Chemical composition, antibacterial, anticancer, and anti-a-glucosidase activities of essential oils from Alpinia nelumboides

Materials

Alpinia nelumboides (Fig. 1) were collected in Di Linh District and cultivated in Da Lat City, Lam Dong Province (Vietnam). Taxonomic authentication utilized morphological assessment for the plant was conducted [10], https://www.ipni.org/n/77315593-1), and type specimen (PHH1004920, PHH1004921) was deposited at the PHH herbarium of the University of Science, Ho Chi Minh city. Before experimental use, the materials underwent washing with water, cleaning, and storage in clean air.

The Center for Research and Application in Bioscience in Ho Chi Minh City, Vietnam, provided clinically isolated antibiotic-resistant bacterial strains, including Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans. The Department of Genetics at VNUHCM-University of Science provided NCI H460, HepG2, and fibroblast cell lines. A procurement of apramycin was made from GoldBio (USA). The following items were purchased from Sigma (USA): dimethyl sulfoxide, ethanol, trichloroacetic acid, sulforhodamine B, PBS, DPPH, 4-nitrophenyl-α-D-glucopyranoside, trolox, and α-glucosidase.

Extraction of essential oils

The freshly chopped rhizomes and leaves (400 g each) underwent a 6-hour hydrodistillation process using a 2,000 ml capacity Clevenger-esque apparatus loaded with 1,000 ml of water, according to the Vietnamese Pharmacopoeia (The Committee of Vietnamese Pharmacopoeia 2017). Following separation from the hydrodistillation, the hydrophobic essential oil layers were dehydrated using anhydrous sodium sulfate and then kept refrigerated for preservation before impending analysis. The essential oil yields were averaged over three repeated experiments and calculated based on dividing the weight (g) of the essential oil by the weight (g) of the fresh weight of plant materials.

Analysis of the essential oils by GC-FID/MS

Gas chromatography of the essential oils utilized an Agilent 8,860 GC with an Agilent 122-5532 DB-5MS column (30 m × 0.25 mm × 0.25 μm). The GC oven program ramped from 60°C to −240°C at 3°C/minute with the injector and detector maintained at 250°C. The essential oil sample was diluted with hexane solvent at a ratio of 0.1 mg/ml. Samples were injected in splitless mode with 1.00 ml/minute nitrogen carrier gas flow at 11.724 psi. For GC/MS, an Agilent 8,890 GC coupled to a 5977B MSD was equipped with a 19091S-433UI HP-5MS column (30 m × 0.25 mm × 0.25 μm). Helium carrier gas flow was 1.0509 ml/minute at 8.8085 psi. The injection volume was 1.0 μl with a 15:1 split ratio and 70.007 eV ionization voltage [12].

Figure 1. Alpinia nelumboides Nob. Tanaka, T.T.K. Van & V. Hoang (A. Leaves, B. Rhizomes). [Click here to view]

Constituent identification initially utilized comparisons of acquired mass spectra against the NIST 2020 compound library. Supplementary confirmation involved matching retention indices for each constituent with indices reported in the literature, specifically the compilation published by Adams in 2007 [13]. Peak area normalization was used to determine the relative percentage of each component in the oil. Response factors were not computed.

Antimicrobial activity

An agar well diffusion assay assessed essential oil bioactivity against select microbial pathogens [14]. The pathogenic cultures were incubated in nutrient broth at 37°C for 18 hours to reach the exponential growth phase. The cultures were diluted with sterile 0.9% NaCl to produce 1.5 × 10⁸ CFU/ml microbial solutions for each pathogen. On Mueller–Hinton agar (MHA) plates, 100 µl of every microbial solution was distributed. On the MHA plates, 8 mm-diameter wells were created by inserting sterile points. In each well, 25 µl of each essential oil was added. Following 37°C, 16–18 hour incubation of inoculated MHA plates, the potency of test essential oils was assessed by measurements of inhibition zone diameters surrounding wells, compared to apramycin and DMSO as positive and negative controls, respectively.

Each essential oil was added to MHA at a concentration spanning from 0.5 mg/ml to 32 mg/ml at a two-fold dilution to determine the minimum inhibitory concentration (MIC) [15]. On the MHA plates containing a serial dilution of the essential oil, 5 × 10⁵ CFU of each microbial pathogen were deposited. After an 18-hour incubation at 37°C, the MIC was determined as the lowest dilution of oil on MHA that completely inhibited the development of the pathogen. The complete inhibition of microbial growth was defined as the absence of any visible colonies on the agar plate at the respective essential oil concentration. This conclusion was based on comparing the appearance of colonies to the reference wells containing apramycin and DMSO.

