GC-MS analysis of Tasmannia lanceolata Extracts which Inhibit the Growth of the Pathogenic Bacterium Clostridium perfringens

Introduction: Clostridium perfringens is the etiological agent of clostridial myonecrosis and enteritis necroticans. Infections result in exotoxin production, tissue necrosis and unless promptly treated, often result in death. Methods: Tasmannia lanceolata extracts were investigated for C. perfringens growth inhibitory activity by disc diffusion analysis and MIC determination. Toxicity was evaluated by Artemia nauplii bioassay and the most potent extracts were phytochemically evaluated by GC-MS headspace analysis. Results: All T. lanceolata berry and leaf extracts displayed potent C. perfringens growth inhibition. The berry extracts were more potent growth inhibitors than the corresponding leaf extracts, although the leaf extracts were also potent growth inhibitors. The berry aqueous, methanolic and ethyl acetate extracts were particularly potent growth inhibitors, with MIC values of 654, 65 and 329 μg/mL respectively. T. lanceolata leaf also displayed good efficacy, with an MIC of 839, 1255 and 625 μg/mL for the aqueous, methanolic and ethyl acetate extracts respectively. All extracts were nontoxic in the Artemia franciscana bioassay, with LC50 values substantially > 1000 μg/mL. Non-biased GC-MS analysis of the aqueous, methanolic and ethyl acetate berry extracts revealed the presence of high relative levels of a diversity of terpenoids. Conclusions: The lack of toxicity of the T. lanceolata extracts and their potent growth inhibitory bioactivity against C. perfringens indicates their potential as medicinal agents in the treatment and prevention of clostridial myonecrosis and enteritis necroticans. GC-MS metabolomic profiling studies indicate that these extracts contained a diversity of terpenoids, with monoterpenoids being particularly abundant.


INTRODUCTION
Clostridium perfringens is an endospore-forming Gram-positive, obligate anaerobic bacterium that is found in a variety of habitats, including soils, sewage or as a naturally occurring inhabitant of the intestinal microflora of humans. 1 It is classed as an opportunistic pathogen and is an etiological agent of clostridial gastroenteritis as well as several other diseases including myonecrosis. 2 The species C. perfringens is divided into five type strains (A-E), each capable of producing exotoxins that are linked to different illnesses of varying severity.These can range from mild food-poisoning to the potentially fatal clostridial myonecrosis (gas gangrene). 3ostridial myonecrosis is a rapidly progressive infection of the soft tissue.The disease is characterised by the necrosis of local muscle and surrounding tissue and can lead to shock and ultimately death, even when promptly treated. 1 Although several Clostridium spp.can cause gas gangrene, C. perfringens is the primary

Minimum inhibitory concentration (MIC) determination
The minimum inhibitory concentrations (MIC) of the extracts was determined as previously described. 16,17 riefly, the plant extracts were diluted in deionised water and tested across a range of concentrations.Discs were infused with 10 µL of the extract dilutions, allowed to dry and placed onto inoculated plates.The assay was performed as outlined above and graphs of the zone of inhibition versus concentration were plotted.Linear regression was used to calculate the MIC values.

Toxicity screening Reference toxin for toxicity screening
Potassium dichromate (K 2 Cr 2 O 7 ) (AR grade, Chem-Supply, Australia) was prepared as a 4 mg/mL solution in distilled water and serially diluted in artificial seawater for use in the Artemia franciscana nauplii bioassay.

