Two Sesquiterpenes from n -Hexane Fraction of Curcuma soloensis Rhizomes and Their Antimicrobial Activities

Curcuma soloensis is one of the medicinal plants that has the potential to be a source of bioactive compounds. The antimicrobial study of the bioactive compounds from C. soloensis was still limited. This study aimed to isolate the bioactive compounds from C. soloensis rhizomes and to evaluate their potential as antimicrobial agents. C. soloensis rhizome extraction was done using the maceration method with acetone and then fractionated with n -hexane: methanol (1:1). The compounds were separated and purified using vacuum liquid chromatography and radial chromatography. The structure of the isolated compounds was determined using the nuclear magnetic resonance ( 1 H and 13 C NMR) spectroscopy analysis and comparison with literature data. Antimicrobial activity assays of the extract, n -hexane fraction, and isolated compounds were carried out by microdilution methods against Escherichia coli , Staphylococcus aureus , Candida albicans


INTRODUCTION
Antibiotics, chemical compounds of microorganisms' metabolic products, are antimicrobial drugs that have been seen as a 'blessing' for mankind since more than half a century ago.But lately, the ability of these antibiotic compounds has begun to decline because the targeted microorganisms have developed immunity or resistance gradually 1 .On the other hand, the human need for antibiotics is not only focused on treating diseases but also on their prevention, particularly concerning their transmission through food.This fact motivates intense research efforts to obtain novel antibiotics.Antimicrobial compounds derived from natural products are now again being used.Natural antimicrobial agents are distinguished by their harmless nature, safety, and diversity of structures 2 .
Indonesia as a region with a diversity of plant species allows the discovery of antimicrobial active compounds from Indonesian medicinal plants, one of which is from the genus Curcuma (family Zingiberaceae).The Curcuma genus is an important medicinal plant in Indonesia because more than 2,031 traditional medicine formulas (6.34% of the formulas identified) used species of Curcuma rhizomes 3 .5] .Slightly more than 20 of the 110 species in the genus Curcuma have been investigated phytochemically; C. longa, C. xanthorrhiza, and C. zedoaria were the most extensively researched. 4.
One species of Curcuma that has not been studied scientifically but has potential as a medicinal plant is Curcuma soloensis.This plant thrives in Indonesia with the local name 'temu tis' or 'temu glenyeh'.C. soloensis rhizome is usually used with other herbs for the treatment of skin infections 6 .The rhizomes of C. soloensis are also used by people for dermatological disorders, especially wounds and burns 7 .
Some studies report that the n-hexane extract of C. soloensis rhizomes has biological activity as a gastroprotective 7 , and anticancer 8,9 .Acetone extract of C. soloensis rhizomes also has antifungal activity against Aspergillus fumigatus, Candida albicans, Epidermophyton sp, Penicillum sp, and Trichophyton rubrum 10 .The essential oil of C. soloensis rhizomes was also reported to have an antiproliferative effect against human carcinoma cell lines 6 and inhibit the growth of Staphylococcus aureus, S. epidermis, and S. haemolyticus 11 .The ethanol extract of C. soloensis showed antimicrobial activity against Streptococcus mutans and C. albicans 12 .Some compounds successfully isolated from the rhizomes of C. soloensis are bisacurone, curcumin 13 , and ar-turmerone 14 .These compounds have been isolated from the C. domestica rhizomes and showed antimicrobial activity against some bacteria 15 .Based on some previous research, it appears that there was a potential for bioactive compounds of C. soloensis rhizomes as antimicrobials, but has not received more specific studies, related to bioactive compounds that act as antimicrobials.
In this research, the antimicrobial activity of bioactive compounds isolated from rhizomes of C. soloensis has been studied.Antimicrobial testing was carried out on Escherichia coli, S. aureus, C. albicans, and Malassezzia furfur using the microdilution method.Identification of isolated compounds was carried out using NMR (Nuclear Resonance Magnetic) spectroscopy, so we have the information on bioactive compounds that have the potential as antimicrobial.

Materials and Instruments
Plant material (C.soloensis rhizomes) was collected from Bandung of West Java, Indonesia, and identified at the Environmental Laboratory of the Faculty of Biology, Universitas Jenderal Soedirman.The tested microbes (E.coli, S. aureus, C. albicans, M. furfur), were collected from the Microbiology Laboratory Faculty of Medicine, Universitas Jenderal Soedirman.The instruments used in this research were a microplate spectrophotometer for antimicrobial analysis (Automatic Microplate Laser: All Sheng, AMR-100), 1 H NMR and 13 C NMR spectrometer for the identification of isolated compounds (Agilent DD2: 500 ( 1 H) MHz and 125 ( 13 C) MHz, solvent CDCl3).

