MATERIALS AND METHODS

Curcuma aeruginosa was collected from cultivation unit of Tropical Biopharmaca Research Center, Bogor Agriculture University, located at Bogor, Indonesia. The C. aeruginosa was identified at Research Center for Biology, Indonesian Institute of Science prior to use. The S. mutans was collected from Microbiology Laboratory, Faculty of Medicine, University of Indonesia (ATCC^® 35668^TM).

Isolation of C. aeruginosa’s essential oil: Each 5 kg of fresh rhizomes, stems and leaves of C. aeruginosa was distilled by steam distillation for 6 h. Distillation was carried out at 100-105°C. Yield of distillation process was calculated based on weight of each sample¹³.

Antibacterial assay: Streptococcus mutans was cultured in Tryptic Soy Broth (TSB) at 37°C. The potency of essential oil of C. aeruginosa as antibacterial toward S. mutans was analyzed by micro-dilution method at 96 well plates. One hundred microliters samples with concentration of 15.63-2000 μg mL^–1 were prepared in 96 well plates. One hundred microliters of TSB and 20 μL of S. mutans inoculants at the concentration of 10^–2 CFU mL^–1 were added to each well of 96 well plates. The aliquot was incubated at 37°C for 24 h. The extract concentration at which there was no visually detectable bacterial growth was described as the Minimum Inhibitory Concentration (MIC). Further, 100 μL of aliquot with no visually detectable bacterial growth was inoculated in new medium and incubated at 37°C for 24 h. The extract concentration at which there was no visually detectable bacterial growth was described as the Minimum Bactericidal Concentration (MBC). The DMSO 20% was used as control-negative and tetracycline was used as control-positive¹⁴.

Biofilm degradation assay: Biofilm degradation activity of C. aeruginosa essential oil was analyzed by micro-dilution method in 96 well plates¹⁵. Biofilm was formed from 100 μL synthetic saliva (Mc Dougall solution) with TSB medium, 3% glucose and bacterial inoculants. The mixture was incubated for 24 h at 37°C. Once a biofilm is formed, the remaining medium is discarded. Essential oils are added at a concentration of 16-2000 mg mL^–1 and then incubated 24 h at temperature of 37°C. Biofilms attached to the wall of the wells is washed using phosphate buffer. Crystal violet 1% was added to the wells and left for 15 min. Well rinsed with sterile water 3 times and 95% ethanol was added. The suspension was incubated for 45 min and the solution was transferred to a new micro-plate. Suspension absorbance of each well was measured using a micro-plate reader at a wavelength of 595 nm to determine the percentage degradation. Chlorhexidine was used as positive control and 20% DMSO as a negative control. Degradation (%) is given as in Eq. 1:

(1)

where, A_{corrected sample} is absorbance (Essential oil or chlorhexidine+inoculants S. mutans) and A_{corrected blank} is absorbance (DMSO 20%+inoculants S. mutans).

Identification of chemical constituents of C. aeruginosa’s essential oil: Component of C. aeruginosa’s essential oils was identified using gas chromatography-mass spectroscopy (GC-MS) agilent 5873. The HP-5 MS (30 m×0.25 mm×250 μm) and helium gas at flow rate of 20 mL min^–1 were used as stationary and mobile phase system in GC. Injector and detector temperature were set at 80 and 250°C, respectively. The separation temperature started at 80°C for 5 min and gradually increased in rate of 10°C min^–1 to reach 250°C, the temperature keep at 250°C for 45 min. The electron impact ionization (EI-MS) was carried at 70 eV. The MS data were collected and compare with library index MS. Component of C. aeruginosa’s rhizome essential oils were further identified by Nuclear Magnetic Resonance (NMR) JOEL ECA-600, CDCl₃ was used as solvent.

RESULTS

Moisture content and yield of essential oil from fresh rhizomes, stems and leaves of C. aeruginosa were presented in Fig. 1. The moisture content of fresh C. aeruginosa was in range of 84-95%. The highest yield of essential oil was obtained from rhizome distillation (1.99 %), meanwhile the yield of essential oil derived from leaves and stems distillation was a 10th and 20th smaller than rhizome, respectively. The color of essential oil obtained from rhizome, stems and leaves of C. aeruginosa were similar, dark brown.

