Variations in chemical constituents and bioactivities of essential oils within Elatostema stewardii Merr. revealed by GC-MS-based metabolomics

2.1. Chemicals and reagents

For EO extraction, the ultra-pure water was produced by a Milli-Q apparatus (Millipore Corporation, Billerica, MA, USA), and the anhydrous sodium sulphate (Na₂SO₄) was produced by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). For GC-MS analysis, HPLC-grade n-hexane was sourced from CNW Technologie (Düsseldorf, Germany), and the saturated alkanes standard covering C₇ to C₄₀ was obtained from Sigma-Aldrich (St. Louis, MO, USA). In bioactivity assays, dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS), cisplatin (DDP), NG-monomethyl-L-arginine (L-NMMA), penicillin/streptomycin mixture, and Griess Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DME/F-12), Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), and phosphate buffer saline (PBS) were produced by Wuhan Pricella Biotechnology Co., Ltd (Wuhan, China). Cell Counting Kit-8 (CCK-8) was acquired from Beyotime Biotechnology Co., Ltd (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), iron chloride hexahydrate (FeCl₃·6H₂O), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), iron (II) sulfate hexahydrate (FeSO₄·H₂O), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), and analytical methanol and ethanol were obtained from Macklin (Shanghai, China). Acetate buffer (pH 3.6, 300 mM) was produced by Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China), while ascorbic acid (VC), potassium persulfate (K₂S₂O₈), and 2,6-di-tert-butyl-4-methylphenol (BHT) were obtained from Aladdin (Shanghai, China).

2.2. Plant materials

The whole plant of E. stewardii was collected from Lushan City of Jiangxi Province with GPS coordinates 1073 m (above sea level), 115°59′05.50′′ E, 29°32′ 51.67′′ N. Plant identification was conducted by Xingxing Chen from Lushan botanical garden, Jiangxi Province. The voucher specimens (LBG-ES-01) were stored in the herbarium at Lushan Botanical Garden. The collected plant materials were then manually separated into root (ES-R), leaves (ES-L), and stems (ES-S). The plant materials were air-dried, crushed into fine powder, and filtered through a 40-mesh screen. The powdered materials were maintained at -20°C before additional analysis.

2.3. EO collection

EO extractions of pulverized samples from different plant parts of E. stewardii were processed by hydrodistillation in a Clevenger extraction system, following the protocol outlined in the Chinese Pharmacopoeia [14]. Approximately 200 g of pulverized material of each plant part was placed in a round-bottom flask (2000 mL) containing 1000 mL of ultrapure water and distilled for 6 h. The EO extraction for each sample was performed four times. Subsequently, the resulting EOs were dehydrated using anhydrous Na₂SO₄ for 24 h and maintained at 4°C until further chemical and bioactivity assessments. EO yield, expressed in percentage, was computed as the oil weight in grams relative to the dry sample weight in grams (w/w, %).

2.4. Metabolite characterization by GC-MS-based metabolomics

The chemical constituents of E. stewardii EOs were detected by a GC-2030 equipment combined with a QP2020 NX mass spectrometer (Shimadzu, Kyoto, Japan). Each EO sample was diluted with n-hexane to a final concentration of 1.0 mg mL^-1. Equal volumes of 30 μL from each sample were pooled to create a quality control (QC) group. For analysis, 5 μL of the analyte was loaded chromatographically separated on a DB-5 MS column (30 m × 250 μm, 0.25 μm film thickness; J&W Scientific, Folsom, CA, USA). Helium was used as the carrier gas, flowing at a rate of 1 mL min^-1. The front inlet purge flow was set to 3 mL min^-1. The column temperature program began at 50°C (held for 1 min), increased to 310°C at a rate of 8°C min^-1, and was kept at 310°C for 11.5 min. The ion source, transfer line, and injection port temperatures were maintained at 230°C, 280°C, and 280°C, respectively. Mass spectrometric data were detected using the mode of electron impact (EI) at 70 eV, operating in full-scan mode across an m/z range of 50-500 with a scan speed of 12.5 spectra per second. Analysis of raw data, such as baseline correction, alignment, peak picking, deconvolution, and integration, was conducted using Leco Chroma TOF software (V4.3X). Linear retention indices (LRIs) of the EO components were determined by referencing n-alkanes (C₇-C₄₀). Identification of EO components relied on matching mass spectra with the NIST23 database, alongside comparison of LRIs with published data [15] and the NIST Chemistry WebBook (https://webbook.nist.gov/chemistry/). The relative abundance of each determined metabolite was represented by the ratio of individual peak area to the total ion chromatogram (TIC) area.

