Metabolomic profiling and bactericidal effect of Polyalthia longifolia (Sonn.) Twaites. stem bark against methicillin-resistant Staphylococcus aureus

Objective. The present study was carried out to establish the chemical profile of the methanolic extract of Polyalthia longifolia stem bark and investigate its antibacterial property against some human pathogenic bacteria. Methods. The extract was analysed using liquid and gas chromatography coupled to mass spectrometry. Antibacterial activity of P. longifolia extract against some human pathogenic bacteria was screened using the AlamarBlue method, and MIC and MBC were determined. Results and Conclusion. Liquid chromatography-mass spectrometry (LC-MS) revealed the presence of 21 compounds among which 12 were identified. Gas chromatography-mass spectrometry (GC-MS) allowed identification of 26 compounds, the three major ones being the following: cis vaccenic acid (17.79 %), 3-ethyl-3-hydroxyandrostan-17-one (13.80 %) and copaiferic acid B (12.82 %). P. longifolia extract was active against Gram-positive bacteria with MIC ranging from 1 to 2 mg ml−1 and MBC from 2 to 6 mg ml−1. This study demonstrated the bactericidal effect of the methanolic extract of Polyalthia longifolia stem bark against some human pathogenic bacteria, including methicillin-resistant S. aureus . This effect could be related to the presence in the extract of a broad diversity of well-known compounds with established pharmacological properties. These results support the ethnomedicinal use of P. longifolia stem bark in Cameroon for the management of methicillin-resistant S. aureus (MRSA)-related infections.


INTRODUCTION
Although pharmaceutical industries have produced a considerable number of commercial antibiotics, resistance of pathogens to these drugs has risen to a point of global concern [1,2]. Much like the situation in human medicine, intensive use of antibiotics in agriculture, livestock and poultry has accelerated the development of antibiotic-resistant strains, potentially complicating treatments for plants, animals and humans. Bacteria have the genetic ability to acquire and transmit resistance towards drugs to many different bacterial strains through horizontal gene transfer. Therefore, there has been an increased mortality rate due to complications of infectious diseases and treatment failure. This explains why resistance to antibiotics has been recognized by the World Health Organization as the greatest threat in the treatment of infectious diseases [3,4]. For example, methicillin-resistant Staphylococcus aureus (MRSA) has been found to be the major cause of hospital-acquired infections and community-acquired infections [5]. Because of such concern, the constant need to find new antimicrobial agents is of paramount importance, alongside the necessity to seek and design alternative approaches to manage resistant bacteria. One of the possible strategies is rational OPEN ACCESS valorization of bioactive phytochemicals with evidence-based antibacterial activity, as medicinal plants are important natural resources and are increasingly gaining wide spread interest for the treatment of numerous diseases.
Polyalthia longifolia (Sonn.) Twaites. (Annonaceae) originates from India and has been afterwards introduced to many tropical countries around the world, including Cameroon. It is an evergreen tree with a height of over 30 ft, which exhibits symmetrical pyramidal growth with willowy pendulous branches and long narrow lanceolate leaves. Due to its aesthetically pleasing structure, this tree is often used as ornamentation in households [6,7]. Almost all parts of P. longifolia are used in traditional Indian medicine for the treatment of various ailments such as uterine disorders, fever, skin diseases, diabetes, hypertension, helminthiasis, gonorrhoea [8]. Several pharmacological properties were reported by researchers about the stem bark and leaves of this plant. These include antimicrobial [9], cytotoxic [10], hypotensive [11] and antiulcer [12] activities. Although numerous studies have been carried out with Asian P. longifolia, its antibiotic activity has not, to our knowledge, been carried out on Cameroonian P. longifolia. Thus, the aim of the present study was to investigate the chemical composition and to test the antibacterial effect of the stem bark extract of P. longifolia from Cameroon.

Plant material
Fresh stem bark of Polyalthia longifolia (Sonn.) Thwaites. were harvested in Yaoundé (Cameroon) (3.861057 N; 11.506323 E) and identified at the National Herbarium of Cameroon by comparison with existing voucher number 67474HNC. The stem barks were chopped into small pieces, shade-dried and then crushed, using an electric blender to obtain a fine powder.

