Antimicrobial Activity and Microbial Transformation of Ethyl p-Methoxycinnamate Extracted  from Kaempferia galanga

INTRODUCTION

The need for a new antibiotic against pathogenic microorganism has increased due to the current problems of resistance associated with them¹. Plant based antimicrobial agents have enormous therapeutically potential as they can serve the purpose without any side effect, that often associated with synthetic  agents.  Malaysian natural product plants have been studied extensively. These include medicinal plant species from Andrographis^2,3,4,5, Musa⁶, Plumeria⁷, Citrus^8.9, Cymbopogon¹⁰,  Artocarpus¹¹, Alpinia¹² and Centella¹³. However, plant species of Zingiberaceae continue to attract much phytochemical interests due to their culinary uses, besides their biological and pharmaceutical activities.  Kaempferia galanga Linn., belongs to the Zingiberaceae family,  is one of the plants that mainly distributed in tropics and subtropics of Asia. Besides Malaysia, it can be found in Southern China, Indochina and India¹⁴. It has been known from ancient times that the essential oil of K. galanga  plays an important role in native medicine as it possesses interesting biological activities such as antimicrobial¹⁵, antihypertensive¹⁶, anticancer¹⁷  and antiproliferative activity¹⁸.

The emergence of bio transformation technique to produce new natural drug as an antimicrobial agent was successfully reported. According to Muller¹⁹, fungi are effective in increasing the chemical diversity by producing new derivatives with lower toxicity but improved biological activity. While Kim et al²⁰, described that the biotransformation afforded another metabolite that has higher antimicrobial activity compared to the parent metabolite. Thus, this approach successfully enhances the antimicrobial activity of the compound. For example, rhapontigenin produced from rhapontin by biotransformation showed 4-16 times higher antimicrobial activity than rhapontin. The activity was higher against Gram-positive strains than Gram-negative strains²⁰. Another study by Srivastava et al²¹,  indicates that A. flavus can transform artemisinin to deoxyartemisinin (Figure 1) and shows antibacterial activity against S. aureus, S. epidermidis and S. mutans at a minimum inhibitory concentration (MIC) of 1 mg/mL compared to artemisinin whose MIC was >2 mg/mL. Siddhardha et al.²² showed biotransformation of (-)-α-santonin using A. parasiticus resulted in the production of 3,4-epoxy-α-santonin, 4,5-dihydro-α-santonin and 1,2-dihydro-α-santonin and evaluated for the biological activity of the transformed products. 3,4-epoxy-α-santonin exhibited higher degree of antibacterial and antifungal activity. 4,5-dihydro-α- santonin showed slight increase in the activity, compared to (-)-α-santonin. 1,2-dihydro-α- santonin is equipotent to the substrate (-)-α-santonin. This is the first report on the bio transformation of (-)-α-santonin by A. parasiticus and evaluation of the biological activities of the bio transformed products.

 

Figure1: Bio transformation of artemisinin to deoxyartemisinin by Aspergillus flavusClick here to View Figure

 

Therefore, this present study was initiated to screen the antimicrobial activity of ethyl p- methoxycinnamate (EPMC), extracted from K. galanga Linn., and  compare the bio activity with its bio transformation  metabolite, ethyl p- hydroxycinnamate (EPHC) produced   by A. niger.

EXPERIMENTAL

Materials

The rhizomes of K. galanga L. were collected at different locations throughout Malaysia in December 2013  and stored at -20 °C prior to analysis. Five reference strains of human pathogens used for antimicrobial assay were Staphylococcus aureus (ATCC25923), Bacillus cereus (ATCC11778), Pseudomonas aeruginosa (ATCC27853) and Escherichia coli (ATCC8739) while one fungal strains was Candida albicans (IMRC533/11 A). The bacteria were maintained on Muller-Hinton Agar (MHA) and Muller- Hinton Broth (MHB) and stored in the refrigerator at  4°C.Meanwhile, C.  albicans was maintained on Sabouraud’s Dextrose Agar (SDA) and stored in the refrigerator at 4°C. Both bacteria and fungus C. albicans were sub cultured onto fresh media at regular interval.

Extraction of K. galanga oil and purification of ethyl p-methoxycinnamate

The fresh rhizomes of K. galanga  were washed  and cut into smaller pieces. The rhizomes were then extracted using steam distillation and supercritical fluid extraction (SFE) according to the previously reported method²³. The essential oil was collected and stored at -4°C for future analyses. For purification of ethyl p-methoxycinnamate (EPMC, 1) the essential oils were stirred with boiling water and then recrystalized at cold temperature      (-4°C). After crystallization, the mixture was filtered through What man  No 1 and then the crystal was kept in the desiccator for 24 hours prior to further analysis.

Fungus culture preparation and bio transformation procedure.

