Chemical Composition, Antibacterial, and Anti-Inflammatory Activities of Essential Oils from Flower, Leaf, and Stem of Rhynchanthus beesianus

Rhynchanthus beesianus is a medicinal, ornamental, and edible plant, and its essential oil has been used as an aromatic stomachic in China. In this study, the chemical constituents, antibacterial, and anti-inflammatory properties of flower essential oil (F-EO), leaf essential oil (L-EO), and stem essential oil (S-EO) of R. beesianus were investigated for the first time. According to the GC-FID/MS assay, the F-EO was mainly composed of bornyl formate (21.7%), 1,8-cineole (21.6%), borneol (9.7%), methyleugenol (7.7%), β-myrcene (5.4%), limonene (4.7%), camphene (4.5%), linalool (3.4%), and α-pinene (3.1%). The predominant components of L-EO were bornyl formate (33.9%), borneol (13.2%), 1,8-cineole (12.1%), methyleugenol (8.0%), camphene (7.8%), bornyl acetate (6.2%), and α-pinene (4.3%). The main components of S-EO were borneol (22.5%), 1,8-cineole (21.3%), methyleugenol (14.6%), bornyl formate (11.6%), and bornyl acetate (3.9%). For the bioactivities, the F-EO, L-EO, and S-EO exhibited significant antibacterial property against Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli with the inhibition zones (7.28–9.69 mm), MIC (3.13–12.50 mg/mL), and MBC (6.25–12.50 mg/mL). Besides, the F-EO, L-EO, and S-EO significantly inhibited the production of proinflammatory mediator nitric oxide (NO) (93.15–94.72%) and cytokines interleukin-6 (IL-6) (23.99–77.81%) and tumor necrosis factor-α (TNF-α) (17.69–24.93%) in LPS-stimulated RAW264.7 cells at the dose of 128 μg/mL in the absence of cytotoxicity. Hence, the essential oils of R. beesianus flower, leaf, and stem could be used as natural antibacterial and anti-inflammatory agents with a high application potential in the pharmaceutical and cosmetic fields.


Introduction
Essential oils are a mixture of natural volatile compounds from different parts of plants and have been widely used in cosmetic, perfume, agriculture, food, and medicine fields [1,2]. Essential oils have been used as complementary and alternative therapies to treat cancer, high blood pressure, pain, rheumatoid arthritis, and so on [3]. The side effects of syn-thetic drugs, the high resistance rate of pathogen strains, and the limitations of existing antibiotics/drugs have motivated people to seek and use alternative or complementary therapies, including the use of essential oils [3,4]. The family Zingiberaceae consists of approximately 52 genera and 1600 species, many of which are rich in essential oils [5,6]. According to the previous studies, the essential oils of Zingiberaceae plants have a great variety of pharmacological activ-  [13,14]. R. beesianus is a wild edible spice, and its tender leaf and rhizome are used as vegetables in Yunnan Province, China. R. beesianus flower with brilliant color and peculiar brush shape is used as a fresh cut flower. Its rhizome has been used as an aromatic stomachic in traditional Chinese medicine to treat stomachache and indigestion [15][16][17]. Additionally, the essential oils from R. beesianus have been used as an aromatic stomachic in China [17]. According to the previous study, the essential oil of R. beesianus rhizome was mainly composed of 1,8-cineole (47.6%), borneol (15.0%), methyleugenol (11.2%), and bornyl formate (7.6%) and was found to possess antibacterial, anti-inflammatory, α-glucosidase, and acetylcholinesterase activity inhibitory properties [18]. R. beesianus mainly relies on the vegetative propagation of rhizome for population expansion. Only harvesting the aerial parts (flower, leaf, and stem) of R. beesianus can reduce its damage, which is conducive to its sustainable use. However, there are no reports on the chemical components and antibacterial and anti-inflammatory properties of essential oils from R. beesianus flower, leaf, and stem.

Essential
Oils' Extraction. The fresh, finely chopped R. beesianus flower, leaf, and stem (1.0 kg) were separately extracted by hydrodistillation using a Clevenger-type apparatus. After 4 h, the flower, leaf, and stem essential oils were 3 BioMed Research International separately obtained and dried over anhydrous Na 2 SO 4 . Then, all essential oils were kept at 4°C in the amber bottle for further tests.

Chromatographic Analysis.
The essential oils were analyzed by an Agilent 6890 gas chromatograph (GC) equipped with an HP-5MS capillary column (60 m × 0:25 mm, 0.25 μm film thickness) and a flame ionization detector (FID) (Agilent Technologies Inc., CA, USA). The split ratio was 1 : 20 (injection volume: 1 μL) with helium as carrier gas (1 mL/min). The GC oven temperature was as follows: held at 70°C (2 min), 2°C/min to 180°C (55 min), 10°C/min to 310°C (13 min), and kept at 310°C (4 min). The GC-MS analysis was carried out using an Agilent 6890 gas chromatograph equipped with an Agilent 5975C mass selective detector. The Agilent 6890 gas chromatograph (GC) coupled to an Agilent 5975C mass selective detector (MS) was used to identify the chemical composition of the essential oils. The parameters of GC and capillary column were the same as in GC-FID. The MS was operated in the electron ionization mode at 70 eV and the mass range (m/z 29 to 500). The ion source temperature and interface temperature were 230°C         [19]. The essential oils and streptomycin (positive control) were separately dissolved in ethyl acetate (100 mg/mL) and distilled water solution (100 μg/mL). 100 μL of bacterial suspensions (10 6 CFU/mL) was evenly inoculated on the Mueller-Hinton agar plate. Then, filter paper discs of 6 mm diameter containing sample solution (20 μL) were added. After 24 h incubation at 37°C, the inhibition zone diameters were measured.

