CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITIES OF HOMALOMENA VIETNAMENSIS BOGNER & V. D. NGUYEN (ARACEAE)

Homalomena vietnamensis is a rare species of the Homalomena genus and only found in Middle region of Vietnam. In this study, we found 10 compounds in ethanol extracts of leaf and rhizome of H. vietnamensis, such as cadinane-4β,5α,10α-triol, oplopanone, 4-epi-oplopananol, 2α-hydroxy homalomenol A, 1β,4β,7β-Trihydroxyeudesmane, homalomentetraol, 4-acetoxyoplopananol, 5,7-diepi-2a-acetoxyoplopanone, eudesma 4β, 7α- diol-1β-fumarate), and homalomenol F, via liquid chromatography-mass spectrometry (LC/MS). Moreover, the antibacterial activity of ethanol extracts of leaf and rhizome from this species has been evaluated by disc diffusion method for the first time. The results showed that rhizome extract of could inhibit the growth of 5 tested micro-organisms, including of Bacillus cereus (28.3 ± 1.5 mm), Salmonella enteritidis (19.5 ± 1.5 mm), Staphylococcus aureus (16.3 ± 1.5 mm), Escherichia coli (14.7 ± 1.2 mm), and Pseudomonas aeruginosa (8.2 ± 0.8 mm), while the leaf extract showed antibacterial effect against Bacillus cereus (22.0 ± 2.0 mm), S. enteritidis (14.7 ± 0.6 mm), and S. aureus (12.5 ± 1.8 mm).


INTRODUCTION
Homalomena Schott is a genus of the Araceae family and comprises of 250 species growing over the world (Boyce et al., 2012). Several members of the Homalomena genus are extensively used as traditional remedies in Vietnamese medicine (Pham, 2000). Chemical composition, antimicrobial and antioxidant activities of the compounds extracted from many species of Homalomena genus have been well-documented in literature (Singh et al., 2000;Rana et al., 2009;Liliwirianis et al., 2011;Yang et al., 2016). For example, the essential oil of H. aromatica contains 55 compounds, such as linalool, terpene-4-ol, δ-cadinene, Tmuurolol, viridiflorol, α-cadinol, α-selinene, M-cymene, γ-Muurolene, and spatulenol… and exhibits a strong antifungal effect against dermatophytes and yeasts, such as Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum fulvum, Microsporum gypseum, Trichosporon beigelii and Candida albicans (Policegoudra et al., 2012). Furthermore, H. aromatica oil also has antibacterial activity against five common and significant pathogens such as S. aureus, E. coli, P. aeruginosa, Klebsiella pneumoniae, and Proteus vulgaris (Laishram et al., 2006). These data suggest the essential oil of H. aromatica as a potential antimicrobial agent or the bioactive component of pharmaceutical preparations. Among them, H. vietnamensis is an extremely rare species and is described for the first time by Bogner and Nguyen in Bach Ma National Park, Thua Thien-Hue province, Vietnam (Bogner and Nguyen, 2008). Nowadays, the presence of this species also has only been recorded in some provinces in Middle region of Vietnam, such as Thua Thien-Hue, Khanh Hoa and Quang Nam Provinces, Vietnam (Bogner and Nguyen, 2008). Due to the limit of the number of specimens, the bioactivity this species is still unknown. In this study, we identifies the chemical composition and proves the antibacterial activity of ethanol extracts of leaf and rhizome from this species for the first time, which will support the information for further application of this species in future.

Bacterial strains
Five bacterial strains, including two Gram-positive bacteria, Bacillus cereus (ATCC 11774) and Staphylococcus aureus (ATCC 25923), and three Gramnegative bacteria, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Salmonella enteritidis (ATCC 13976), were used to evaluate the antibacterial activity of ethanol extracts. Microorganisms were kindly provided from the microbiology collection, Department of Biotechnology, Institute of Food and Biotechnology, Industrial University of Ho Chi Minh city, Viet Nam. All bacterial strains were cultured in Luria-Bertani broth at 37 o C for 24 h to be re-activated again before using in further experiments.