2.5. Cytotoxicity evaluations

Cytotoxicity effects of EOs samples were assessed using the CCK-8 assay on three human cancer cell lines: A549 (lung), HeLa (cervical), and HGC-27 (gastric). A549 and HeLa cells were cultured in DME/F-12 medium, whereas HGC-27 cells were maintained using RPMI 1640 medium. All media were supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin, and cultures were kept at 37°C with 5% CO₂. EO samples were weighed and dissolved in DMSO to prepare stock solutions at a concentration of 100 mg mL^-1. The culture medium was replaced with EO solutions at a final concentration of 100 μg mL^-1 in triplicate after 24 h of incubation. DDP (5 μg mL^-1) and 0.1% DMSO served as the positive and negative controls, respectively. The supernatant was discarded after 48 h of treatment, then 100 μL of 10% (v/v) CCK-8 reagent was added to each well and incubated for 2-3 h. The absorbance at 450 nm was recorded using an Epoch microplate spectrophotometer (BioTek, Winooski, VT, USA). The EO samples, showing an inhibition rate exceeding 50%, were further analyzed to estimate their half-maximal inhibitory concentration (IC₅₀).

2.6. Anti-inflammatory determination

The effect of anti-inflammation was performed using the RAW 264.7 murine macrophages induced by LPS [16]. RAW 264.7 cells at a density of 8×10⁴ per well were placed into 96-well plates and maintained for 6 h in DMEM medium enriched with 10% FBS and 1% penicillin/streptomycin, at 37°C with 5% CO₂. Stock solutions of EOs at 100 mg mL^-1 were prepared by weighing and dissolving them in DMSO. After removing the medium, EO solutions (100 μg mL^-1) were dispensed at 100 μL per well in triplicate. L-NMMA (12.4 μg mL^-1) was used as a positive control, while 0.1% DMSO was employed as a negative control. After 24 h of treatment, 50 μL of supernatant from each well was combined with equal volumes of Griess reagents A and B (50 μL each) to determine nitric oxide (NO) production by measuring absorbance at 540 nm. For assessing the cytotoxic effects of EOs, 10% CCK-8 solution (100 μL) was added after the removal of the remaining supernatant. EOs that inhibited NO production by 50% without exhibiting toxicity toward RAW 264.7 cells were subjected to IC₅₀ value determination.

2.7. Determination of antioxidant potential

2.8. Statistical analysis

Analysis of variance (ANOVA) was conducted via IBM SPSS Statistics (IBM SPSS Inc., Chicago, IL, USA). PCA and partial least-squares discriminant analysis (PLS-DA) were carried out on Soft Independent Modeling of Class Analogy (SIMCA, version 14.1) (MKS Umetrics AB, Umeå, Sweden). The PLS-DA model was validated by a permutation test with 200 permutations. Differential EOs were identified based on an ANOVA p-value below 0.05 and a PLS-DA variable importance in projection (VIP) score exceeding 1.5 [17]. For biological activity assessments, assays were conducted in triplicate. Statistical significance was determined by one-way ANOVA followed by Tukey’s test in SPSS, with p-values < 0.05 considered statistically significant. IC₅₀ values were determined, and the heatmap was generated by GraphPad Prism 8.01 (San Diego, CA, USA).

