Antimicrobial Activity of Coronarin D and Its Synergistic Potential with Antibiotics

Coronarin D is a labdane-type diterpene from the rhizomes of Hedychium coronarium. In the view of our ongoing effort to explore its novel biological activity, antimicrobial activity study of coronarin D was performed. The results showed that coronarin D was active against tested Gram-positive bacteria, inactive for tested Gram-negative bacteria, and weakly active against tested fungi. The antibacterial effect of the combination of coronarin D with nine classical antibiotics against four Gram-positive bacteria was also evaluated. The fractional inhibitory concentration indices (FICI) of coronarin D-antibiotics combinations, calculated from the checkerboard assay, were used as synergism indicator. Out of 36 combinations, 47% showed total synergism, 33% had partial synergistic interaction, 17% showed no effect, and 3% showed antagonism. By combination with coronarin D at concentration of 0.25 minimal inhibitory concentration (MIC), the activities of antibiotics were boosted to 4- to 128-fold. These finding suggested an attractive approach to combat the infectious diseases by using coronarin D-antibiotic drug combination.


Introduction
During the past to present, infectious diseases are the leading cause of death worldwide particularly in developing countries [1]. The problems are from unrecognized emerging infections, reemerging infections, more virulent pathogens, and drug-resistance bacterial infections. The infectious microorganisms that cause major public health problems can be divided into three main groups; those are opportunistic pathogens, nosocomial pathogens, and gastrointestinal pathogens. Opportunistic pathogens are infectious microorganisms that are potentially harmful for immunodeficiency or immunosuppressed patients [2]. Nosocomial pathogens are infectious microorganisms that cause problem to patients admitted to hospital for a long time [3]. For example, P. aeruginosa is a leading Gram-negative opportunistic and nosocomial pathogen that is seriously problematic for patients in ICUs [4]. S. aureus and S. epidermidis are Grampositive pathogenic bacteria that cause common nosocomial infection [3,5]. A. niger is the common opportunistic and nosocomial species associated with invasive pulmonary aspergillosis in human [6]. Gastrointestinal pathogens are infectious microorganisms that cause gastrointestinal disease such as diarrhea, typhoid fever, and cholera [7]. B. cereus is one of gastrointestinal pathogens usually found in canned food industries and causing serious foodborne disease [8]. Nowadays, the infectious diseases become more dangerous and more infections become resistant to classical antibiotics. Therefore, the continuing efforts to search for new antibacterial substances are necessary and urgently needed. Natural products still remained as important source in drug discovery, providing crucial and unmatched chemical diversity. Most of antibiotics used today are derived from natural products or natural product scaffolds [9].
In this study, the pathogens we selected are those commonly found and those that lead to the major health problems. The selected pathogens comprised of P. aeruginosa, C. albicans, C. albidus, Acremonium sp., A. flavus, A. niger, and Penicillium sp. as opportunistic pathogens, S. aureus, S. epidermidis, P. aeruginosa, E. faecalis, and A. niger as nosocomial pathogens, and E. coli, S. typhimurium, and B. cereus as gastrointestinal pathogens.
Coronarin D (1) (Figure 1) is a labdane-type diterpene isolated mainly from the rhizomes of Hedychium coronarium [10,11], which is known in Thai as "Mahahong. " Various biological activities of coronarin D were observed, for example, cytotoxic activity against cancer cell [10,11] and inhibiting both constitutive and inducible nuclear factorkappa B pathway, a key mediator of inflammation, apoptosis, invasion, and osteoclastogenesis [12]. Recently, antifungal activity of coronarin D against Candida albicans has just been reported [13]. This encouraged coronarin D to be of interest for studying its activity against different microorganisms.
For many years until now, combination therapy with two or more antibiotics is used in special cases to prevent or delay the emergence of resistant strain, to treat emergency case during the process of diagnosis, and to take advantage of antibiotic synergism [14]. Recently, the combinations of natural products and antibiotics were studied and reported to enhance the activity of classical antibiotics [15][16][17]. From data above, the synergistic effects between natural products and standard antibiotics were an alternative potential approach to treat infectious diseases. In the view of our ongoing effort to search for promising antimicrobial agents and explore the novel biological activity of coronarin D, we decided to study antimicrobial activity as well as synergistic effects of coronarin D to classical antibiotics.

Coronarin D Isolation.
Coronarin D was isolated from the rhizomes of Hedychium coronarium according to the previous report [11] and its structure was confirmed by the spectroscopic methods ( 1 H and 13 C NMR, MS, and FTIR).   hispida Dennst), Thailand, Aspergillus flavus (A. flavus) TISTR 3366, isolated from compost, and Aspergillus sp. TISIR 3105, isolated from hair pomade. The latter three strains were isolated by dilution plating technique and identified by morphological characterization.

Tested
The bacteria were maintained on nutrient agar (NA) at 37 ∘ C and fungi were maintained on potato dextrose agar (PDA) at 28 ∘ C.