Aspergillus niger fungal culture preparation

Pure fungal culture of  A. niger was obtained from Microbiology Laboratory Kulliyyah of Science, International Islamic University Malaysia. The pure fungus was streaked on Sabouraud Dextrose Agar slant (SDA) at 30 °C for a week and stored at 4 °C. After  cultivation, the well grown mycelia on the agar slants were placed in the conical flask (250 ml) and  inoculated  with 10.0 ml of sterilized medium broth containing glucose, glycerol, peptone, yeast extract, KH₂PO₄, NaCl and distilled H₂O. The flask was incubated using an incubator shaker for 48 hr  at 120 rpm and 30°C. Then, mycelia suspensions were transferred into 17 flasks containing sterile broth medium (100 ml of each) and incubated for 24 hour using the similar condition as reported earlier²⁴.

Biotransformation of the bioactive compounds

Ethyl p-methoxycinnamate, 1 (480 mg, ) was dissolved in dimethyl sulfoxide (DMSO) (24 ml) and distributed among 48 flasks containing 24 hour stage culture (10 mg/0.5 ml in each flask) and continuously shaken for 24 h using a rotary shaker (120 rpm) at 30 °C. At the same time, a control flask having substrate without fungus and a control flask containing fungus without substrate were analysed in order to check the substrate ability and to determine the endogenous metabolite, respectively.

Separation and purification of biotransformed product

After incubation, the culture media and mycelium were separated using cotton in a funnel. Then, mycelium was washed with ethyl acetate (1.5 liter) while the culture media were extracted 3 times with ethyl acetate (2 liter). The combined organic extract was dried with anhydrous sodium sulphate (Na₂SO₄) and evaporated using a rotary evaporator. The similar procedures were employed for the control group.  The residues from both experimental and control group were analysed by TLC to confirm the presence of biotransformed product. The bio transformed products were isolated by column chromatography using silica gel column (200-300 mesh, Merck Ltd.) with hexane: ethyl acetate as solvent.

Structural elucidation of the biotransformed product

Gas Chromatography/Mass Spectrometry (GC/MS)

The crystal was further analyzed using  an Agilent 5975C  GC-MSD system (Agilent, Avondale, USA) equipped with a 30 m x 0.25 mm i.d. x 0.25 μm , HP-5 capillary column (SGE, Australia) and 70 eV of the electron impact technique. The carrier gas was helium at a flow rate of 1.3 mL/min. The crystal was dissolved in dichloromethane (DCM) at concentration of 1%. Then, 1 μL of sample was injected into the column. The injector and detector temperatures were 250 °C and 230 °C, respectively. The other analytical conditions were as follows: Temperature programming: 60 °C, as initial temperature, for 5 min, 8 °C/min to 180 °C at 10 °C/min to 240 °C, holding for 5 min. Mass spectroscopy analysis was carried out using positive electron ionization method in order to obtain fragments of the compounds. The identification of compounds were based on a comparison of their retention times with those of authentic standards and by comparison of their mass spectra with those of data in the Wiley Registry of Mass Spectral Data and National Institute of Standards and Technology (NIST) libraries¹⁸. Low resolution mass spectrometry (m/z) was carried out on a VG Biotech Quattro II triple quadrupole instrument.

Nuclear Magnetic Resonance (NMR) analysis

The nuclear magnetic resonance (NMR) spectroscopy was used to elucidate the structure of compound (1 & 2) using   1D (1H- and ¹³C-NMR) and 2D (COSY 45°, HSQC, HMBC and NOESY) experiments   according to the standard protocols. The NMR analysis was carried out at the Department of Chemistry, Universiti Kebangsaan Malaysia.  ¹H and ¹³C NMR spectra were recorded on a Bruker 400 MHz FT-NMR spectrometer. The crystal was dissolved in CD₂Cl₂ and chemical shifts (δ) are reported in ppm downfield of tetramethylsilane. Coupling constants (J) are quoted in Hz.

Antimicrobial Assays

Minimum Inhibitory Concentration (MIC)

The test was performed using 96-well plates and prepared in a three-fold dilutions manner, where the concentration in each well is three times less than the preceding well. The first well for each sample (single compounds) was prepared in a 1000 µg/mL concentration by mixing 30 µL of the single compounds (10 mg/mL concentration) with 270 µL broth media. After mixing the samples with the broth, the mixture (100 µL) was transferred to the next well, in the same column,  containing 200 µL broth media. The similar procedure was repeated for the subsequent well until the  end of each column. The final concentrations formed in each well after this process were 1000 (first well), 333, 111, 37, 12.3, 4.1, 1.37, and 0.45 µg/mL²⁵.