3.1. EO yield

The EO content from different plant parts of E. stewardii varied notably, ranging from 0.009% (w/w) to 0.070% (Figure 1). The highest EO content was found in the roots (0.070%), followed by the leaves (0.024%) and stems (0.009%), suggesting that the root tissue is the most abundant source of EOs in E. stewardii. Previous studies reported EO yields of 0.0016% and 0.0018% from the aerial parts of E. laetevirens and E. umbellatum, respectively [11], which are comparable to the combined yield obtained from the leaves and stems of E. stewardii in the present study. Statistical analysis revealed highly significant differences in EO yields among all sample pairs (p < 0.001 or p < 0.01) (Figure 1), indicating the organ-specific variability in EO biosynthesis and accumulation within E. stewardii. These pronounced differences in EO yields across plant organs are consistent with observations in other aromatic and medicinal species, including Laportea aestuans [18] and Cuminum cyminum [19]. This study, to our knowledge, was the first to investigate EO yield differences across various plant parts of Elatostema species. Although E. stewardii is relatively abundant in some regions of China, such as Jiangxi Province, its low EO yield may limit large-scale exploitation. Previous studies have shown that EO yields of a given plant species can be significantly influenced by multiple factors, including extraction methods [20], harvesting time [21], and environmental conditions such as soil type, temperature, and humidity [22]. Therefore, future studies are warranted to optimize extraction procedures (e.g., microwave-assisted or supercritical CO₂ extraction) and cultivation practices to enhance EO yield and support the sustainable utilization of E. stewardii.

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Figure 1.

EO yield from different plant parts of E. stewardii. (ES: E. stewardii; R: roots; L: leaves; S: stems.) Asterisks above the columns indicate statistical differences in EO yield between plant parts (**p < 0.01, ***p < 0.001).

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3.2. EO characterization by GC-TOF-MS analysis

The compositions of EOs from three plant parts of E. stewardii were first uncovered using GC-MS, identifying a total of 96 constituents, which accounted for 70.37% to 86.83% of the total EO content. The 30 most abundant EO compositions have been listed in Table 1, with the complete dataset provided in Table S1. These annotated EOs were classified into 11 classes, with the most prominent being fatty acids (trace-61.56%), diterpenes (3.47-32.39%), alkanes (7.79-16.32%), ketones (1.46-12.00%), and sesquiterpenes (2.60-6.22%) (Table S1). Several compound classes, including fatty acids, alkanes, ketones, and sesquiterpenes, are also commonly found in many aromatic plants, such as Ruta graveolens [23]. In the ES-R sample, fatty acids were the most abundant metabolites with a relative abundance of 61.56%, followed by alkanes (7.79%), diterpenes (3.47%), esters (2.83%), and sesquiterpenes (2.60%). In the ES-S group, fatty acids (24.00%), diterpenes (22.44%), and alkanes (16.32%) were predominant, along with notable levels of ketones (6.44%) and sesquiterpenes (4.34%). In contrast, the ES-L sample was dominated by diterpenes (32.39%), ketones (12.00%), alkanes (11.55%), and sesquiterpenes (6.22%) (Table S1). Analysis of individual metabolites revealed both similarities and differences among the three EO profiles. In the ES-R EO, the dominant constituents were n-hexadecanoic acid (60.58%), 2-methyloctadecane (5.67%), squalene (4.44%), α-epi-7-epi-5-eudesmol (1.60%), and 6,10,14-trimethyl-2-pentadecanone (1.39%). In the ES-S sample, n-hexadecanoic acid (23.45%) and 2-methyloctadecane (12.31%) were also predominant, followed by 3,5,11,15-tetramethyl-1-hexadecen3-ol (9.40%), phytol (8.58%), and 6,10,14-trimethyl-2-pentadecanone (6.40%). The abundant constituents in the ES-L sample included phytol (12.00%), neophytadiene (10.30%), 6,10,14-trimethyl-2-pentadecanone (9.92%), isophytol (8.84%), and 2-methyloctadecane (4.63%) (Table 1, Table S1). Although some predominant EOs, such as neophytadiene, phytol, and isophytol, were also abundant in EOs of E. laetevirens and E. umbellatum [11], key constituents like n-hexadecanoic acid and 2-methyloctadecane characterized in this study were not detected in these two species, indicating significant interspecific variations in EO compositions within Elatostema species.

