Chemical Composition and Antistaphylococcal Activity of Essential Oil of Curcuma mangga Rhizome from Indonesia

: This study assessed the antistaphylococcal activity of essential oil (EO) hydrodistilled from the rhizome of Curcuma mangga grown in Indonesia using the broth microdilution volatilization method and standard broth microdilution method modified for evaluation of volatile agents, as well as described its chemical composition using gas chromatography (GC) with mass spectrometry (MS). A fused-silica HP-5MS column and a DB-17MS column were used to separate the components into two columns. The results demonstrated that the EO exhibited antistaphylococcal activity at the minimum inhibitory concentration (MIC) ranging from 128 to 1024 µ g/mL. In contrast, the clinical isolate of tetracycline-resistant Staphylococcus aureus was the most sensitive strain (MIC 128 µ g/mL). The major constituents of the EO were 15,16-dinorlabda-8(17),11-dien-13-one (24.63/15.78%), followed by ambrial (16.12/10.97%), 13-nor-eremophil-1(10)-en-11-one (7.16/6.21%), 15,16-dinorlabda-8(17),12-dien-14-al (6.61/11.57%), and aromadendrene oxide (5.98/3.77%). These results propose C. mangga rhizome EO as a promising agent for developing natural-based anti-infective preparations.


Introduction
Infectious diseases continue to pose significant challenges to public health, particularly in low-and middle-income countries, where the escalating prevalence of microorganisms resistant to antimicrobial agents is a pressing concern [1,2].Among these pathogens, Staphylococcus aureus stands out as a human threat, contributing substantially to the burden of morbidity associated with community and hospital-acquired infections.These infections range from lower respiratory tract infections to pneumonia and nosocomial bacteremia [3,4].
Historically, S. aureus infections were effectively treated with β-lactam antibiotics [5].However, the emergence of resistant strains, particularly methicillin-resistant S. aureus (MRSA), has become a significant impediment to successful treatment [6].The evolution of resistance mechanisms, including the expression of enzymes that hydrolyze the βlactam ring and the acquisition of the mecA gene, encoding a modified penicillin-binding protein 2a, has rendered conventional antibiotics ineffective [7].The urgent need for novel therapeutic agents capable of combating staphylococcal infections has become paramount in the face of this escalating challenge.
Researchers have increasingly focused on plant-derived products, such as extracts and essential oils (EOs), as potential alternatives for treating staphylococcal infections.While these natural remedies have a long history of use in traditional medicine for infectious diseases, the renewed interest in EOs gained momentum in the late 20th century [8,9].eri [28] identified sesquiterpenes, including caryophyllene and caryophyllene oxide, as major compounds, demonstrating their antimicrobial efficacy against S. aureus and C. albicans.Despite these findings, there remains a notable gap in our understanding, particularly regarding the EO derived from C. mangga rhizome grown in Indonesia.Limited reports exist on its chemical composition and antimicrobial activity against a broad spectrum of standard and clinical strains of S. aureus.
This research endeavours to bridge this knowledge gap by comprehensively investigating the chemical composition and antistaphylococcal effects of C. mangga rhizome EO from Indonesia.Through detailed analyses and experiments, we aim to uncover the potential of this essential oil as a valuable resource in the quest for novel therapeutic agents against staphylococcal infections.Therefore, this research determined the chemical composition and antistaphylococcal effect of C. mangga rhizome EO from Indonesia and the implications of this research, showing the promising role of C. mangga rhizome EO in antimicrobial therapeutics.

Plant Materials and Sample Preparation
C. mangga (Voucher specimen no.Ar-0069) was obtained from the Biopharmaca collection garden at Bogor Agricultural University (IPB) in Dramaga-Bogor (West Java Province, Indonesia).Plant material was dried and homogenized using a Grindomix apparatus (GM 100 Retsch, Haan, Germany).

Hydrodistillation of EO
The essential oil was extracted by hydrodistillation of 50 g of C. mangga rhizome powdered samples in 1 L of distilled water for 3 h using a Clevenger apparatus (Merci, Brno, Czech Republic).The collected EO was dried using anhydrous sodium sulphate (Merck, Darmstadt, Germany) and stored at 4 • C in airtight glass vials.