Extract preparation
In total, 100 g of Polyalthia longifolia stem bark powder was macerated in 3 l of methanol for 48 h at room temperature. The mixture was filtered through Whatman filter paper № 3 and the resulting filtrate was then concentrated at (40 °C) using a rotative evaporator (Heidolph, Germany). The paste obtained was dried in a convection oven (Jencons-PLS, UK) set at 40 °C. The resulting extract [5.3 g (5.3 % yield)] was stored at 4 °C for later use.

Gas chromatography-mass spectrometry analysis
The GC-MS characterization of the methanolic extract of Polyalthia longifolia stem bark was done using Agilent Technologies GC systems with GC-7890A/MS-5975C models (Agilent Technologies, Santa Clara, CA, USA) equipped with HP-5MS column (30 m in length ×0.32 mm in diameter ×0.25 µm in thickness of film). After solubilization in methanol, the extract was filtered through a 0.45 µm filter (Millipore, USA) and then injected in splitless mode. Spectroscopic detection by GC-MS involved an electron ionization system, which utilized high-energy electrons (70 eV). The carrier gas was helium with a flow rate of 1.0 ml min −1 . The initial temperature was set at 50 °C with increasing rate of 5 °C min −1 and holding time of about 10 min. Finally, the temperature was increased to 300 °C at 10 °C min −1 . The compounds were identified by comparison of their mass spectra with standards available in National Institute of Standards and Technology (NIST) (Gaithersburg, USA) mass spectral library.

Liquid chromatography-mass spectrometry analysis
LC-MS characterization of the methanolic extract of Polyalthia longifolia stem bark was realized using a Hewlett-Packard 1100 chromatograph (Agilent Technologies, Santa Clara, CA, USA) with diode array detector (DAD), and equipped with a C 18 column (4.6 mm × 150 mm × 5 µm particle size). The mobile phases were 0.1 % formic acid in water (A) and 0.1 % formic acid in methanol (B) at a flow rate of 0.5 ml min −1 . The chromatographic method consisted of the following elution gradient: 10 % B for 1.00 min; 10-100 % B for 6.00 min, 100 % B for 1.0 min. An MSD Ion Trap XCT mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with an electrospray ionization (ESI) interface was used in positive ionization mode (between 100 and 1200 m/z) for the MS analysis using data-dependent automatic switching between MS and MS/MS acquisition modes. Identification of compounds was conducted using a cross comparison of their mass spectra with standards contained in three online spectral databases, namely, spectral database for organic compounds (SDBS) ( sdbs. db. aist. go. jp), mass bank Europe ( massbank. eu) and metlin database ( metlin. scripps. edu).

