Identification of Volatile Components of Liverwort (Porella cordaeana) Extracts Using GC/MS-SPME and Their Antimicrobial Activity

Chemical constituents of liverwort (Porella cordaeana) extracts have been identified using solid-phase microextraction-gas chromatography mass spectrometry (SPME-GC/MS). The methanol, ethanol and ethyl acetate extracts were rich in terpenoids such as sesquiterpene hydrocarbons (53.12%, 51.68%, 23.16%), and monoterpene hydrocarbons (22.83%, 18.90%, 23.36%), respectively. The dominant compounds in the extracts were β-phellandrene (15.54%, 13.66%, 12.10%) and β-caryophyllene (10.72%, 8.29%, 7.79%, respectively). The antimicrobial activity of the extracts was evaluated against eleven food microorganisms using the microdilution and disc diffusion methods. The minimum inhibitory concentration (MIC) varied from 0.50 to 2.00 mg/mL for yeast strains (Saccharomyces cerevisiae 635, Zygosacharomyces bailii 45, Aerobasidium pullulans L6F, Pichia membranaefaciens OC 71, Pichia membranaefaciens OC 70, Pichia anomala CBS 5759, Pichia anomala DBVPG 3003 and Yarrowia lipolytica RO13), and from 1.00 to 3.00 mg/mL for bacterial strains(Salmonellaenteritidis 155, Escherichia coli 555 and Listeria monocytogenes 56Ly). Methanol extract showed better activity in comparison with ethanol and ethyl acetate extracts. High percentages of monoterpene and sesquiterpene hydrocarbons could be responsible for the better antimicrobial activity.

To the best of our knowledge there are no reports on the GC/MS-SPME analysis and antimicrobial properties of the extracts from the liverwort P. cordaeana. The aim of this study was to analyze volatile compounds of different extracts of P. cordaeana by using the GC/MS-SPME technique and evaluate the extracts' activity against important food spoilage and potential pathogenic microorganisms.