Preparation of Inoculum.
The tested bacteria were cultured in nutrient broth (NB) and incubated for 18-24 h at 37 ∘ C. The tested yeast, C. albicans and C. albidus, were made by growing on PDA or Sabouraud dextrose agar (SDA) for 48 hours at 28 ∘ C. The colonies were harvested, suspended in sterile saline, and their concentrations were adjusted to a 0.5 McFarland standard, the equivalence of 1-2 × 10 8 cfu/mL. The samples were then diluted 1 : 10,000 in Muller Hinton broth (MHB) or Sabouraud dextrose broth (SDB) to 1 × 10 4 cfu/mL. For fungi, Penicillium sp., A. flavus, Aspergillus sp., Acremonium sp., and A. niger, spore suspension were adjusted from 0.4 × 10 4 to 5 × 10 4 spore/mL in sterile saline. ). Antimicrobial agents were prepared as stock solutions at concentrations of 4 and 16 mg/mL in dimethylsulfoxide for susceptibility testing and checkerboard method, respectively.
MICs. The minimal inhibitory concentration (MIC) was determined using the twofold broth microdilution method in accordance with NCCLS guideline [20]. Concentrations of coronarin D from 0.39 to 200 g/mL were used. After

Results
The antimicrobial activity of coronarin D was tested in vitro against microorganisms by using broth microdilution technique and were compared with antibiotics. All MIC and MBC values of coronarin D and antibiotics were summarized in Table 1 To evaluate the synergistic effects of coronarin D and antibiotics, the checkerboard assay was employed. The fractional inhibitory concentration index (FIC I ) value was utilized to assess the synergism (total synergism, FIC I ≤ 0.5; partial synergism, 0.5 < FIC I ≤ 0.75; no effect, 0.75 < FIC I ≤ 2; and antagonism FIC I > 2) [23]. Synergistic effects were investigated only in Gram-positive bacteria. Antibiotics in synergistic testing included penicillin G (PNG), oxacillin (OX), polymyxin B (PMB), ciprofloxacin (CIP), rifampicin (RIF), chloramphenicol (CRP), tetracycline (TTC), gentamicin (CN), and erythromycin (ERY). Antibiotics were selected based on their mode of action.
The results of synergism indicated by FIC index were displayed in Table 3. Out of 36 combinations tested between coronarin D and nine antibiotics, 47% showed total synergism, 33% had partial synergistic interaction, 17% showed no effect, and 3% showed antagonism.