Anticancer activity

The cytotoxicity of essential oils was assessed on three animal cell lines utilizing the Sulforhodamine B (SRB) method [16]. Cells were seeded in 96-well plates at a density of 10⁴ cells per well for Hep G2 and fibroblast cell lines and at 7.5×10³ cells per well for NCI-H460. After 24 hours of culture, the cells were exposed to different concentrations of each essential oil and incubated for an additional 48 hours. The cells were fixed for 1–3 hours with a cold 50% (w/v) trichloroacetic acid solution, rinsed, and stained for 20 minutes with 0.2% (w/v) SRB. Following five treatments with a 1% acetic acid solution, the solubilization of the protein-bound dye was carried out using a 10 mM Tris base solution. At 492 nm and 620 nm, optical density values were determined using a 96-well microtiter plate reader. The growth inhibition percentage (I%) was determined using the formula: I% = (1 - [ODt/ODc]) × 100%, where ODt and ODc denote the optical density values of the test sample and the control sample, respectively. Camptothecin served as the positive control, while DMSO was utilized as the negative control.

Alpha-glucosidase inhibition activity

In a 96-well plate, 120 μl of each essential oil and 20 μl of α-glucosidase at a concentration of 1 unit/ml were dispensed into each well. The mixture was then incubated for 15 minutes at 37°C. Subsequently, 20 μl of a 5 mM p-Nitrophenyl-α-D-glucopyranoside solution was introduced into each well, and the incubation continued for an additional 15 minutes at 37°C. The reaction was terminated by adding 80 μl of a 0.2 M Na2CO3 solution to each well. The quantitative measurement of enzyme activity was performed at an absorbance of 405 nm. All samples were evaluated at five concentrations around the IC50 values. The following equation was used to determine the percentage of inhibition: I (%) = [1—(Asample/Acontrol)] × 100% [17]. The assay used acarbose as the standard control.

Statistical analyses

Statistical analyses were conducted using GraphPad Prism 9. Each experiment was independently replicated at least three times. The data were presented as mean values accompanied by standard deviations and error bars.

Chemical composition of essential oils

Essential oils from the fresh rhizomes and fresh leaves of A. nelumboides, growing in Vietnam were yellow and obtained a yield of 0.20% and 0.22% ± 0.02%, respectively. Table 1 and Table 2 (Supplementary Figures 1 and 3) indicate the chemical constituents and their percentages present in the rhizomes and leaves of A. nelumboides essential oils. Results revealed 99.75% and 88.19% of detectable substances in rhizomes and leaves essential oils, respectively. There were 14 compounds detected in rhizomes essential oils, while this number was 74 in leaves essential oils.

Notes: The percentage values were determined through FID peak area normalization, employing the response factor exclusively for main components (≥ 10%). LRIs (linear retention indices) on HP-5MS Ultra Inert [Lit: literature [13], Obs: observed].

Compared with rhizome essential oil, leaves essential oil contains a chemical composition with more abundant components. Eucalyptol is known as one main components in the oil with the highest percentage in both rhizome and leaves essential oil (71.23% in rhizome essential oil and 32.82% in leaves essential oil). A. nelumboides was morphologically most similar to A. kwangsiensis, but the essential oils of their rhizomes were different which was the main constituent of rhizome A. kwangsiensis essential oil was terpinene-4-ol (14.3%) [9]. Differences in the number of compounds detected in the rhizome and leaves essential oils were similar to previous research on Alpinia vietnamica with 45 compounds detected from leaves and 36 compounds detected from the rhizome [18]. The essential oil from leaves and rhizomes of Kaempferia galanga Linn also showed that 108 and 81 compounds were detected, respectively [19]. Interestingly, the essential oil from this species in the publication of Nguyen Hoang Tuan et al. [11] shows that rhizome essential oil contains the main components of 8-cineole, linalool, (E)-citral, (Z)-citral,α-pinene, limonene, and β-pinene which was different from our study. Also in the study of Nguyen Hoang Tuan et al. [11], the essential oils of rhizome and pseudo-stem had a large amount of polyphenols, which had high antioxidant capacity, while the essential oils in our study showed weak antioxidant activity (IC50 for DPPH assay was over 35 mg/ml). These differences could be explained by the influence of environmental factors. As reported in previous studies, seasonal variations, geographical locations, and weather conditions can affect the chemical compositions and biological activities of essential oils extracted from medicinal plants. Variations in soil properties, sunlight exposure, rainfall patterns, and so on, contribute to changes in the biosynthesis and accumulation of secondary metabolites in plants grown under different environmental conditions [20]. However, it should be noted that the quantitative data reported in this study were obtained through peak area normalization, which provides only semi-quantitative results without considering potential response factor variations.