3.3. Metabolite variations among EO samples

As shown in Table 1 and discussed above, notable variations in both chemical classes and individual metabolites were observed among the EO samples from different parts of E. stewardii. To globally visualize these metabolite differences, all detected molecules were analyzed using PCA and PLS-DA methods (Figure 2). The PCA plot yielded cumulative R²X and Q² values above 0.5 (Table S2), indicating good model reliability [24]. PLS-DA analysis revealed R²Y (cum) and Q² (cum) values over 0.9, and permutation testing with 200 permutations yielded a negative Q² intercept (Table S2, Figure S1), confirming the robustness of the established model. Both PCA and PLS-DA plots exhibited clear separations among the three sample groups (Figure 2), underscoring significant differences in EO compositions across plant parts of E. stewardii. These findings are consistent with previous reports, such as in Hypericum scabrum, where distinct EO profiles were also found between plant parts [25]. The observed variations may result from tissue-specific biosynthetic pathways, which are affected by genetic, developmental, and environmental conditions [26]. Our results supported the observation that plant part-specific variations in EO profiles are common across different species, highlighting the complexity and specificity of EO production in E. stewardii.

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Figure 2.

Metabolite variations of EOs from different plant parts of E. stewardii were analyzed by (a) PCA and (b) PLS DA. ES: E. stewardii; R: roots; L: leaves; S: stems; QC: quality control.

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The integration of VIP scores from PLS-DA analysis and p-values obtained from ANOVA assay enables the identification of key compounds that contribute significantly to biochemical variations observed between samples [17]. In the present work, we adopted this approach to characterize discriminative compounds among EO samples of E. stewardii. Metabolites with VIP values exceeding 1.5 and p-values < 0.05 [17] were considered as the most significant differential molecules, accounting for the chemical variations observed across the EOs from different plant parts. The analysis identified 15 discriminative metabolites such as diterpenes, fatty acids, ketones, and sesquiterpenes. Notably, n-hexadecanoic acid exhibited the highest VIP value (8.54), followed by linoleic acid (6.57), 3,5,11,15-tetramethyl-1-hexadecen3-ol (5.97), 2-methyloctadecane (5.21), and neophytadiene (4.35) (Table 2). This study, to our knowledge, firstly reported the application of multivariate analysis to identify variations in EO constituents in Elatostema species. Among these discriminative compounds, several metabolites, such as n-hexadecanoic acid, neophytadiene, and phytol, are known for their biological activities, including anti-inflammatory, antioxidant, cytotoxic, and neuropharmacological properties [27-29]. Variations in the abundance of these bioactive compounds may partially account for differences in the therapeutic potential of E. stewardii EOs. These compounds may serve as potential chemical markers for distinguishing and authenticating EO products or raw plant materials derived from specific plant parts of E. stewardii.