Artemia franciscana nauplii toxicity screening
Toxicity was tested using a modified Artemia franciscana nauplii lethality assay. 18,19 riefly, 400 µL of seawater containing approximately 43 (mean 43.2, n = 155, SD 14.5) A. franciscana nauplii were added to wells of a 48 well plate and immediately used for bioassay.A volume of 400 µL of diluted plant extracts or the reference toxin were transferred to the wells and incubated at 25 ± 1 °C under artificial light (1000 Lux).A negative control (400 µL seawater) was run in triplicate for each plate.All treatments were performed in at least triplicate.The wells were checked at regular intervals and the number of dead counted.The nauplii were considered dead if no movement of the appendages was observed within 10 seconds.After 24 h all nauplii were sacrificed and counted to determine the total % mortality per well.The LC 50 with 95% confidence limits was calculated using probit analysis.is a medium to large shrub that varies between 2-5 m in height.Individual plants are unisexual, having either male or female flowers.The stems, branches and twigs are red in colour.The aromatic leaves are lanceolate to narrowly elliptical in shape (4-12 cm long, 0.7-2 cm wide) with a distinctly pale under surface.Small creamy-white unisexual flowers appear during the summer months.These develop into small fleshy black 2 lobed berries (5-8 mm wide) during autumn.The berries, leaves and bark of this species have historical uses as a food and as a medicinal plant. 6,7 hen the berry is air dried, it forms a small, hard peppercorn which is suitable for milling or crushing.The berry has a pleasant spicy flavour and sharp aroma.T. lanceolata was used as flavouring agent by Australian Aborigines and more recently by European settlers.Historically, the leaves have been used as an herb and the berries have been used as a spice.Australian Aborigines also used T. lanceolata as a therapeutic agent to treat stomach disorders and as an emetic, as well as general usage as a tonic. 7T. lanceolata was also used traditionally for the treatment and cure of skin disorders, venereal diseases, colic, stomach aches and as a quinine substitute. 7Later, European colonists also recognized the therapeutic potential of T. lanceolata and the bark was used as a common substitute for other herbal remedies (including those derived from the related South American Winteraceae species, Drimys wintera (winter bark) 7 to treat scurvy due to its high anti-antioxidant content. 7,8 spite its ethnobotanical usage, there have been limited rigorous scientific studies into the therapeutic properties of T. lanceolata.Recent studies have demonstrated the high antioxidant content of T. lanceolata fruit and leaves. 8It has been postulated that this high anti-oxidant content may provide therapeutic effects for this plant. 7Indeed, studies within our laboratory have reported potent inhibition of bacterial growth by T. lanceolata berries, leaves and peppercorns against panels of pathogenic and food spoilage bacteria. 9T. lanceolata extracts can also inhibit the growth of a bacterial trigger of rheumatoid arthritis (P.mirabilis). 10owever, despite the documented ability of T. lanceolata to inhibit the growth of many bacterial species, to our knowledge there have been no similar studies against Clostridium perfringens.The current study was undertaken to test T. lanceolata berry and leaf extracts for the ability to inhibit the growth of this pathogen.

T. lanceolata samples and extraction
Dried Tasmannia lanceolata (Poir.)A.C.Sm berry (without seed) and leaf materials were obtained from Go Wild Harvest, Australia.The material was thoroughly dried using a Sunbeam food dehydrator and stored at -30 o C until use.The dried plant materials were thawed and freshly ground to a coarse powder prior to extraction.Individual 1 g quantities were extracted by weighing each powdered plant part into each of 5 tubes and adding 50 mL of methanol, water, ethyl acetate, chloroform or hexane respectively.All solvents were obtained from Ajax, Australia and were AR grade.The berry and leaf material was extracted in each solvent for 24 h at 4 o C with gentle shaking.The extracts were filtered through filter paper (Whatman No. 54) under vacuum followed by drying by rotary evaporation in an Eppendorf concentrator 5301.The resultant dry extracts were weighed and redissolved in 10 mL deionised water (containing 1% DMSO).

Qualitative phytochemical studies
Phytochemical analysis of the extracts for the presence of alkaloids, anthraquinones, cardiac glycosides, flavonoids, phenolic compounds, phytosterols, saponins tannins and triterpenoids were conducted by previously described assays. 11,12 armacognosy Journal, Vol 9, Issue 5, Sep-Oct, 2017 Non-targeted GC-MS head space analysis Separation and quantification of phytochemical components were performed using a Shimadzu GC-2010 plus (USA) linked to a Shimadzu MS TQ8040 (USA) mass selective detector system as previously described. 20riefly, the system was equipped with a Shimadzu auto-sampler AOC-5000 plus (USA) fitted with a solid phase micro-extraction fibre (SPME) handling system utilising a Supelco (USA) divinyl benzene/carbowax/ polydimethylsiloxane (DVB/CAR/PDMS).Chromatographic separation was accomplished using a 5% phenyl, 95% dimethylpolysiloxane (30 m x 0.25 mm id x 0.25 um) capillary column (Restek USA).Helium (99.999%) was employed as a carrier gas at a flow rate of 0.79 mL/min.The injector temperature was set at 230°C.Sampling utilised a SPME cycle which consisted of an agitation phase at 500 rpm for a period of 5 sec.The fibre was exposed to the sample for 10 min to allow for absorption and then desorbed in the injection port for 1 min at 250 °C.The initial column temperature was held at 30°C for 2 min, increased to 140 °C for 5 min, then increased to 270 °C over a period of 3 mins and held at that temperature for the duration of the analysis.The GC-MS interface was maintained at 200 °C with no signal acquired for a min after injection in split-less mode.The mass spectrometer was operated in the electron ionisation mode at 70 eV.The analytes were then recorded in total ion count (TIC) mode.The TIC was acquired after a min and for duration of 45 mins utilising a mass range of 45 -450 m/z.