The essential oil from rhizomes of C. aeruginosa showed good antibacterial activity toward S. mutans. The MIC value of rhizome oil was 15.63 μg mL^–1. The MIC value of rhizome oil was same with the MIC of synthetic antibacterial tetracycline (Table 1). This result indicated that rhizome oil of C. aeruginosa was potential as antibacterial agent. On the contrary, the antibacterial potency of stems and leaves oil were smaller than rhizome oil.

Biofilm degradation activity of stems oil was higher than rhizomes and leaves oil, the IC₅₀ value was 1347.27 μg mL^–1 (Table 1). Otherwise, fractions from column chromatography of rhizome oil exhibited higher biofilm degradation activity than stem oil (IC₅₀ value was in range of 228-508 μg mL^–1). Meanwhile, the biofilm degradation activity of all crude oil and purified fractions were lower than positive control, chlorhexidine (Table 1).

Rhizomes, stems and leaves oil of C. aeruginosa were identified using GC-MS. The results showed that 2,2,5-trimethyl-2'(H)-5',6'-dihydropyrano[3',4',9]indan-1-one (40%) and 1,8-cineol (13%) were the major compounds in rhizome oil (Table 2). The major compounds in stem and leave oil almost similar, namely β-elemene, caryophyllene and epicurzerenone. Trans-6-ethenyl-4,5,6,7-tetrahydro-3, 6-dimethyl-5-isopropenyl was identified in stem oil at higher amount (7%) than in leaves oil.

Fraction 1 from rhizome oil indicated the potency as antibacterial toward S. mutans. Its biofilm degradation activity also stronger than stem oil. The GC-MS analysis of fraction 1 showed that 2,2,5-trimethyl-2'(H)-5', 6'-dihydropyrano[3',4',9]indan-1-one is the major component with relative abundance more than 50%.

Fig. 1(a-b):	(a) Moisture content and (b) Yield of essential oil of C. aeruginosa rhizomes, stems and leaves

Table 1:	Antibacterial activities toward S. mutans and biofilm degradation of essential oil of C. aeruginosa rhizomes, stems and leaves
*>2000 μg mL–1

Table 2:	Component of C. aeruginosa essential oil analyzed by GC-MS
-: Unidentified, +: Identified at abundance of 1-6%

Further fractionation of fraction 1 of rhizome oil produced three fractions, namely F1.1, F1.2 and F1.3. Fraction F1.2 gave highest antibacterial activity with MIC value of 500 μg mL^–1. The GC-MS analysis of fraction F1.2 showed single dominant peak with molecular weight of 230 g mol^–1 (Fig. 2). The ¹³C NMR spectrum revealed 15 carbon atoms with chemical shift of 195.0, 145.6, 141.1, 139.6, 119.3, 115.7, 113.0, 64.1 and 50.8 ppm. Meanwhile, ¹H NMR spectrum showed 18 hydrogen atoms with various chemical shifts (Table 3).

DISCUSSION

Distillation is a suitable technique for separation of volatile compound and essential oil from sample. Yield of distillation process will depend on type of distillation, time and temperature operation. Yield of essential oil from rhizome of C. aeruginosa was 1.99%, higher than yield of C. aeruginosa rhizome reported by Kamazeri et al.¹⁶. The difference could be due to the separation of oil using dichloromethane after distillation by Kamazeri et al.¹⁶ method.

Fig. 2(a-b):	(a) Chromatogram and (b) MS spectrum of fraction F1.2

Table 3:	Chemical shift of 1H and 13C NMR of F1.2 MR (solvent: CDCl3)

In this study separation step using dichloromethane did not conducted since the oil was easily separated from water using separation funnel attached to distillation apparatus. Yield of rhizome oil distillation was a 10 and 20 times higher than leaves and stem oil, denoted that oil content in rhizome was higher than leaves and stem oil. The color of essential of rhizome, leaves and stem are similar (dark brown) indicated possibility of similarity constituent in each oil.