Microorganisms and Media
In the context of this investigation, standard strains from the American Type Culture Collection (ATCC) were employed, including Candida albicans ATCC 10231, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 27853, S. aureus ATCC 29213, S. aureus ATCC 33591, S. aureus ATCC 33592, and S. aureus ATCC BAA 976.Additionally, clinical isolates comprising methicillin-resistant S. aureus (MRSA 1, MRSA 2, MRSA 3, and MRSA 4) and tetracycline-resistant S. aureus (TRSA 1 and TRSA 2) were obtained from agar plates sourced from Motol University Hospital in Prague, Czech Republic.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as detailed in Rondevaldova [29], was employed for the identification of clinical isolates.Cultures of microorganisms were maintained on Mueller-Hinton agar (MHA) and stored at 4 • C until use.Before testing, stock cultures were cultivated in Mueller-Hinton broth (MHB) at 37 • C for 24 h, with pH adjusted to 7.6 using Trizma base (Sigma-Aldrich, Prague, Czech Republic).The ATCC strains and media were procured from Oxoid (Basingstoke, UK).To standardize the inoculum, the turbidity of the microorganism suspension was adjusted to the 0.5 McFarland standard (1.5 × 108 CFU/mL) using a Densi-La-Meter II (Lachema, Brno, Czech Republic) spectrophotometric device.Tetracycline and tioconazole (Sigma-Aldrich, Prague, Czech Republic) were employed as positive controls.

Antimicrobial Assay
The antibacterial potential of the EO in liquid and vapour phases was subjected to the broth microdilution volatilization method [30].The antistaphylococcal activity was determined using the standard broth microdilution method according to the Clinical and Laboratory Standards [31] modified by Rondevaldova [32].The experiments were conducted in white 96-well immunoplates (total well volume = 400 µL) covered by tight-fitting lids with flanges designed to reduce evaporation (SPL Life Sciences, Naechon-Myeon, Re-public of Korea).Initially, 30 µL of MHA (Oxoid, Basingstoke, UK) was pipetted into every flange on the lid and inoculated with 5 µL of bacterial suspension after agar solidification.Then, the EO was dissolved in 100% DMSO (Sigma-Aldrich, Prague, Czech Republic) and diluted in MHB.The final concentration of DMSO in the microtiter plate wells did not exceed 1%. 100 µL two-fold serial dilution of each sample concentration starting from 1024 µg/mL was distributed into the plates.The plates were then inoculated with bacterial suspensions.Finally, the clamps (Lux Tool, Prague, Czech Republic) with the handmade wooden pads (size 8.5 × 13 × 2 mm) were used for fastening the plate and lid together.An experimental protocol was implemented to determine antistaphylococcal activity; the plates were covered by the EVA Capmat (Micronic, Aston, PA, USA) immediately after inoculating the bacterial suspension to prevent evaporation of the EO.Then, the plates were incubated at 37 • C for 24 h for bacteria (48 h for C. albicans).The minimum inhibitory concentration (MIC) values were evaluated by visual assessment of microbial growth after colouring of the metabolically active colonies of microorganisms with thiazolyl blue tetrazolium bromide (MTT) dye (Sigma-Aldrich, Prague, Czech Republic) when the interface of colour changed from yellow to purple (relative to that of the colours in control wells) was recorded.The DMSO at the concentration of 1% did not inhibit any of the strains tested either in the broth or agar media.All experiments were performed in triplicate in three independent experiments, and results were expressed as the median/mode of MICs.
The identification of chemical compounds was based on comparing their mass spectra with the National Institute of Standards and Technology Library ver.2.0.f(NIST, Gaithersburg, MD, USA) [33], retention indices (RI) [34] and co-injection of authentic standard compounds (Sigma-Aldrich, Prague, Czech Republic).The relative percentage contents of essential oil components were determined from FID, indicated on both columns.