Bacterial susceptibility testing procedure (Microplate AlamarBlue assay)
The microplate AlamarBlue assay was used to check the antibacterial activity of Polyalthia longifolia against the bacterial strains under study. This assay is based on resazurin dye (7-hydroxy-10-oxidophenoxazin-10-ium-3-one), which is an oxidationreduction (REDOX) weakly fluorescent indicator that undergoes colorimetric change (from blue to pink/red) in response to cellular metabolic reduction. Its reduced form, resorufin, is pink and highly fluorescent, and the intensity of fluorescence produced is proportional to the number of living cells. One colony of each of the aforementioned strains (except Helicobacter pylori), grown in Tryptone Soya Agar (TSA) (Oxoid, UK), was inoculated in Mueller-Hinton Broth (MHB) (Oxoid, UK) and incubated at 37 °C for 22 h. Bacterial cultures obtained were diluted using MHB and adjusted to 0.5 McFarland turbidity index (~1.5×10 8 c.f.u. ml −1 ). A stock solution (40 mg ml −1 ) of P. longifolia extract was prepared in 10 % DMSO and 10 µl of this stock solution were placed in all the wells of a sterile 96-well microplate except positive (bacteria+medium), negative (medium +10 % DMSO) and drug (bacteria+medium+standard antibiotics) control wells. After adding appropriate volume of MHB in all the wells, bacterial suspensions (5 µl) were then introduced, except in negative control wells. The concentration of extract in the final 200 µl solution was 2 mg ml −1 . Tetracycline was used as the control drug with a stock solution prepared at 1 mg ml −1 in distilled water. After 22 h of incubation at 37 °C, 20 µl of 0.02 % AlamarBlue dye (Chem-Impex-International, USA) solution was added in all the wells, and the plates incubated at 37 °C in a shaking incubator at 80 r.p.m. for 2 h. Helicobacter pylori was cultured in Brain Heart Infusion Agar (BHIA) supplemented with Laked Horse Blood (Oxoid, UK), and H. pylori selective supplement (Dent) (Oxoid, UK). Bacterial suspensions were diluted in Brain Heart Infusion Broth (BHIB) and incubated for 48 h at 37 °C in 10 % CO 2 incubator. Amoxicillin and metronidazole, prepared at 1 mg ml −1 each, were used in mixture (1 : 1) as the control drug. For quantitative analysis, absorbance was read at two wavelengths (570 and 600 nm) using a spectrophotometer (Thermo Scientific, USA). The percentage of inhibition of bacterial growth due to extract/control drug treatment was calculated as described by Al-Nasiry et al. [13]. Each experiment was run in triplicate.

Determination of minimum and maximum inhibitory concentrations
MIC was determined using the standard broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [14]. P. longifolia extract was serially diluted twofolds in MHB with final concentrations ranging as follows: 0.062, 0.125, 0.25, 0.50, 1, 2, 4 and 8 mg ml −1 . Thereafter, 5 µl of respective bacterial suspensions (except Helicobacter pylori) prepared as described above, were added in all the wells, and plates incubated for 22 h at 37 °C. For H. pylori, MHB was replaced by BHIB. The lowest concentration of extract, capable of inhibiting visible growth with no turbidity, was then recorded as the MIC.
Minimal bactericidal concentration (MBC) was determined by sub-culturing 50 µl of each well content, from serial dilutions described above, in TSA plates. TSA plates were incubated at 37 °C for 22 h. For H. pylori, TSA plates were replaced by BHIA

GC-MS analysis
The GC-MS spectra (Fig. 1) allowed identification of 26 compounds being reported, alongside their retention time, monoisotopic mass and abundance (peak area %), in Table 1.

LC-MS analysis
The total ion chromatogram (TIC) of P. longifolia methanolic extract is represented by Fig. 3. Twelve (12) compounds were identified and summarized in Table 2, with their chemical structures illustrated by Fig. 4.

Antibacterial activity
Antibacterial activity of P. longifolia methanolic extract against Gram-positive and Gram-negative bacteria is described in Tables 3  and 4, respectively. Percentages of inhibition were ranged from 79.05-97.81 % (except Enterococcus faecalis) for Gram-positive bacteria and from 1.76-7.64 % for Gram-negative bacteria.
MIC and MBC of P. longifolia methanolic extract against some Gram-positive bacteria are presented in Table 5. MIC values were comprised between 1000 µg ml −1 and 2000 µg ml −1 , and MBC values between 2000 and 6000 µg ml −1 .