The essential oil from rhizomes of C. aeruginosa exhibited strong antibacterial activity toward S. mutans. The MIC value of rhizome oil was equal with tetracycline (Table 1). Kamazeri et al.¹⁶ reported that rhizome oil of C. aeruginosa possess antibacterial activity toward Gram-positive and Gram-negative bacteria and also fungi. The constituent of rhizome oil predicted to be act as antibacterial agent through one or combination of the following mechanisms: Ruin the cell wall of bacteria, hamper the protein synthesis and obstruct the DNA and RNA replication. Trombetta et al.¹⁷ reported that mechanism of antibacterial action of monoterpene compounds was through perturbation of the lipid fraction of microorganism plasma membrane, changing membrane permeability and leaking intracellular materials. Furthermore, the drugs might interacting with intracellular sites critical for antibacterial activity after cross the cell membranes and penetrating into the interior of the cell¹⁷.

Streptococcus mutans produce exopolysaccharides and use it to form biofilm as their habitat on teeth^2,3. Degradation of biofilm correlated with the destruction of S. mutans habitat and expected to inhibit its growth. Stem oil of C. aeruginosa exhibited weak activity in biofilm degradation, meanwhile the fraction of rhizome oil indicated stronger activity in biofilm degradation. On the other hand, leaves oil denoted no activity in biofilm degradation. Batubara et al.¹⁸ reported that leaves oil of other Curcuma plants showed biofilm degradation activity with IC₅₀ in range of 289 and 689 μg mL^–1.

Essential oil from leaves, stems and rhizome of C. aeruginosa contained some terpenoid compounds. Rhizome oil contained 1,8-cineol (13%), higher than reported by Sirat et al.¹⁰. Beside terpoid, essential oil from rhizome also contained nonterpenoid compounds as the main component, namely 2,2,5-trimethyl-2'(H)-5',6'-dihydropyrano[3',4',9]indan-1-one (40%).

Fig. 3:	Structure of 2,2,5-trimethyl-2'(H)-5',6'-dihydropyrano[3',4',9]indan-1-one (δ ppm 13C NMR and 1H NMR)

Sirat et al.¹⁰ reported that essential oil of C. aeruginosa rhizome from Malaya contained curzerenone (24.6%), 1,8-cineole (11.0%), camphor (10.6%), zedoarol (6.3%), isocurcumenol (5.8%), curcumenol (5.6%) and filranogermenone (5.5%). Stem and leaves oil of C. aeruginosa possessed similar major terpenoid compounds such as β-elemene, caryophyllene and epicurzerenone.

The NMR spectrum indicated pointed a shift at 195 ppm indicated carbonyl group of conjugated ketone. Another chemical shift was monitored at 145.6, 141.1, 139.6, 119.3, 115.7 and 113.0 ppm related with substituted benzene. The NMR data referred that the compound in F1.2 suggested to be 2,2,5-trimethyl-2'(H)-5',6'-dihydropyrano[3',4',9]indan-1-one (C₁₅H₁₈O₂) (Fig. 3).

CONCLUSION

Rhizome of C. aeruginosa provided the highest yield of essential oil (1.99%) compare with stems and leaves. The rhizome essential oil showed good antibacterial activity toward S. mutans with the same MIC value with synthetic antibacterial tetracycline. 2,2,5-trimethyl-2'(H)-5',6'-dihydropyrano[3',4',9]indan-1-one and 1,8-cineol were the major compounds in rhizome oil. Biofilm degradation activity of stems oil was higher than rhizomes and leaves oil. Otherwise, fractionation of rhizome oil produced fractions with higher biofilm degradation activity than stems oil. This fraction also could maintain its antibacterial activity. Further purification toward the selected fraction yielded F1.2. According to MS and NMR spectrum, the identity of compound in F1.2 was 2,2,5-trimethyl-2'(H)-5',6'-dihydropyrano[3',4',9]indan-1-one.

SIGNIFICANCE STATEMENT