Results and Discussion
The hydrodistilled EO underwent an initial assessment of its antimicrobial properties utilizing the broth microdilution volatilization method [30].However, with the exception of the oil's growth-inhibitory impact on S. aureus in the liquid phase, no discernible positive results were observed for the other tested microorganisms.In response to this, a more detailed evaluation of the EO's antistaphylococcal efficacy was conducted using the modified broth microdilution method specifically designed for volatile antimicrobial agents [32].Additionally, its chemical composition was characterized by GC-MS and GC-QTOF-MS.The results of the broth microdilution volatilization method (Table 1) showed that S. aureus was susceptible to the C. mangga EO, exhibiting in vitro growth-inhibitory effect in the liquid phase at an MIC of 256 µg/mL.Still, no inhibition activity was observed during the vapour phase (MIC > 1024 µg/mL).The EO did not exhibit inhibitory activity against other microorganisms (C.albicans, E. faecalis, E. coli, and P. aeruginosa) in both liquid and vapour phases (MIC > 1024 µg/mL, respectively).In the modified broth microdilution method, C. mangga EO exhibited antistaphylococcal activity at the MIC ranging from 128 to 1024 µg/mL (Table 2).Of particular note is the heightened sensitivity of the tetracycline-resistant S. aureus (TRSA) 2 strain to the EO, displaying the lowest MIC of 128 µg/mL.This observation aligns with findings reported by Kamazeri et al. [28], where the C. mangga rhizome EO demonstrated growth-inhibitory activity against S. aureus ATCC 25923 at an MIC of 1.2 µL/mL.Furthermore, these results find support in studies conducted by Romulo [35] and Renisheya [36], both indicating the inhibitory activity of C. mangga rhizome extracts against S. aureus.However, what sets this study apart is its novel contribution-the demonstration of the EO's antistaphylococcal effect against a broad spectrum of standard and clinical strains, encompassing both methicillin-resistant S. aureus (MRSA) and tetracycline-resistant S. aureus (TRSA).This signifies a valuable extension of our understanding of the potential therapeutic applications of C. mangga EO in addressing infections caused by diverse strains, particularly those resistant to conventional antibiotics.
The composition of C. mangga rhizome essential oil (EO) has exhibited notable variations in this study compared to previous reports, highlighting the influence of factors such as geographical origin, growing conditions, and isolation methods.β-pinene, identified as the predominant compound in the EO extracted from wild populations in Indonesia (>15%) [37], was found to constitute a minor percentage in the current study (<3%).Conversely, Kamazeri [28] reported caryophyllene oxide, a sesquiterpenoid, as the principal component in C. mangga rhizome EO obtained through steam distillation, constituting 18.71%.However, our study detected this compound in lower amounts, accounting for less than 6% of the EO composition.The uncommon dinorlabdanic diterpenoids have not yet been described in C. mangga essential oil.15,16-dinorlabda-8(17),11-dien-13-one and other structurally similar diterpenes were isolated from the methanol and acetone extracts of the rhizomes of Alpinia calcarata [38] and Alpinia formosana [39], genus Alpina, belonging to the Zingiberaceae family.13-nor-eremophil-1(10)-en-11-one was previously found in wood oil from Xanthocyparis vietnamensis known as Vietnamese gold cypress [40], and as a component of oil of fragrant grass vetiver (Vetiveria zizanioides, Gramineae) together with many other sesquterpens [41].
This shared presence of certain diterpenoids across different plant species within and outside the Zingiberaceae family underscores the potential conservation of these compounds with different biological significance.
Ambrial, another compound of interest, emerged as one of the major constituents in the EO of C. mangga rhizome from Indonesia.Intriguingly, only a minimal quantity (2.3%) of this compound was reported in the literature about plant material obtained in Malaysia [25].These observed disparities underscore the complex nature of the chemical compositions in C. mangga EOs and emphasize the need to consider multiple variables in their analysis.
The pronounced differences between the chemical profiles presented in this study and the literature mentioned above could be attributed to several factors.Firstly, the geographical origin of the plants plays a pivotal role in determining the chemical composition of essential oils, as variations in soil composition, climate, and altitude can significantly impact the plant's secondary metabolite production.Additionally, growing conditions, such as temperature, humidity, and sunlight exposure, can exert a substantial influence on the synthesis of specific compounds within the plant.
Furthermore, the isolation methods employed in EO extraction can contribute to variations in the final chemical profile.Different extraction techniques, such as hydrodistillation or steam distillation, may selectively capture certain compounds while excluding others, thereby influencing the overall composition of the essential oil.
In conclusion, the significant disparities observed in the chemical compositions of C. mangga EOs between this study and previous reports underscore the dynamic and multifaceted nature of plant chemistry.These variations offer valuable insights into the intricate interplay of environmental and methodological factors shaping the chemical profile of essential oils.Consequently, a comprehensive understanding of these factors is essential for elucidating the true therapeutic potential of C. mangga EO and ensuring its effective utilization in various applications.[42].
In order to enhance the precision in identifying the constituents of the essential oil (EO), two distinct columns, namely the non-polar HP-5MS and the middle-polarity DB-17MS, were employed.The result of using the DB-17MS column revealed 16 additional compounds identified in C. mangga rhizome EO.Enhancing the quantification and identification of essential oil (EO) components is crucial, and the utilization of dual-column/dual-detector systems contribute to improved accuracy and serves as a preventive measure against false-positive identifications of compounds.Employing such dual systems enhances the reliability of the analytical process, providing a more comprehensive and accurate depiction of the EO's composition.This approach mitigates the risk of misidentification, thereby fortifying the validity of results in EO analysis.[43].Additionally, the sample was also analyzed using GC-QTOF-MS.This analytical approach provides fast scanning, higher sensitivity, and mass accuracy compared to the common quadrupole MS (GC-MS), as it can deconvolute the overlapping peaks and detect some minor compounds [44,45].Based on this approach, 15 additional minor constituents were found in the C. mangga rhizome EO.