Procedures Isolation of compounds
The rhizome of C. soloensis (8 kg) was washed, cut into thin and small pieces then dried by drying or oven at low temperature (30-40 ºC), and then mashed until it became powder.The powder (1.1 kg) was macerated with acetone for 3x24 hours.Every 24 hours the extract was taken by filtering, and the residue from the filtering was macerated again until three repetitions.The acetone extract was concentrated using a rotary evaporator.The concentrated acetone extract (107 g) was partitioned with n-hexane: methanol (1:1), and the fraction of n-hexane was accommodated and then concentrated with a rotary evaporator.The n-hexane fraction (18 g) was fractionated by vacuum liquid chromatography (VLC) using silica gel, and eluent of nhexane, a mixture of n-hexane and chloroform (8:2, 6:4, 4:6, 2:8), and chloroform.The fractions that showed the same spot profile in the TLC that were observed with UV lamps were then combined and obtained four subfractions namely Fr.1(8.9 g), Fr.2 (2.25 g), Fr.3 (0.7 g) and Fr.4 (0.9 g).Two main fractions (Fr.1 and Fr.2) were further separated through radial chromatography until the pure compound was obtained.A compound was said to be pure when it showed a single spot on the TLC.The fraction of Fr.1 was purified by radial chromatography using n-hexane eluents, and a pure compound (1) in the form of colorless oil was obtained (56 mg).While the Fr.2 fraction was purified using an eluent of n-hexane: chloroform (9:1), a pure compound (2) in the form of yellowish clear oil was obtained (207 mg).Both isolates were then analyzed for their chemical structure by NMR spectroscopy and tested for antimicrobial activity.

Determination of the structure of the compound
The structure of the isolated compound was determined by 1 H NMR, 13 C NMR, and HSQC (Heteronuclear Single Quantum Coherence) spectrometers.The isolated compound has been known, so the data obtained was compared with literature data. 16ntimicrobial activity assays were carried out by liquid dilution method.The concentration of the sample stock solution was 1000 μg /mL.The concentration of test samples used was 500, 250, 125, 62.5, 31.25,15.62, and 7.81 μg/mL.The sample was dissolved in 10% DMSO.

Antimicrobial assays by microdilution method
Microbes were cultured for 24 hours for bacteria and 48 hours for fungi at 27 ºC under aerobic conditions in an agar medium (Mueller Hinton Agar (MHA) for bacteria and Potato Dextrose Agar (PDA) for fungi).The microbes were then suspended in 0.9% NaCl solution (w/v) and the density was equalized to 0.5 Mc Farland.
A total of 200 μL of liquid media (Mueller Hinton Broth (MHB) for bacteria and Potato Dextrose Broth (PDB) for fungi was filled into each microplate well (96 wells).The sample's stock solution (1000 μg/mL) was introduced in 200 μL to the first well.To conduct the solution concentration series, 200 μL of solution was transferred from the first well to the second, and then 200 μL of solution was withdrawn from the second well and put into the third, and so on up to the eighth well.The amount of solution in each well was 200 μL.Further, into each well was inserted 10 μL of microbial suspension.The microplate was then incubated at 37 ºC for 24 hours.Microbial growth was determined using a microplate spectrometer at a wavelength of 600 nm.The minimum inhibitory concentration (MIC) is the lowest concentration that can inhibit microbial growth.This experiment was carried out twice.As positive controls, amoxicillin was utilized for bacteria, and miconazole was used for fungus.

Isolated Compounds
The results of physical properties observations and spectroscopic measurements ( 1 H and 13 C NMR) of isolated compounds obtained two known sesquiterpene compounds, namely ar-curcumene (1) and ar-turmerone (2), in Figure 1.

ar-Curcumene
ar-Curcumene (1) is a sesquiterpene compound with a bisabolene-type skeleton (Figure 1).This compound is in the form of colorless oil.