Discussion
The obtained results showed that coronarin D was active against Gram-positive bacteria but inactive against Gramnegative one. These could be rationalized by the capability of compound to cross or damage bacterial cell membrane. Gram-negative bacteria possess the outer membrane, in addition to cell wall, as the barrier to restrict the hydrophobic substances diffusion into the cell. On the other hand, the absence of the outer membrane in Gram-positive bacteria allowed the hydrophobic compounds to penetrate and/or damage cell membrane more easily [24]. Coronarin D, a labdane-type diterpene consisting of decalin ring and unsaturated lactone ring with one hydroxyl group was considered as a hydrophobic molecule; therefore, it could penetrate more easily into and interrupt the cell membrane of Gram-positive bacteria than that of Gram-negative bacteria. From Table 1, coronarin D showed remarkable activity against B. cereus at MIC value of 6.25 g/mL and moderate activity against S. aureus and S. epidermidis at MIC value of 12.5 g/mL. Isolated compounds that possess MIC value lower than 10 g/mL were considered to be a very promising anti-infection agent [25,26]; therefore, coronarin D could be regarded as a candidate for anti-infection agent against B. cereus.
Drug synergy is well known and was used for a long time such as the herbal formulation in traditional medicine. Moreover, in the defense mechanism against infectious diseases of plants, diverse small molecules are produced. It is interesting to note that although most of these small molecules showed weak antibiotic activity, they are successful to combat infections via synergistic mechanisms [27]. Therefore the investigation of synergistic effect particularly between natural products and classical antibiotics is an alternative potentiated approach to fight microorganisms. Various combinations of plant metabolites with antibiotics displayed promising synergistic results such as combination of epigallocatechin gallate (EGCg) with various antibiotics [15], carnosic acid (benzenediolabietanediterpene)tetracycline combination for the inhibition of MDR pumps [16], and baicalin (flavone glucuronide)-beta-lactam combination as beta-lactamase inhibitor [17]. As mentioned above, the synergistic combinations between coronarin D and nine antibiotics were carried out in this study.
Due to the low MIC value of coronarin D against Grampositive bacteria, the synergistic combinations only on these strains were studied. As shown in Table 3    a gain >1: decrease concentration of antibiotic, <1: increase concentration of antibiotic, and 1: no effect. b The best fractional inhibitory concentration index; FIC I ≤ 0.5 means total synergism, 0.5 < FIC I ≤ 0.75 means partial synergism, 0.75 < FIC I ≤ 2 means no effect, and FIC I > 2 means antagonism.
were downed to ng/mL level. A decrease in MIC value of antibiotics due to the combination was very beneficial because (i) toxicity and/or side effects from antibiotics usage were reduced, (ii) the therapeutic cost was reduced, and (iii) the emerging of resistance strains was prevented or prolonged. Moreover, the process for developing a new drug is very expensive which is not possible in most developing countries and using a new drug also has risks of unknown side effects. Therefore, using well-known drugs in combination with herbal substances is quite elegant alternative method to combat infectious diseases.
Recently, in Urzúa et al. 's work [28], fifteen diterpenes were tested against B. cereus and S. aureus. They suggested two structural requirements for antimicrobial activity; those are a hydrophobic moiety and a hydrophilic region possessing one hydrogen-bond-donor group (HBD). These observations were confirmed in published reports [29,30]. Coronarin D contains both hydrophobic part (decalin ring) and hydrophilic moiety (hydroxyl attached on lactone ring) which is fulfilled with those two requirements. In order to test the Urzúa et al. 's suggestion, coronarin D acetate (2) (Figure 2) was prepared. Coronarin D acetate (2) in which the hydroxyl group on the lactone ring was acetylated gave no activity against all tested bacteria (result from disc diffusion experiment, data not shown). This confirmed the necessity of the hydrophilic moiety in the active antimicrobial molecule.
Bacterial membranes compose of 40 percent phospholipids and 60 percent proteins [31]. The phospholipids are amphoteric molecules. It comprises two parts, that is, a polar hydrophilic glycerol containing phosphate group and a nonpolar hydrophobic fatty acid tail. In aqueous environment, it forms bilayer with the polar ends at the outmost and innermost surface and the nonpolar ends at the center of the membrane. Urzúa et al. simulated the insertion of diterpene kaurenoic acid (3) (Figure 2) into a phosphatidylcholine bilayer. The results revealed that kaurenoic acid (3) incooperated itself in the bilayer interface. The decalin ring was surrounded by hydrocarbon chains of the lipid and carboxylic group interacted with the phosphorylated group through hydrogen bonding. When kaurenoic acid (3) was methylated into methyl ester, the hydrogen-bond interaction to bilayer phosphorylated group was suppressed. MIC values of kaurenoic acid (3) were 0.16 and 0.32 g/mL against B. cereus and S. aureus, respectively, while kaurenoic acid methyl ester was inactive.
Villalaín's group studied the interactions of two diterpenes, (+)-totarol (4) and abietic acid (5) (Figure 2), against phospholipid model membrane. By using high resolution magic angle spinning-nuclear magnetic resonance (MAS-NMR), the results indicated that (+)-totarol molecule was situated in the upper region of the membrane and phenolic group was placed in the vicinity of the C-3/C4 carbon atoms of the phospholipid acyl chain [32]. Using steady-state fluorescence anisotropy measurement, it was shown that (+)totarol (4) promoted the changes in physical properties of the model membranes [33]. An intrinsic fluorescent property of this molecule was also investigated in order to obtain information in location and interaction of (+)-totarol (4) in biomembrane model system. The results suggested that it was incorporated very efficiently into membranes and located in the inner region of the membrane far away from the phospholipid/water interface [34]. The results mentioned above demonstrated that the antibacterial action of (+)totarol (4) was mediated by perturbing the membrane structure and weakening the Van der Waals interactions between the phospholipid chains. In the study of abietic acid (5), by using MAS-NMR, it was found that the molecule of abietic acid (5) was located in the upper part of the palisade structure of the membrane. The carboxyl group was in close proximity to the phospholipid ester groups and did not extend beyond C4/C7 carbons of the phospholipid molecule [35]. By using differential scanning calorimetry and 13 P-nuclear magnetic resonance spectroscopy, it was found that abietic acid (5) greatly affected the phase transition of the model membrane [36]. These results clearly revealed that abietic acid (5) drastically changed the structural and polymorphic properties of the model membrane. In addition, the mode of action of two labdane diterpenes, (E)-labda-8(17),12-diene-15,16-dial (6) and (E)-8 ,17-epoxylabd-12-ene-15,16-dial (7) (Figure 2), was recently investigated. The results revealed that these molecules caused significant damage and disintegration to the bacterial cell membranes and cell leakage was found [37].
(+)-Totarol (4), abietic acid (5), and coronarin D (1) share some structural similarities, that is, containing hydrophobic part (hydrocarbon ring) and hydrophilic moiety (phenolic, carboxylic and hydroxyl groups attached on lactone ring in (+)-totarol, abietic acid, and coronarin D, resp.). From the data mentioned above, it was anticipated that decalin ring of coronarin D (1) was embedded in the acyl chains of phospholipid bilayers whereas the hydroxyl group on lactone ring interacted with the phospholipid ester groups. It could be postulated that the antibacterial activity of coronarin D (1) may be from, at least in part, its capability to disrupt the membrane integrity and/or damage the cell membrane of Gram-positive bacteria. The mode of action in detail needs to be investigated.

Conclusion
Coronarin D (1) was a good antibacterial agent. It was active against Gram-positive bacteria. The promising activity was found against B. cereus. Synergistic effect was observed in the combination of coronarin D (1) to various classical antibiotics. From the literature data, the mode of action of this molecule may involve the cell membrane disruption.