Statistical analysis
Data is expressed as the mean ± SEM of at least three independent experiments.

RESULTS
Extraction of 1 g of the T. lanceolata plant materials with various solvents yielded dried plant extracts ranging from 17 mg (T.lanceolata leaf ethyl acetate extract) to 171 mg (methanolic T. lanceolata fruit extract) (Table 1).Aqueous and methanolic extracts generally gave higher yields of dried extracted material compared to ethyl acetate extracts.The dried extracts were resuspended in 10 mL of deionised water (containing 1 % DMSO) resulting in the extract concentrations shown in Table 1.Qualitative phytochemical studies showed little difference between the aqueous and methanolic extracts, however there were notable differences between these extracts and the ethyl acetate extracts.High levels of phenolics (both water soluble and insoluble) were extracted in the aqueous and methanolic samples.There were substantially lower levels detected in the corresponding ethyl acetate extracts.Similarly, there was a lower level of flavonoids detected in the ethyl acetate extracts than the corresponding aqueous and methanolic extracts.Triterpenes were detected in both methanolic and ethyl acetate extracts although they were absent in the aqueous extracts.To measure the inhibitory activity of the crude plant extracts against C. perfringens, 10 µL aliquots of each extract were screened using a disc diffusion assay.The bacterial growth was inhibited by all of the 6 extracts tested (Figure 1).The methanolic berry extract was the most potent inhibitor of growth, with inhibition zones of 16.3 ± 0.3 mm.This compares favourably with the penicillin and ampicillin controls, which had inhibitory zones of 12.3 ± 0.3 mm and 13.0 ± 1.0 mm respectively.The aqueous and methanolic extracts showed greater zones of inhibition than the ethyl acetate extracts, with inhibitory zones ≥ 11 mm.The antimicrobial efficacy was further quantified by determining the MIC values (Table 2).All the extracts were determined to be potent inhibitors     of C. perfringens growth, with MIC <1000 µg/mL for all extracts except the aqueous leaf extract.Even that extract had a relatively low MIC (1255 µg/mL), indicating antibacterial efficacy.The T. lanceolata berry extracts were more potent inhibitors than were the corresponding leaf extracts.Indeed, a MIC of 65 µg/mL was determined for the methanolic T. lanceolata berry extract.This is particularly noteworthy as it equates to a mass of less than 0.7 µg infused into the disc (compared with 2 and 10 µg for the penicillin and ampicillin controls respectively).All extracts were initially screened at 2000 µg/mL in the assay (Figure 2).For comparison, the reference toxin potassium dichromate (1000 µg/mL) was also tested in the bioassay.The potassium dichromate reference toxin was rapid in its onset, promoting nauplii death within the first 3 h of exposure with 100 % mortality evident following 4-5 h (unpublished results).Similarly, all the T. lanceolata extracts displayed significant mortality rates following 24 h exposure (>50%).
Pharmacognosy Journal, Vol 9, Issue 5, Sep-Oct, 2017  Continued... To further quantify the effect of toxin concentration on the induction of mortality, the extracts were serially diluted in artificial seawater to test across a range of concentrations in the Artemia nauplii bioassay.Table 2 shows the LC 50 values of the extracts towards A. franciscana.LC 50 values >1000 µg/mL towards Artemia nauplii have been defined as being nontoxic. 21Based on these results, all extracts tested were deemed nontoxic.
Optimised GC-MS parameters were developed and used to examine the T. lanceolata berry extracts.The resultant gas chromatograms are presented in Figure 3. Numerous overlapping peaks were evident in the aqueous berry extract chromatogram (Figure 3a).A total of 79 peaks were detected in the aqueous T. lanceolata berry extract, with the peak eluting at 20.9 min being the most prominent.Comparison with a phytochemical library putatively identified this peak as the sesquiterpenoid polygodial (Table 3).Numerous overlapping peaks were also evident throughout the chromatogram, with a broad range of retention times between 10-40 min.The presence of peaks throughout the chromatogram