Antimicrobial Activity
According to the results given in Table 2, all three extracts of P. cordaeana showed inhibition effects on the growth of all the strains tested. In particular, a notable antimicrobial activity was observed against all the strains of the tested yeasts species, which were more sensitive than bacteria to the effect of the extracts. For the yeast strains the MIC varied from 0.50 mg/mL to 2.00 mg/mL, while MFC varied from 1.00 mg/mL to 3.00 mg/mL. The most sensitive yeast was Yarrowia lipolytica RO13 with a MIC of 0.50-1.00 mg/mL and a MFC of 1.5 mg/mL. Zygossacharomyces bailii 45 (MIC of 1.50-2.00 mg/mL and MFC of 3.00 mg/mL) and P. membranaefaciens OC 70 (MIC 2.00 mg/mL and MFC 2.50 mg/mL) were the most resistant ones to the extracts tested. The strongest anti-yeast activity was displayed by the methanol extract of P. cordaeana. Commercial antibiotic cycloheximide showed inhibition for concentrations lower than 0.05 mg/mL for all micromycetes except against Y. lipolytica RO13 that was completely inhibited at 0.02 mg/mL ( Table 2). The methanol extract exhibited the highest activity against Y. lipolytica RO13. In general the methanol extract was the most effective against all the strains (bacteria and yeasts). Methanol extract contained high amount (8.02%) of acetic acid which has shown excellent bactericidal effect [16]. Data relative to the antibacterial effects of P. cordaeana are presented in Table 3. The data showed the higher antimicrobial efficacy of methanol extract than the other two. MIC ranged between 1.00-3.00 mg/mL, while MBC was 1.50-3.00 mg/mL. The most sensitive bacterium was Listeria monocytogenes 56Ly. In particular, the methanol extract of P. cordaeana presented a MIC of 1.00 mg/mL against L. monocytogenes 56Ly. Furthermore, the growth of L. monocytogenes 56Ly was completely inhibited at 1.50 mg/mL for the methanol extract. The most resistant bacterial strain was E. coli 555 with a MIC of 2.00-3.00 mg/mL and MBC of 3.00 mg/mL. MIC for streptomycin was 0.02-0.05 mg/mL and MBC 0.1 mg/mL. According to the MICs and MBCs, the Gram-negative bacterial strains were more resistant than the Gram-positive one. S. enteritidis 155 and E. coli 555 (Gram-negative) showed MICs of 1 mg/mL, 2.00 mg/mL and MBCs of 2.00 mg/mL, 3.00 mg/mL respectively, for the methanol extract. Nevertheless, it should be taken into consideration that quite a high variability could be found in the sensitivity to antimicrobials among strains of the same microbial species.    Results obtained from disc diffusion method followed by measurements of minimal inhibitory concentration (MIC), confirmed that Y. lipolytica RO13 was the most sensitive microorganism with the highest inhibition zone (24 mm) ( Figure 3) and the lowest MIC value (0.5 mg/mL) ( Table 2). The results demonstrated that Porella methanol extract was more effective against the yeast strains than the bacterial ones. The methanol extract of Porella showed the maximum zones of inhibition against yeast Y. lipolytica RO13 (15, 18 and 24 mm), followed by S. cerevisiae 635 (13, 16 and 18 mm) and P. membaranifaciens OC71 (13, 14 and 16 mm), whereas P. anomala CBS 5759 and P. anomala BVPG 3003 showed the same results (12, 14 and 16 mm). On the other hand, P. membranifaciens OC70 (11, 11 and 12 mm) and Z. bailli 45 (9, 10 and 13 mm) presented the lowest inhibition haloes due to 20, 30 and 40 μL of Porella extract. Z. bailli was the most resistant yeast (zone of inhibition was 9 mm), whereas Y. lipolytica RO13 was the most susceptible also at the lowest concentration tested.
The zones of inhibition detected for bacteria due to 40 μL of the methanol extract varied from 11 mm (E. coli 555) to 17 mm (L. monocytogenes 56Ly). Results summarized in Figure 2 indicate that L. monocytogenes 56Ly was the most susceptible bacterium for the three volumes of the extract (20, 40 and 60 μL). Based on the results of the chemical composition of the methanol extract it can be concluded that the antimicrobial activity is apparently attributed to its high content of sesquiterpenes and monoterpenes. P. cordaeana extracts are rich in sesquiterpenes hydrocarbons components as caryophyllene, neoisolongifolene, aromadendrene, γ-muurolene, and monoterpene hydrocarbons such as β-phellandrene, D-limonene and cymene. The results confirmed that the antimicrobial activity of P. cordaeana could be correlated to the presence of β-phellandrene and caryophyllene. These constituents have been reported to possess significant antimicrobial activity. Caryophyllene showed activity against the microorganisms Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans [17].
In all the tested extracts β-phellandrene and caryophyllene were found as the major compounds which could be mainly responsible for the antimicrobial effect of the tested extracts. An antimicrobial activity of caryophyllene has been previously reported [18,19]. Plant extracts and essential oils that contain sesquiterpene hydrocarbons are reported to have high antimicrobial activity, and the monoterpenes found in the extract analysed in this work may act as antimicrobial agents [20].
Our results showed that Porella extracts could be used as natural preservative in food or pharmaceutical industries. Several liverworts display antifungal activity [21]. Moreover Porella extracts contain β-elemene (3.94%; 2.77%; 1.39% in methanol, ethanol and ethyl acetate extract, respectively), i.e., compounds which in last recent years are known as strongly novel antitumor agent against ovarium cancer [22] and lung cancer [23]. D-limonene, which is a natural monocyclic monoterpene that has also been detected in significant percentages in all the Porella extracts (3.68%, 2.93%, 3.31%), has been shown to possess chemopreventive and therapeutic properties against many human cancers [24].
It is important to outline that the antibiotic cycloheximide, used in comparison with the P. cordaeana extracts and endowed with a strong activity against the test strains, is one of the most powerful antimicrobial agents. However, although it is widely used in biomedical research it is not suitable for human use both as therapeutic or antimicrobial agent as its health risks have become better known [25]. The bottleneck of the toxicity against humans and animals of the major part of the antimicrobial molecules or antibiotics delays antifungal discovery. The occurrence of antimicrobial compounds, which are known as safe for human consumption, in P. cordaeana could be successfully exploited particularly for the control of food borne yeasts or pathogenic yeasts like Candida albicans.

Plant Material
The samples of Porella cordaeana (Hueb.) Moore were collected in April 2009, locality Zlatar Mt. (Serbia). A voucher specimen (No. 16620) has been deposited in the Herbarium at the Institute of Botany and Botanical Garden "Jevremovac", University of Belgrade (BEOU). Material was dried at room temperature.

Extracts Preparation
Dried plants were pulverized into a fine powder using an electric blender. Powdered material (10 g) was extracted with methanol, ethanol or ethyl acetate (100 mL) for 24 h at room temperature. After 24 h, the mixture was filtered through Whatman No. 1 filter paper. Extracts were prepared on a rotary evaporator (Laborota 4001, Heidolph). The solvent was removed with a rotary vacuum evaporator at 40 °C. The yields of the extracts were 1.23%, 0.92% and 1.06% for methanol, ethanol and ethyl acetate extract, respectively. The obtained extracts were stored at 4 °C until further tests.