Extraction procedure
Fresh rhizomes of H. vietnamensis were peeled and subsequently cut into slices. The fresh leaves and sliced rhizomes were moderate dried at 50-55 o C until masses of samples were unchanged. The samples were pulverized by an electric grinder into fine powder and kept at 4 o C. 50 g of the dried powder of leaves and rhizomes of H. vietnamensis were immersed with 450 mL of 98% ethanol for 5 weeks. The extracts were filtrated via Whatman filter paper, and subsequently concentrated in reduced pressure at 60 o C until the residue remained ½ volume of the initial filtrate (Altemimi et al., 2017), then sublimation drying was performed to remove ethanol in extracts. The obtained residue was stored at 4 o C until further use.

Liquid chromatography mass spectrometry (LC/MS)
Ethanol extracts were sent to the Central Laboratory for Analysis, University of Science, Vietnam National University of Ho Chi Minh City to conduct LC/MS analysis and elucidate the chemical composition of ethanol extract. In brief, aliquot of ethanol extract was injected to HPLC Agilent 1200 infinity liquid chromatography system (Agilent Technologies, CA, USA) coupled with MicroTOF-QII mass spectrometer (Bruker Daltonics, Germany). The chromatographic separation was carried out in an ACE3-C18 analytical column (4.6 ×150 mm, 3.5 µm). In mobile phase, deionized water with 0.1% formic acid was used as solvent A and acetonitril with 0.1% formic acid was used as solvent B. Gradient elution program for the chromatographic separation was presented in Table 1 with the flow rate at 0.3 mL/min. The mass spectrometer was implemented with electrospray ionization source (ESI) at positive mode and mass spectra data were recorded for a mass range 50-2000 m/z. Data analysis was performed using Data Analysis software (Bruker, Germany). To determine the compounds in extract, the mass spectra of compounds were compared with mass spectra of reference compounds which were identified in other species of Homalomena genus from previous studies (Wang et 0  90  10  15  0  100  30  0  100  31  90  10  40 90 10 (  ): presented as the percentage of volume of mobile phase

Antibacterial activities
The antibacterial activity of ethanol extracts of leaf and rhizome of H. vietnamensis was analyzed according to Bauer protocol (Bauer et al., 1996). The bacteria were inoculated in LB Broth until reached a turbidity of 0.5 McFarland standard. 100 µl of bacterial suspensions were inoculated on Mueller Hinton plate, and a sterilized 6 mm diameter disc was placed on the plate. 10 µl of sample were put onto each disc and the plate was kept at 4 o C for 2 hours to fully diffuse extract into the medium. Diameters of zones of inhibition of extracts against tested bacteria were observed and measured after inoculation at 37 o C for 24 hours. Sterile distilled water was used as negative control and Gentamycin antibiotic disc (Nam Khoa BioTek, Viet Nam) was used as positive control.

Data analysis
The experiments were repeated in triplicate. The average and standard deviation of measurements were calculated using The Excel 2010 software. The data of experiments were expressed as mean ± standard deviation (SD).

Antibacterial activity
Antibacterial activity of ethanol extracts from rhizome and leaf of H. vietnamensis was evaluated by the diameter of inhibition zone against tested bacteria (Table 3 and Figure 3). Ethanol extract of rhizome of this species showed the antibacterial effect against 5 tested microorganisms while those from leaf inhibited the growth of 3 bacterial strains, including of B. cereus, S. enteritidis, and S. aureus. We observed that diameters of inhibition zones of the rhizome extract against B. cereus, S. enteritidis, S. aureus, E. coli, and P. aeruginosa were 28.3 ± 1.5, 19.5 ± 1.5, 16.3 ± 1.5, 14.7 ± 1.2, and 8.2 ± 0.8 mm, respectively. On the other hand, the leaf extract showed the strongest antibacterial effect against B. cereus (22.0 ± 2.0 mm), following by S. enteritidis (14.7 ± 0.6 mm) and S. aureus (12.5 ± 1.8 mm). The results suggested that antibacterial effect of rhizome extract was stronger that of leaf extract, both the number of bacterial strains and the diameter of inhibition zone of each strain.