DISCUSSION
Whilst all the T. lanceolata extracts screened displayed potent C. perfringens growth inhibitory activity, the berry extracts generally had greater efficacy than the corresponding leaf extracts.Therefore, the berry extracts were further analysed to determine their phytochemical composition.An examination of the GC-MS headspace metabolomics profile analysis of the aqueous, methanolic, and ethyl acetate berry extracts highlights several interesting compounds.An obvious feature was the number and diversity of terpenoids present in all extracts.The monoterpoid α-terpineol, as well as the sequiterterpenoids polygodial and caryophyllene oxide, was prevalent in all T. lanceolata berry extracts.Indeed, polygodial was the major compound detected in the methanolic and aqueous extracts (based on peak area).This agrees with previous studies which frequently cite polygodial as a major component in T. lanceolata berries (6).Indeed, some studies have reported that polygodial may account for nearly 40 % of commercial T. lanceolata essential oil components. 22he bacterial growth inhibitory activity of polygodial has been reported in several studies.Polygodial isolated from Warburgia salutaris was reported to be a potent inhibitor of the growth of a panel of bacteria. 23ndeed, MIC's of 12.5 µg/mL were reported for polygodial against Staphylococcus aureus and Bacillus subtilis in that study.Whilst less potent, the same study also reported good growth inhibition for polygodial against Staphylococcus epidermidis, Micrococcus luteus, Escherichia coli and Klebsiella pneumoniae, with MIC values generally ≤100 µg/mL.Other studies have reported little or no antibacterial activity for polygodial against limited panels of bacteria, although many of these studies only tested at relatively low concentrations (100 µg/mL)). [24]In contrast, more recent studies have demonstrated good bactericidal activity against both Gram-positive and Gram-negative bacteria. 25Antifungal efficacy and mechanistic studies of polygodial have been more definitive, with several publications highlighting its potent fungicidal activity. 24,26,27Polygoidal appears to exert its antifungal activity by several mechanisms.It nonspecifically disrupts/denatures fungal integral membrane proteins by functioning as a non-ionic surfactant. 25It also readily reacts with amino acids (especially cysteine and aromatic amino acids), resulting in further denaturation.As an additional antifungal mechanism, polygoidal may permeate cells by diffusing across the cell membrane.Once inside the cell, polygoidal interacts with various intracellular components and affects metabolic processes.It is possible that polygodial also interacts with bacterial cells in a similar way.The monoterpene α-terpineol was also a common component across all T. lanceolata extracts.It is generally believed that monoterpenoids have the most potent broad spectrum bacterial inhibitory activity of all terpenoids compounds, and that this activity is closely linked to their lipophilic character. 28A variety of monoterpenoids including α-terpineol have been shown to have potent antibacterial activity against a panel of Gram-positive and Gram-negative bacteria. 28,29The small, hydrophobic nature of monoterpenoids allow them to insert into cytosolic membranes, altering the fluidity and permeability of the membrane, thereby changing the conformation and function of membrane proteins.This consequently interrupts crucial cellular processes including the respiratory chain.Furthermore, the cytoplasmic membrane comprises a cellular barrier to protons and larger ions. 30Interestingly, bacteria respond to monoterpenoid exposure by modulating membrane fluidity. 31Despite this, the specific antimicrobial mechanisms of monoterpenoids are not yet fully understood.
Interestingly, the non-specific growth inhibitory mechanism of monoterpenoids is inherently difficult for bacteria to counteract/develop resistance to.Indeed, to the best of our knowledge, no bacteria have yet attest to the wide range of compounds of widely varying polarity extracted with water.
The methanolic berry extract GC-MS chromatogram (Figure 3b) was more complex than the aqueous extract chromatogram.Indeed, a total of 129 peaks were detected in this chromatogram, with major peaks at approximately 14.1, 15.1, 16.4, 19.1, 21.0, 31.3,31.8 and 35.0 min.As for the aqueous extract, polygodial (eluting at approximately 21 min) was a major component.In addition, a further major peak was evident in the methanolic extract at approximately 16.5 min.A comparison the phytochemical database putatively identified this peak as linalool (Table 3).This compound was also present in the aqueous extract, albeit at a much lower level.Numerous overlapping peaks were also evident throughout the chromatogram, many at retention times corresponding to peaks in the aqueous extract.This indicates that methanol and water extracted many similar components, although many of the lower polarity compounds appear to be more effectively extracted into methanol than water Fewer peaks were evident in the ethyl acetate extract chromatogram (Figure 3c).Indeed, only 61 unique mass signals were detected in the ethyl acetate extract.As for the other extracts, polygodial was detected in the ethyl acetate berry extract, albeit in substantially lower levels.The ethyl acetate chromatogram also had a major peak present at 19.7 min, which was putatively identified as 2-methyl-2-phenyl-oxirane. Whilst this compound was also detected in both the aqueous and methanolic extracts it was only present in relative abundance in the ethyl acetate extract.Multiple other peaks were also noted in the ethyl acetate extract, many corresponding to peaks in the aqueous and methanolic extracts.However, several peaks in this extract were at different retention times than seen for the more polar methanolic and aqueous extracts.