DISCUSSION
The methanolic extract of P. longifolia stem bark showed good antibacterial activity against Gram-positive bacteria, irrespective of whether they were sensitive or methicillin-resistant strains, and was ineffective on Gram-negative bacteria. In Gram-positive bacteria, the MBC/MIC ratio was found to be less than 4, which classifies the extract as bactericidal against these bacteria as per the Marmonier [15] recommendations. The difference in sensitivity to Gram-negative and Gram-positive bacteria may be due to the variation in their cell-wall structure. Most Gram-positive bacteria are surrounded by a coarse peptidoglycan cell wall. The Gram-positive bacterial cell wall consists of 70-100 layers of peptidoglycans, which are comprised of two polysaccharides, N-acetyl-glucosamine and N-acetyl-muramic acid, cross-linked by peptide side chains and cross bridges. This structure, although mechanically strong, appears to offer little resistance to the diffusion of small molecules such as antibiotics. In contrast, Gramnegative bacteria, surround themselves with a second membrane, the outer membrane, which functions as an effective barrier [16,17]. In a study conducted by [9], the methanolic extract of P. longifolia leaves, harvested in Karachi (Pakistan), exhibited good antibacterial activity against both Gram-positive and Gram-negative bacteria. MIC values were ranging from 125 µg ml −1 (Pseudomonas aeruginosa) to 500 µg ml −1 (Salmonella typhi) for Gram-negative bacteria, and 125 µg ml −1 for the three Grampositive bacteria tested. On the other hand, another study [18] investigating antimicrobial activity of the methanolic extract of P. longifolia leaves, harvested in India, reported MICs of 3125 µg ml −1 against Staphylococcus aureus, and higher than 25 000 µg ml −1 against Salmonella typhimurium. These results are closer to those of the present study, with both extracts seeming less active that the one harvested in Karachi, Pakistan. This difference between the results of the Karachi study and those of the present study could be due to a plethora of factors among which an uneven distribution of active metabolites between the plant's organs and/or between plants located in different ecological zones. These factors could include seasonality, temperature, water availability, UV radiation, nutrients present in soil, altitude, air pollution and even induction by mechanic stimuli or attack by pathogens.
LC-MS analysis allowed identification of 12 compounds, some with tested antibacterial properties. For example, heliotrine has been reported to be effective against bacteria (E. coli, S. pneumoniae, B. subtilis and B. anthracis) and fungi (Aspergillus fumigatus, Aspergillus niger and Penicillium chrysogenum) [19]. Heliotrine-N-oxide is a pyrrolizidine alkaloid (PA) and   [20] using a diallel cross of Senecio jacobaea revealed that 48 % of the variation in total PA content was caused by genotypic variation. This variability is further increased by the possible formation of monoesters at different positions and open or cyclic diesters. The effect of each single PA is dependent on its chemical structure and physical properties such as lipophilicity, hydrophilicity and pharmacokinetics [20]. These reasons may explain a possible low level of heliotrine in the whole extract, and thus, a decreased potency against Gram-negative bacteria in the present study. Apomorphine was found to be bactericidal against E. coli by targeting the pleiotropic bacterial regulator Hfq [21]. Pinoresinol showed good antibacterial activities against most of bacteria studied in the current work [22], while propiconazole and mebendazole demonstrated their potency against several fungi strains including Cryptococcus neoformans and Sclerotium rolfsii [23,24]. Among the 26 compounds identified by GC-MS, some were equally reported in the literature for their antimicrobial properties. For instance [25], demonstrated antibacterial potential of coniferol against E. coli and B. cereus, while the bactericidal effect of hexadecenoic acid against S. aureus was also evidenced [26]. All these compounds present in the methanolic extract of P. longifolia stem bark, and possibly acting either synergistically or additively, could be responsible for the observed bactericidal effect of this extract. Antibacterial activity of plants is directly linked to the diversity and complexity of their secondary metabolites. Major groups of phytochemicals, which are known to possess antimicrobial properties are the following: polyphenols (flavonoids, quinones, tannins, coumarins), terpenoids, alkaloids,  lectins and polypeptides [27]. Therefore, the relatively high MIC and MBC values of P. longifolia stem bark extract could be explained by its low content of bioactive compounds such as heliotrine, apomorphine or pinoresinol.

CONCLUSION
LC-MS and GC-MS analysis of methanolic extract of P. longifolia stem bark permitted the identification of valuable compounds with well-established antibacterial properties, which would be responsible of the extract's bactericidal activity. These compounds would act either synergistically or additionally to produce the bactericidal effect. Further studies are planned to investigate the underlying mechanisms involved to this end.

Conflicts of interest
The authors declare that there are no conflicts of interest. Values are presented as mean percentage ±sem of three replicates. Amoxicillin and metronidazole in combination were tested at 25 µg ml −1 each. ***P<0.001=significant statistical difference by comparison with tetracycline. NA=non applicable.