Minimum Bactericidal/Fungicidal Concentration (MBC)

The minimum bactericidal/fingicidal concentration (MBC/MFC) test was carried out according to Betts et al²⁶,  with slight modifications in order to determine whether the MIC values  only inhibit the growth or  even kill the microorganism. 100 µL of each broth in the well that represents MIC values for all bacteria and fungus was spread in MHA and SDA, respectively. Plates were kept in incubator for 24 hr. The  MBC/MFC could be determined when  the microorganisms were killed and unable to regrow back.

RESULTS AND DISCUSSION

Microbial transformation of ethyl p-methoxycinnamate (1) by A. niger afforded a main compound, identified as ethyl p-hydroxycinnamate (2) (Figure 2) in good yield (24%). The molecular formula of 2 proved to be C11H12O3 from ¹H-NMR and ¹³C-NMR data. The NMR spectral data of compound 2 are summarized in the Table 1. These finding presented a clear correlation to support the proposed structure and identified by comparing their spectra with literature²⁷.

Figure2: Bio transformation pathway of ethyl p-methoxycinnamate into ethyl p-hydroxycinnamate acid by Aspergillus nigerClick here to View Figure

 

Table1: 1H- NMR, 13C-NMR, COSY and HMBC Spectral Data of EPHC, 2 .Click here to View table

 

The antimicrobial assay in terms of  the minimum inhibitory concentrations (MIC) of   of ethyl p- methoxycinnamate (EPMC) and ethyl p-hydroxycinnmate (EPHC) against different pathogenic bacteria and fungus  are shown in Table 2.

Table2: Minimum Inhibitory Concentration (MIC) of ethyl p-methoxycinnamate (EPMC)  and ethyl p-hydroxycinnamate (EPHC).Click hereto View table

 

Table3: Minimum Bactericidal/Fungicidal  Concentration (MBC/MFC) of ethyl p-methoxycinnamate (EPMC)  and ethyl p-hydroxycinnamate (EPHC).Click here to View table

 

MIC tests were done selectively against four types of bacteria which were B. cereus, S. aureus, E. coli, P. aeruginosa and one fungus which was C. albicans.  The result showed that both compounds have good antimicrobial activities against all tested bacteria and fungus. The MIC values also indicated that both compounds (EPMC & EPHC) were able to inhibit Gram-negative bacteria at lower concentrations compared to Gram-positive bacteria. The  MIC value of  ethyl p-hydroxycinnamate (EPHC) against both  E. coli   and P. aeruginosa was  111 µg/mL, which were more active than the  value  exhibited by  ethyl p- methoxycinnamate (EPMC, MIC = 333 µg/mL) (Table 2).  Meanwhile, the MIC values of EPHC  against both B. cereus and S. aureus  was 333 µg/mL, which were also lower than the  value  exhibited by  EPMC (MIC=1000 µg/mL). Furthermore,     EPHC was able to inhibit the growth of C. albicans with  the concentration that exhibited inhibition potential (MIC) was  111 µg/mL which were lower than the  value  exhibited by  EPMC (MIC= 333 µg/mL).

In terms of minimum bactericidal concentration (MBC), there were bactericidal effect of EPHC  against S. aureus, B. cereus, E. coli and  P. aeruginosa  at 1000 µg/mL and fungicidal effect (MFC) against   Candida albicans at  333 µg/mL (Table 3). However, there were no MBC was obtained for ethyl p-methoxycinnamate (EPMC) against all tested microorganisms. Hence, ethyl p-hydroxycinnamate (EPHC) has great potential to inhibit and killing the growth of Gram-positive bacteria, Gram-negative bacteria and fungus compared to ethyl p- methoxycinnamate (EPMC). The present of hydroxyl group in structure may increase the antimicrobial activities of compound²⁸. The result has proved that the folkloric use of this plant in treating microbial infections and suggested that ethyl p-hydroxycinnamate (EPHC) could be exploited in future as one of the new antibiotics in line with the current drug development.

CONCLUSION

Bio transformation has been successfully utilized as a tool to generate pharmaceutical compounds from natural products. Through this process, ethyl p-methoxycinnamate (EPMC), extracted from Kaempferia galanga plant, was transformed using Aspergilus niger to ethyl p-hydroxycinnamate (EPHC), which shown antimicrobial activities.

 Looking at the antimicrobial activities, ethyl p-hydroxycinnamate (EPHC) has an inhibitory potential effect  against S. aureus, B. cereus, P. aeruginosa, E. coli  and C. albican  better than the effect by ethyl  p-methoxycinnamate  (EPMC).  EPHC has also shown bactericidal and fungicidal effects  against all tested bacteria and fungus  while EPMC did not show killing potential toward them.

ACKNOWLEDGEMENT

The authors would like to express greatest appreciation and gratitude to the International Islamic University of Malaysia (IIUM) and The Ministry of Higher Education (MOHE) for financial support (RMGS). Also thank to all Kulliyyah of Science (KOS) and IIUM staff for technical supports.

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