Conclusions
In this comprehensive article, we present a thorough analysis of the chemical composition and antistaphylococcal activity exhibited by the essential oil (EO) extracted from the rhizome of C. mangga, cultivated in Indonesia.Our findings underscore the significant antistaphylococcal potential of C. mangga EO, demonstrating efficacy against a diverse spectrum of S. aureus strains, encompassing antibiotic-resistant variants such as MRSA and TRSA.
The robust antistaphylococcal activity observed in our study positions C. mangga EO as a promising candidate for the development of anti-infective preparations rooted in traditional medicine.However, a cautious approach is warranted, acknowledging the preliminary nature of our findings.The journey from the current promising insights to a de-

Conclusions
In this comprehensive article, we present a thorough analysis of the chemical composition and antistaphylococcal activity exhibited by the essential oil (EO) extracted from the rhizome of C. mangga, cultivated in Indonesia.Our findings underscore the significant antistaphylococcal potential of C. mangga EO, demonstrating efficacy against a diverse spectrum of S. aureus strains, encompassing antibiotic-resistant variants such as MRSA and TRSA.
The robust antistaphylococcal activity observed in our study positions C. mangga EO as a promising candidate for the development of anti-infective preparations rooted in traditional medicine.However, a cautious approach is warranted, acknowledging the preliminary nature of our findings.The journey from the current promising insights to a dependable therapeutic agent is multifaceted and requires further exploration.
Our study serves as a catalyst for future research endeavours, emphasizing the need for additional investigations into the safety profile and in vivo efficacy of C. mangga EO.Before considering its potential application in clinical settings, it is imperative to conduct rigorous assessments to ensure safety and efficacy.
In conclusion, while our study unveils promising avenues for leveraging C. mangga rhizome EO in combatting staphylococcal infections, the translation of these findings into a reliable therapeutic agent necessitates a holistic approach.Ongoing research initiatives, encompassing safety evaluations and in-depth efficacy studies, will pave the way for the integration of C. mangga EO into traditional medicine-based anti-infective preparations.By doing so, we contribute to the global efforts to address the challenges posed by antibioticresistant pathogens.

( a )
Retention indices taken from the literature [33,34]; (b) RI, retention indices calculated on DB-5 capillary column.(c) MS, identification based on mass spectra matching (MSD quadrupole); QTOF, identification based on mass spectra matching (QTOF-MS); RI, identification based on retention index; Std, identification based on co-injection of authentic standard; (d) data not available.

Table 1 .
In vitro antimicrobial activity of Curcuma mangga rhizome essential oil in liquid and vapour phase tested in broth and agar media, respectively.

Table 2 .
In vitro antistaphylococcal activity of Curcuma mangga rhizome essential oil determined by EVA capmat cover modified method.

Table 3 .
Chemical composition of Curcuma mangga rhizome essential oil.