ar-Turmerone
ar-Turmerone (2) is also a sesquiterpene with a bisabolene skeleton.This compound is a yellowish oil with a chemical structure as shown in Figure 1.The signals from the NMR spectrum indicate that compound 2 has the same skeleton as compound 1, namely bisabolene.The difference between compounds 1 and 2 is that there are substituents in the aliphatic skeleton, compound 2 has a carbonyl group (C=O) at C-9, which is characterized by the absence of a proton signal at C-9 and the appearance of a carbon signal at δc of 19.7 ppm.Based on the NMR data, it was suggested that compound 2 was ar-turmerone (C15H22O).Conformance may be seen when 1 H NMR, 13 C NMR, and HSQC data of compound 2 (Table 2) are compared to ar-turmerone data that have been published 14 The bisabolene skeleton is a monocyclic sesquiterpene with a six-ring, which is formed from the cyclization of trans-farnesyl cations to form bisabolyl cations 18 .The bisabolyl cation is a precursor forming sesquiterpene compounds with a bisabolene skeleton.arcurcumene compound formed from the dehydrogenation reaction of bisabolene, then ar-curcumene undergoes oxidative reactions to form ar-turmerone 17 .

Antimicrobial Activity
The antimicrobial activity of extracts, fractions, and compounds isolated from C. soloensis rhizomes showed diverse activity.The data showed that acetone extract, n-hexane fraction, and isolated compounds have the potential to be developed as antimicrobial agents.The minimum inhibitory concentration (MIC) of a test sample indicates its capacity to inhibit bacteria; the lower the MIC value, the higher the antimicrobial activity.Until recently, it has not been known with certainty what the standard value for the level of activity of antimicrobial compounds.According to some studies, a compound has a very high potential for usage as an antimicrobial if its MIC value is between 0.02-10.0μg/mL 19 , On the other hand, a compound can be considered antimicrobial if its MIC value is between 100-1000 μg/mL 20 .In general, the antimicrobial activity of isolated compounds (ar-curcumene and ar-turmerone) has higher activity than acetone extracts and n-hexane fractions.The highest activity was shown by arturmerone against S. aureus with a MIC value of 15.6 μg/mL, while ar-curcumene showed the same activity in all four test microbes with a MIC value of 62.5 μg/mL.Previous research has shown that ar-turmerone was inactive against S. aureus 21 .This is likely due to differences in the assay methods used, the environment where bacteria grow, and the type of bacterial culture used.In addition, ar-Curcumene isolated from the fresh aerial parts of Senecio selloi was found to be effective against some microbial such as S. aureus, Bacillus subtilis, E. coli, Pseudomonas aeruginosa, C. albicans, C. glabrata, and Saccharomyces cerevisiae 22 .
The varying antimicrobial properties for each compound in a particular microbe are not only determined by the type and characteristic of the microbe but also by the physical and chemical properties as well as the structure and functional groups of the compound 23 .The study of the structure and activity of isolated compounds in this study was stated qualitatively and focused on the activity level of isolated compounds for the same microbes.
ar-Curcumene and ar-turmerone compounds are sesquiterpene compounds with a monocyclic bisabolene skeleton that has a benzene ring.The difference between the two compounds is that in ar-turmerone there is a ketone group at the acyclic chain (C-9).The difference in the antifungal activity of the two compounds above against the four test microbes might be caused by the presence of ketone groups in C-9 of ar-turmerone.The antimicrobial activity of ar-turmerone against all four test microbes showed higher MIC values (MIC 15.6 -62.5 μg/mL) than ar-curcumene (MIC 62.5 μg/mL).This suggests that the presence of carbonyl groups in arturmerone was thought to increase antimicrobial activity.
The mechanism of action of terpenoid compounds in inhibiting the growth or killing of microbes has not been widely reported.Some studies reported that terpenoid compounds work by disrupting the function of cell membranes.The lipophilicity of terpenoid compounds can enhance the permeability and fluidity of cell membranes, obstruct ion transport and respiration, and prevent membrane proteins from binding 24 .

CONCLUSIONS
The isolation of bioactive compounds from the nhexane fraction of C. soloensis rhizomes obtained two pure sesquiterpene compounds.Based on 1 H NMR, 13 C NMR, and HSQC spectroscopy analysis, the isolated compounds were identified as sesquiterpene compounds with bisabolene molecular skeletons, namely arcurcumene (C15H22) and ar-turmerone (C15H22O).
The antimicrobial activity of acetone extract, nhexane fraction, and isolated compounds of arcurcumene and ar-turmerone showed diverse activity.The data showed that ar-turmerone had the highest activity against S. aureus with a MIC value of 15.6 μg/mL, while ar-curcumene showed the same activity against all test microbes with a MIC value of 62.5 μg/mL.

Table 3 .
Antimicrobial activities of extract, fraction, and isolated compounds