The shown data is mean ± SD.
Following their cultivation in Mueller-Hinton Broth (MHB, Sigma Co., USA) medium, the bacteria were diluted using MHB media until their density reached 106 CFU/mL. To the 96-well plate, add 100 μL of AVEO, adjusting the concentration step between 1 and 0 mg/mL for half dilution. Afterward, each well received 100μL of diluted bacterial culture, resulting in 105 CFU/well of bacteria. The plate was then incubated at 37 °C for 24 h, during which the MIC value was determined. 57
The bacteria in the wells with various multi-concentration dilutions of AVEO were used to inoculate the MHB agar plate and incubate at 37 °C to determine the minimum bactericidal concentration (MBC). This experiment is similar to the MIC determination method. The appearance of colonies on the agar plates was observed after twenty-four hours. The minimum extraction concentration (MBC) at which colony reemergence cannot be found is measured. 57
Antioxidant Assay
The AVEO was determined through DPPH, 58 ABTS 58 and reducing power assay. 59
DPPH Experiment
A 1.0 mL DPPH (Sigma Co., USA) solution (0.1 mM) was combined with 1.0 mL of essential oil at the given concentrations (1-100 μg/mL), and the mixture was incubated for 30 min at room temperature. The solution's absorbance was measured at 517 nm. The blank solution included DPPH and methanol (Fisher Chemical, USA) but no essential oil. As a positive standard, ascorbic acid was employed. The equation was used to determine the % inhibition of DPPH free radical:
ABTS Experiment
2.0 ml of 2.45 mM ammonium persulfate (Fisher Chemical, USA) and 2.0 ml of 7 mM ABTS (Sigma Co., USA) solution were combined to create a solution ABTS
Reducing Power
0.5 milliliters of phosphate buffer (Sigma Co., USA) (0.2 M, pH-6.6) were combined with 0.5 milliliters of essential oil at varying concentrations (1-100μg/mL). After adding 1.1% potassium ferricyanide (0.5 mL) to the mixture and keeping it standing at 50 °C for 20 min, 0.5 mL of 10% TCA (Fisher Chemical, USA) was added. The aliquot (1.5 mL) of supernatant was filled with an equal volume of distilled water, and then 0.1 mL of ferric chloride (Fisher Chemical, USA) (0.1%) solution was added. At 700 nm, the absorbance was measured. The results were expressed as OD0.5, the concentration of the sample, causing the absorbance of the solution to be 0.5 at 700 nm.
Anti-Inflammatory Activity
Cell Culture
The RAW264.7 cells were cultivated in DMEM supplemented with 10% FBS (Fisher Chemical, USA), non-essential amino acids, penicillin G (100 IU/mL), and streptomycin (50 µg/mL), as well as β-mercaptoethanol (5 × 105 mM) and sodium pyruvate (all from Fisher Chemical, USA) (1 mM). 60
Cell Viability
The Cell Counting Kit-8 (Sigma Co., USA) was used to measure cell viability following the manufacturer's instructions. To summarise, the cells were cultured on a Corning Costar 48-well plate for 60 min after the addition of 10 µL of CCK-8. The mixture's absorbance was calculated at 450 nm.
60
Detection of NO, TNF-α
Before treatment, the Raw 264.7 cells were seeded into 96-well plates within 16 h at a density of 106 cells/mL. After co-culturing the cells for 60 min with AVEO at various dosages, 1 µg/mL of LPS (Sigma Co., USA) was added to elicit cell inflammation within 24 h. The culture supernatants were obtained using centrifugation, and the levels of NO and TNF-α were assessed using detection kits by the instructions provided in the corresponding user manuals. 60
Statistical Analysis
Data from independent experiments were presented as mean ± SD for statistical analysis, which required multiple comparisons using the Student's t-test with ANOVA at p < 0.01, differences have been considered significant. To evaluate the variations in the chemical compositions of essential oils in various locations, Hierarchical Cluster Analysis (HCA) was performed using Minitab 21.4.2. Employing an Euclidean distance, HCA performed Ward's minimum variance strategy.
Supplemental Material
sj-docx-1-npx-10.1177_1934578X241275782 - Supplemental material for Antioxidant, Anti-Inflammatory, and Anti-Bacterial Activities of Artemisia vulgaris L. Essential Oil in Vietnam
Supplemental material, sj-docx-1-npx-10.1177_1934578X241275782 for Antioxidant, Anti-Inflammatory, and Anti-Bacterial Activities of Artemisia vulgaris L. Essential Oil in Vietnam by Pham Thi Nhat Trinh, Le Xuan Tien, Tong Thanh Danh and Dang Thi Le Hang, Nguyen Van Hoa, To Thi Bao Yen, Le Tien Dung in Natural Product Communications
Footnotes
Acknowledgments
This project is partially supported by the Vietnam Academy of Science and Technology (VAST) under grant number VAST04.02/23-24. The authors thank the Institute of Applied Materials Science, Vietnam Academy of Science and Technology for allowing the use of the equipment in the study.
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 received no financial support for the research, authorship, and/or publication of this article.
Statement of Human and Animal Rights
This article does not contain any studies with human or animal subjects.
Statement of Informed Consent
The authors have read the final manuscript and approved the submission.
Supplemental Material
Supplemental material for this article is available online.
References
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