Gaschromatographic (GC)-Mass Spectrometry (MS)-Solid Phase Microextraction (SPME) Analysis
A divinylbenzene-poly(dimethylsiloxane)-coated stable flex fiber (65 μm) and a manual SPME holder (Supelco Inc., Bellefonte, PA, USA) were used in this study after preconditioning according to the manufacturer's instruction manual. Before each headspace sampling, the fiber was exposed to the GC inlet for 5 min for thermal desorption at 250 °C in a blank run.
Samples were put into sealed vials and then equilibrated for 10 min at 40 °C. The SPME fiber was exposed to each sample for 10 min by manually penetrating the septum, and, finally, the fiber was inserted into the injection port of the GC for 10 min sample desorption. GC-MS analyses were carried out on an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to an Agilent 5970 mass selective detector operating in electron impact mode (ionization voltage, 70 eV). A Chrompack CP Wax 52 CB capillary column (50 m length, 0.32 mm i.d., 1.2 μm df) was used (Chrompack, Middelburg, The Netherlands). The temperature program was 50 °C for 0 min, then programmed at 5 °C/min to 230 °C for 10 min. Injector, interface, and ion source temperatures were 250, 250, and 230 °C, respectively.
Injections were performed with a split ratio of 1:50 and helium (1 mL min −1 ) as the carrier gas. Identities were confirmed by searching mass spectra in the available databases (NIST, version 2005; Wiley, version 1996) and according to the Registry of Mass Spectral Data (1998) mass spectra libraries as well as literature MS data and in comparison with authentic chemical compounds when commercially available.

Chemicals and Strains
Different bacterial (Salmonella Enteritidis 155, Escherichia coli 555-Gram-negative and Listeria monocytogenes 56Ly-Gram-positive) and yeast strains (Saccharomyces cerevisiae 635, Zygosacharomyces bailii 45, Aerobasidium pullulans L6F, Pichia membranaefaciens OC 71, Pichia membranaefaciens OC 70 and Yarrowia lipolytica RO13 belonging to the Department of Food Science, University of Bologna, Italy, as well as Pichia anomala CBS5759 and Pichia anomala DBVPG 3003 were used to evaluate the effect of plant extracts. The yeasts and bacteria strains used in this study were grown on Yeast Extract Peptone Dextrose (YPD) and Tryptic Soy Broth (TSB) medium, respectively, at 27 °C for 48 h (yeasts) and 37 °C for 24 h (bacteria). Cells were suspended in sterile distilled water and used immediately.

Determination of Minimum Inhibitory Concentration (MIC) by Microdilution Method
In order to investigate the antimicrobial activity of the extracts, the modified microdilution technique was used [26,27]. Bacterial species were cultured overnight at 37 °C in a Tryptic Soy Broth (TSB) medium. Yeast strains were cultured at 27 °C in YPD medium.
Minimal inhibitory concentrations (MICs) determination was performed by a serial dilution technique using 96-well microtitre plates. The investigated extracts were added in a broth TSB medium (bacteria)/broth YPD medium (yeasts) with inoculum. The microplates were incubated for 48 h at 37 °C for bacteria and 72 h at 28 °C for yeasts, respectively. The lowest concentrations without visible growth were defined as MIC the MBC (minimal bactericidal concentration)/MFC (minimal microbicidal concentration), indicating (99.5%) killing of the original inoculums, have been detected. Generally MBC/MFC values are defined as the minimal concentrations of the tested molecule not allowing the microbial growth when 10 µL of the cultures taken from the wells with no visible growth are plated onto agarised medium and incubated at the optimial temperatre. Dimethyl sulfoxide (DMSO) was used as an effective extraction solvent, and always used as negative control. All tests were performed in triplicate.

Disc Diffusion Method
The following method was used: A portion of each suspension (100 μL) containing approximately 10 5 cfu/mL was spread over the surface of TSB/YPD plates and allowed to dry. A paper disc (diameter 6 mm) was laid on the inside surface of the upper lid and 10 μL was placed on each disc. The plates inoculated with microorganisms were immediately inverted on top of the lid and sealed with parafilm to prevent leakage of the vapour. Plates were incubated at 37 °C/27 °C for 24 h/48 h and the diameter of the resulting inhibition zone in the bacterial/yeasts lawn was measured. The volume of extracts placed on the paper discs varied, i.e., 20, 30 or 40 μL. A negative control was also included in the test using a filter paper disc saturated with DMSO to check possible activity of this solvent against the bacteria/yeasts assayed. All tests were performed in triplicate.

Conclusions
The results of this work demonstrated that P. cordaeana extracts possess significant antimicrobial potential against food microorganisms. The methanol extract showed a higher activity compared to ethanol and ethyl acetate ones. High percentages of monoterpene and sesquiterpene hydrocarbons could be responsible for antimicrobial activity. GC/MS-SPME analysis of volatile compounds showed some interesting substances which may prove useful in further investigations for food preservation purposes or as anticancer components.