Table 1. Chemical composition of rhizomes A. nelumboides essential oil. [Click here to view]

Table 2. Chemical composition leaves A. nelumboides essential oil. [Click here to view]

Antimicrobial activity

The antimicrobial activity of essential oil from A. nelumboides was performed by agar well diffusion method, as shown in Table 3. The essential oil extracted from the rhizomes and leaves of A. nelumboides were effective against Gram-negative bacteria, Gram-positive bacteria, and fungi. Rhizome essential oil was more active than leaves essential oil which was 6/8 test microorganisms sensitive to the leaves essential oil, 8/8 test microorganisms sensitive to the rhizomes essential oil. On E. faecium, S. aureus, and P. aeruginosa, the inhibition zone diameters of the rhizomes essential oil (38, 26, 25 mm) were greater than those of apramycin at a concentration of 1 mg/ml (18, 21, 21 mm).

Table 3. Antimicrobial activity of the rhizomes and leaves essential oil from A. nelumboides. [Click here to view]

In general, the essential oil extracted from the rhizomes and leaves exhibited a range of antibacterial and antifungal properties against a variety of bacterial and fungal species. A similar result was found in the study of E. fimbriobracteata essential oil [21] and A. fragrans essential oil from Iran [22]. According to a study on the antibacterial activity of Z. spectabile Griff, the essential oil from the leaves had no antibacterial activity, but the essential oil from the rhizomes did, which was due to a large variation in the chemical composition of essential oil [23]. The results of our analysis could be explained by the difference in concentrations of antibacterial compounds, such as eucalyptol [24], that accumulate in the rhizomes (71.23%) versus the leaves (32.82%). Moreover, eucalyptol is the primary antibacterial component of many antimicrobial-active plants [24,25]. Therefore, the essential oil of A. nelumboides rhizomes, whose primary constituent was eucalyptol, is a potential alternative for the control of multiple microorganisms. Following this, we determined the antibacterial activity of essential oil from A. nelumboides by determining the MIC of essential oil against susceptible microorganism strains. The MIC values of essential oil against susceptible bacteria strains were obtained in Table 4.

The rhizomes essential oil exhibited antibacterial activity against all 8 microbiological strains, with MIC values ranging from 8 to 16 mg/ml. The antibacterial activity of leaves essential oil ranged from 4 mg/ml to 16 mg/ml against E. faecium, S. aureus, E. coli, K. pneumoniae, and C. albicans. The anti-C. albicans activity of the leaves essential oil was determined to be more impressive than that of the microorganisms examined in this study. The MIC values of rhizomes essential oil from A. nelumboides were stronger than S. abrotanoides essential oil on E. coli (MIC = 64 mg/ml), P. aeruginosa (MIC = 64 mg/ml), but its value was identical on S. aureus (MIC = 8 mg/ml), and C. albicans (MIC = 8 mg/ml) [26].

Alpha-glucosidase inhibition activity

Table 6 displayed the α-glucosidase inhibitory activity results of A. nelumboides essential oils. A. nelumboides essential oil exhibited the α-glucosidase inhibition activity with the IC50 of leaves essential oil was 2.42 ± 0.22 mg/ml while the values of rhizomes essential oil were higher than 3 mg/ml. The IC50 values of A. nelumboides essential oil extracted from leaves were lower than those of Centaurea calcitrapa essential oil from Iraq (IC50 = 4.38mg/ml) [31]. The hydrolysis of nonreducing α-1,4-linked glucose molecules from disaccharides or oligosaccharides is catalyzed by α-glucosidase. α-glucosidase enzymes complete carbohydrate breakdown into monosaccharides until units are taken into the body [32]. Glucosidase inhibitors have the potential to be utilized to treat diabetes. Therefore, our data preliminary find out the potential of A. nelumboides essential oil in diabetes treatment.

The findings of this study demonstrate the potential of Alpinia nelumboides essential oils, particularly from the rhizomes, as promising natural sources for pharmaceutical applications. The observed anticancer, antibacterial, and antidiabetic properties present opportunities for developing novel therapeutic agents or functional ingredients. Additionally, this research enhances our understanding of the applicational capabilities of these essential oils compared to the preliminary findings reported earlier since just focus on antioxidation activity [11]. However, further research is required to isolate and characterize the bioactive components, optimize extraction processes, and conduct comprehensive preclinical and clinical evaluations before practical applications can be realized in the pharmaceutical industry.

Table 6. The α-glucosidase inhibition of the rhizomes and leaves essential oil from A. nelumboides. [Click here to view]

Authorship

VTTK and TTL carried out experiments, performed statistical analysis, and wrote the manuscript. TTNT conducted experiments chemical analysis experiments. VH and CHN designed experiments, supervised experiments, and reviewed the manuscript. The manuscript underwent review by the full author list before obtaining consent for submission from each party for journal publication.