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The presence of a hydroxyl group further enhances the potency of the terpenoids.

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The position of the hydroxyl group also influences the growth inhibitory potency of the terpenoids.

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Alkyl substitutions reduce the surface tension, altering polarity and subsequently altering bacterial selectivity.

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The addition of an acetate moiety further enhances antibacterial efficacy.Our study demonstrates that T. lanceolata berry extracts contain a variety of different compounds which may contribute to the C. perfringens growth inhibitory activity.Furthermore, a comparison between the metabolomics profiles of the extracts has highlighted several compounds of interest.However, it is unlikely that any single molecule is solely responsible for the T. lanceolata berry C. perfringens growth inhibitory activity.Instead, it is more likely that several compounds contribute to this activity.Furthermore, it is possible that synergistic interactions between the various bioactive components may be potentiating the growth inhibitory activity of the individual components, increasing their efficacy.At the very least, the presence of numerous molecules with growth inhibitory activity indicates that these extracts are likely to function by pluripotent pathways.Further studies are warranted to test the activity of the phytochemical compounds, both individually and in combinations.

Figure 3 :
Figure 3: Head space gas chromatograms of 0.5 µL injections of T. lanceolata berry (a) aqueous, (b) methanolic and (c) ethyl acetate extracts.The extracts were dried and resuspended in methanol.Chromatography conditions were as described in the methods section.

Table 1 : The mass of dried extracted material, the concentration after resuspension in deionised water and qualitative phytochemical screen- ings of the T. lanceolata extracts. Extract Mass of Dried Extract (mg) Concentration of Resuspended Extract (mg/ mL) Total Phenolics Water Soluble Phenolics Water Insoluble Phenolics Cardiac Glycosides Saponins Triterpenes Phytosterols Alkaloids (Mayer Test) Alkaloids (Wagner Test) Flavonoids Tannins
+++ indicates a large response; ++ indicates a moderate response; + indicates a minor response; -indicates no response in the assay.BW = aqueous T. lanceolata berry extract; BM = methanolic T. lanceolata berry extract; BE = ethyl acetate T. lanceolata berry extract; LW = aqueous T. lanceolata leaf extract; LM = methanolic T. lanceolata leaf extract; LE = ethyl acetate T. lanceolata leaf extract.

Table 2 : Minimum inhibitory concentration (µg/mL) of the plant extracts and LC 50 values (µg/mL) in the Artemia nauplii bioassay.
Numbers indicate the mean MIC and LC 50 values of triplicate determinations.-indicatesno inhibition.BW = aqueous T. lanceolata berry extract; BM = methanolic T. lanceolata berry extract; BE = ethyl acetate T. lanceolata berry extract; LW= aqueous T. lanceolata leaf extract; LM = methanolic T. lanceolata leaf extract; LE = ethyl acetate T. lanceolata leaf extract; PC = potassium dichromate control; SW = artificial seawater negative control; ND = the indicated test was not performed.