3.4. Cytotoxicity assays

Significant variations in EO metabolites among the three plant parts of E. stewardii were observed (Figure 2), indicating potential differences in their bioactivities. The cytotoxicity of the EOs from E. stewardii was comparatively evaluated on A549, HeLa, and HGC-27 cancer cell lines. The findings revealed marked differences in cytotoxicity, with growth inhibition rates ranging from 9.05% to 88.50% at a test concentration of 100 μg mL^-1 (Figure 3a). Across the samples, the EO derived from leaves (ES-L) exhibited the strongest activity, with inhibition rates of 88.47%, 88.5%, and 58.84% for A549, HeLa, and HGC-27 cells, respectively. In contrast, EOs from roots (ES-R) and stems (ES-S) showed moderate to weak activity, with inhibition rates below 50% (Figure 3a). These findings partly support the traditional consumption of E. stewardii leaves and stems as vegetables with potential nutraceutical value. Statistical analysis confirmed significant differences in bioactivity among these EO samples (p < 0.05). The positive control, DDP, exhibited potent inhibition at 5 μg mL^-1, validating the effectiveness of the bioactivity evaluation system. Given that ES-L EO displayed over 50% inhibition, its IC₅₀ value was further determined, yielding 36.58 ± 1.81, 48.42 ± 2.87, and 69.23 ± 2.86 μg/mL for A549, HeLa, and HGC-27 cells, respectively, in a dose-dependent manner (Figure 3b). Although cytotoxicity has been previously reported in Elatostema species such as E. papillosum [30], this study was the first to explore the antiproliferative activity of volatiles from Elatostema species. Our findings highlight E. stewardii leaf EO as a promising candidate for the discovery of anticancer drugs. These in vitro results indicate that future in vivo studies, including pharmacokinetic evaluations, are warranted to characterize the absorption, distribution, and bioavailability of the active EO, considering factors such as sex and disease model-specific drug behavior [31,32].

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Figure 3.

Cytotoxicity of EO samples from different parts of E. stewardii against three cancer cell lines. (a) Cytotoxic effects of three EO samples at 100 μg mL-1 and cisplatin (DDP) at 5 μg mL-1. (b) Cytotoxic effects of ES-L EO at five different concentrations. Different lowercase letters (a, b, c) in Figure 3(a) indicate statistically significant differences between samples (p < 0.05) within the same cancer cell line, while samples sharing the same letter are not significantly different. (ES: E. stewardii, R: roots, L: leaves, S: stems).

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3.5. Anti-inflammatory activity

The whole plant of E. stewardii has traditionally been used to treat inflammation in China; however, its anti-inflammatory effects have not been previously reported. In this study, EO samples from E. stewardii were detected for their anti-inflammatory potential by calculating NO production in LPS-induced RAW 264.7 macrophages. At 100 μg mL^-1, the leaf-derived EO (ES-L) exhibited the highest inhibition of NO production (97.32%), followed by ES-S (41.08%) and ES-R (16.9%). The positive control, L-NMMA (12.4 μg mL^-1), showed significant inhibition, demonstrating the assay system’s reliability (Figure 4). However, cytotoxicity analysis revealed that ES-L EO at 100 μg mL^-1 induced over 30% growth inhibition in RAW 264.7 cells, suggesting that the observed NO suppression may be partly due to cytotoxic effects rather than specific anti-inflammatory activity. To ensure a more accurate assessment, the EO concentration was reduced to 50 μg mL^-1, a non-toxic dose for RAW 264.7 cells. At this concentration, ES-L EO still demonstrated a notable NO inhibition rate of 52.76%, with an IC₅₀ value of 47.03 μg/mL. Even though previous studies have reported the anti-inflammatory potential of EOs from other Urticaceae species, such as Urtica dioica [33], this study was the first to explore the anti-inflammatory activity of EOs from Elatostema plants. Our findings indicate that EOs from E. stewardii, particularly the leaf EO, possess promising anti-inflammatory potential. These results not only support the ethnomedicinal uses of E. stewardii for inflammatory remedies but also underscore the broader therapeutic potential of Elatostema species for future pharmacological exploration. Since environmental factors such as altitude, temperature, and soil composition, as well as plant growth stages, can significantly influence EO composition and bioactivity [22], future studies involving samples from multiple habitats and developmental stages are needed to further validate these findings.

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Figure 4.

NO inhibition by EOs from different parts of E. stewardii. EO samples and L-NMMA were tested at 100 μg mL-1 and 12.4 μg mL-1, respectively. (ES: E. stewardii; R: roots; L: leaves; S: stems.) The asterisk above the columns indicates statistically significant differences in NO inhibition between plant parts (***p < 0.001). The asterisk (*) in the ES-L column denotes the significant cytotoxic effect of ES-L EO on RAW 264.7 cells at 100 μg mL-1.