Determination of MIC and MBC.
The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were assayed by our previously published microplate dilution method [20]. Briefly, 100 μL of bacterial suspension and twofold serially diluted sample solution (100 μL) were added to each well at a final density of 10 5 CFU/mL and incubated at 37°C for 24 h. Subsequently, resazurin solutions (20 μL, 0.1 mg/mL) were added to each well. After 2 h incubation at 37°C in the dark, the MIC values were determined as the minimum sample concentration without color change. For the determination of the MBC values, 10 μL of samples from the wells without color change was subcultured on Mueller-Hinton agar medium. After 24 h incubation at 37°C, the MBC values were determined as the minimum sample concentration without bacterial growth.
2.5. Anti-Inflammatory Activity 2.5.1. Cytotoxic Assay. The cytotoxicity was evaluated on murine fibroblast cells (L929) and murine macrophages (RAW264.7) by the MTT assay with slight modification [21]. The L929 and RAW264.7 cells were separately maintained in RPMI 1640 medium and DMEM medium (10% fetal bovine serum, 2 mM glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin) and incubated in a humidified incubator at 37°C with 5% CO 2 atmosphere. 100 μL of cell suspensions was added to each well at a density of 2 × 10 4 cells per well. After 24 h incubation, twofold serially diluted essential oil solutions (100 μL) were added to each well and incubated for 24 h. Subsequently, 10 μL of MTT solution (5 mg/mL in PBS) was added and incubated for 4 h. After discarding the supernate, DMSO (150 μL) was added to each well to dissolve the formazan crystal. The absorbance was recorded at 490 nm using a Varioskan Lux Multimode microplate reader (Thermo Fisher Scientific, USA).

Morphology
Assay and Measurement of NO, IL-6, and TNF-α. 100 μL of RAW264.7 cell suspensions was added to each well at a density of 2 × 10 4 cells per well and incubated for 24 h. After discarding the medium, twofold serially diluted essential oil solutions (100 μL) were added and incubated for 2 h. Subsequently, lipopolysaccharide solutions (LPS, 100 μL) were added to each well at a final concentration of 1 μg/mL and incubated for 24 h. Morphological changes were recorded using a Leica DMi8 inverted microscope (Leica Microsystems, Germany). Then, the supernatants were collected and centrifuged. The accumulation of NO in the culture supernatant was determined by a colorimetric NO detection kit following the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Dexamethasone (DXM, 20 μg/mL) was used as a positive reference. The secretion of IL-6 and TNF-α was  2.6. Statistical Analysis. All experiments were performed independently at least three times, and the results were expressed as the means ± SD. SPSS software (version 19.0) was used for statistical analysis. Data were compared by one-way analysis of variance (ANOVA) using Fisher's LSD post hoc tests. Differences were considered significant at the p < 0:05 level.

Results and Discussion
3.1. Chemical Composition. The hydrodistillation of fresh flower, leaf, and stem of R. beesianus separately yielded essential oils at 0.21% (w/w), 0.47% (w/w), and 0.94% (w/w) on a fresh weight basis. The GC-FID/MS analysis showed the identification of forty-six, forty-four, and sixty-three compounds accounting for 98.6%, 98.7%, and 96.0% of the total oil content of flower, leaf, and stem, respectively (Table 1). R. beesianus F-EO was mainly composed of bornyl formate (21.7%), 1,8-cineole (21.6%), borneol (9.7%), methyleugenol  Experiments were performed independently at least three times, and the results were expressed as mean ± standard deviation (SD) values. a-j Different letters in the same column indicate a significant difference (p < 0:05).

Conclusion
To our knowledge, this is the first report on the chemical constituents and bioactivities of essential oils from R. beesianus flower, leaf, and stem. Forty-six, forty-four, and sixtythree compounds were identified in the F-EO, L-EO, and S-EO by using GC-FID/MS, respectively. The F-EO, L-EO, and S-EO exhibited significant antibacterial property against Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli. Besides, the F-EO, L-EO, and S-EO significantly inhibited the production of proinflammatory mediator NO and cytokines (IL-6 and TNF-α) in LPS-stimulated RAW264.7 cells in the absence of cytotoxicity. In particular, the essential oil of the stem showed the highest yield and antiinflammatory activity. Hence, the essential oils of R. beesianus flower, leaf, and stem could be regarded as antibacterial 9 BioMed Research International and anti-inflammatory natural products with a high application potential in the pharmaceutical and cosmetic fields.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.