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3.6. Antioxidant capacity

EOs from various plant species have demonstrated promising antioxidant activity, emphasizing the importance of investigating plant-derived EOs for their antioxidant potential [34]. In the current research, the antioxidant capacity of EOs derived from different parts of E. stewardii was evaluated using DPPH, ABTS, and FRAP assays. The results highlighted significant differences in antioxidant activity among the EOs (p < 0.05). Among them, ES-L EO exhibited the greatest activity, with IC₅₀ values of 0.33 mg mL^-1 and 0.08 mg mL^-1 in DPPH and ABTS radical scavenging assays, respectively, and a FRAP value of 0.30 μmol Fe²⁺/mL at a concentration of 0.5 mg mL^-1. ES-S and ES-R EOs also showed antioxidant activity, with DPPH and ABTS IC₅₀ values varying between 1.38 and 6.12 mg mL^-1 and FRAP values between 0.06 and 0.08 μmol Fe²⁺/mL (Table 3). The positive controls, VC and BHT, exhibited significant antioxidant activity in the DPPH, ABTS, and FRAP assays, confirming the effectiveness of our evaluation system. Previous studies have reported strong antioxidant effects in non-volatile extracts from Elatostema species, such as E. papillosum and E. rugosum [35,36]. For example, the methanol extract of E. papillosum leaves significantly inhibited DPPH radicals, with an IC₅₀ of 32.35 ± 0.68 μg mL^-1 [36]. Nevertheless, this research was the first to uncover the antioxidant capacity of EO extracts from Elatostema taxa. Our findings suggest that the leaf EO of E. stewardii may serve as a promising resource for antioxidant agent discovery, showing superior activity compared to EOs from its stems and roots.

3.7. Correlation between EO constituents and bioactivities

The bioactivity findings highlight significant variations in the bioactivities of EOs derived from different plant parts of Elatostema, emphasizing the significance of exploring the correlation between EO constituents and observed activities to identify potential bioactive molecules. To gain deeper insights into the connections between biological activities and EO compositions in E. stewardii, Pearson’s correlation analysis was performed, considering variations across plant parts. Key metabolites, including differential constituents and the top 30 most abundant EOs, were selected to generate a heatmap.

The results revealed that EO constituents were linked to bioactivities through both positive and negative correlations (Figure 5). Several compounds, such as β-ionone-5,6-epoxide, neophytadiene, and phytol, showed positive associations with the cytotoxic, anti-inflammatory and antioxidant effects observed in E. stewardii EOs (Figure 5). β-Ionone and its analogs are recognized as potential candidates for anticancer therapy [37]. Extracts containing neophytadiene have demonstrated significant growth inhibition in cancer cells like A549, with IC₅₀ values less than 10 µg mL^-1 [38], and have demonstrated efficacy in both in vitro and in vivo models of LPS-induced inflammation [39]. Additionally, phytol has been renowned for its cytotoxic, antioxidant, and anti-inflammatory properties [40]. The higher relative abundance of these metabolites in ES-L EO compared to the other two samples (Table 1) may partly explain why this EO exhibited the strongest biological activity.

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Figure 5.

Heatmap showing Pearson correlation analysis of selected metabolites and bioactivities in E. stewardii EOs. The values on the right side of the figure represent the positive (>0) and negative (<0) relationships between metabolites and biological activities.

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In contrast, some metabolites, such as n-hexadecanoic acid, linoleic acid, and 4,6,8-trimethyl-1-nonene, showed strong negative effects. Although n-hexadecanoic acid is recognized for its cytotoxic, anti-inflammatory, and antioxidant capacity [29,41,42], it exhibited strong negative associations in this study, possibly due to antagonistic interactions with other EO metabolites. This study represents the first exploration of associations between EO constituents and biological activity in Elatostema plants. The findings provide valuable insights for further research into the mechanisms underlying Elatostema EO effects and the identification of pharmacologically active agents.