Bacillus anthracis growth Inhibitory Properties of Australian Terminalia spp.: Putative Identification of low Polarity Volatile Components by GC-MS Headspace Analysis

Introduction: Anthrax is a severe acute disease caused by Bacillus anthracis infections. If untreated, it often results in mortality. Many Terminalia spp. have documented therapeutic properties as general antiseptics, inhibiting the growth of a wide variety of bacterial species. This study examines the ability of selected Australian Terminalia spp. extracts to inhibit B. anthracis growth. Methods: Solvent extracts were prepared from Terminalia carpen­ tariae and Terminalia grandiflora plant material and investigated by disc diffusion assay for the ability to inhibit the growth of an environmental strain of B. anthracis . Their MIC values were determined to quantify and compare their efficacies. Toxicity was determined using the Artemia franci­ scana nauplii bioassay. The most potent extracts were analysed by GC-MS headspace analysis. Results: T. carpentariae and T. grandiflora leaf, fruit and nut solvent extractions displayed good growth inhibitory activity against B. anthracis . Methanolic T. carpentariae leaf and T. grandiflora nut extracts were particularly potent growth inhibitors, with MIC values of 74 and 155 µg/mL respectively. The T. carpentariae leaf ethyl acetate extract was also a good inhibitor of B. anthracis growth (MIC 340 µg/mL). All other extracts were substantially less potent growth inhibitors. Interestingly, the T. carpentariae leaf extracts with growth inhibitory activity were nontoxic in the Artemia fransiscana bioassay, with LC 50 values >1000 µg/mL. In contrast, the LC 50 value 740 µg/mL reported for the methanolic T. grandiflora nut extract indicates low-moderate toxicity. Non-biased GC-MS phytochemical analysis of the most active extracts (methanolic T. carpentariae leaf and T. grandiflora nut) putatively identified and highlighted several compounds that may contribute to the ability of these extracts to inhibit the growth of B. anthracis . Conclusion: The growth inhibitory activity of the metha nolic T. carpentariae leaf and T. grandiflora nutextracts against B . anthracis indicates their potential for the treatment and prevention of anthrax. Furthermore, the lack toxicity of the T. carpentariae leaf and the low-moderate toxicity of the T. grandiflora nut extract, indicates that their use may extend to all forms of the disease (cutaneous, inhalation or gastrointestinal).


INTRODUCTION
Bacillus anthracis is a gram-positive, endospore-forming bacterium and is the etiological agent of the disease anthrax.The disease has extensive implications in the livestock industry through the infection of grazing animals, however it is perhaps most commonly associated with its use in bioterrorism. 1The most notable recent instance of weaponised anthrax occurred in 2001: B. anthracis spores were mailed to several locations in the U.S. and resulted in the infection of many people. 2However, inadvertent mass infections can be traced as far back as ancient Egypt and it is theorised that the plagues described in ancient literature may have been anthrax mass infections. 3Human anthrax is relatively rare compared to other vertebrates, and indeed outbreaks in both wildlife and livestock are a significant health and economic issue in many parts of the world. 4Anthrax infection in humans occurs when B. anthracis endospores enter the body through inhalation, ingestion or through abrasions in the skin. 5,6nce internalised, the body elicits an immune response, however the encapsulating endospore coating provides protection for the bacterium and can contribute to germination (via a process known as macrophageenhanced germination). 7The bacterium then resumes normal metabolic function and toxins are subsequently produced.Inhalation anthrax is the most dangerous of the three forms of the disease and infection via this pathway often results in death unless rapid treatment is administered.
Current strategies for the treatment of anthrax rely on the administration of both oral and intravenous antibiotics.Although vaccines have been available since the 19 th century, they must be administered prior to infection and are generally ineffective in the treatment of anthrax once infection has initiated. 8Whilst current antibiotic treatments are effective, due to the nature of antibiotics there is an inherent risk of B. anthracis conferring drug resistances and thus it is important to search for new antibiotics. 9Antibiotic therapy development may occur via the design and synthesis of new chemical agents, and also through the investigation and discovery of natural resources for use as antimicrobial agents.Furthermore, the development of novel anti-B.Anthracis products that could disinfect contaminated sites without the use of harsh chemicals offers an effective, safe alternative of decreasing the spread of the disease.Plants of the genus Terminalia have extensive therapeutic uses in multiple traditional healing systems, including uses for the prevention andtreatment of pathogenic diseases.Multiple studies have reported the antibacterial properties of Terminalia species used in traditional Indian medicine.Leaf and bark extracts of Terminalia arjuna have growth inhibitory activity against a wide panel of microbes. 10,11Terminalia chebula also has a tradition of use in Ayurveda for the treatment of numerous diseases and conditions [12][13][14] and has also been reported to display potent antibacterial activity against a microbial panel. 15Similarly, Terminalia alata, Terminalia bellirica and Terminalia catappa have also been reported to have broad spectrum antibacterial activity. 11Numerous African Terminalia species also have potent antibacterial activity.One study of the South East African species Terminalia stenostachya and Terminalia spinosa reported strong inhibitory activity against a broad spectrum of medicinally important bacteria including several Mycobacterium species, Streptococcus faecalis, Staphylococcus aureus, Vibrio cholera, Bacillus anthracis, Klebsiella pneumoniae, Salmonella typhi, Pseudomonas aeruginosa and Escherichia coli. 16The Southern African species Terminalia sericea and Terminalia pruinoides have similarly potent inhibitory activity against a broad panel of pathogenic 17 and food spoilage bacteria, 18 as well as against bacteria associated with autoimmune diseases. 19,20Terminalia brownii also has a history of usage in traditional eastern and central African medicinal systems, including usage for the treatment of diverse medicinal conditions including diarrhoea and gonorrhoea. 21,22Interestingly, a recent study also reported that the T. brownie was also a potent inhibitor of B. anthracis growth. 164][25][26] Other Terminalia spp.which are endemic to the tropical northern regions of Australia also have a history of traditional therapeutic usage to treat microbial infections. 27However, few studies have rigorously evaluated their therapeutic potential.This study screened two Australian Terminalia species (T.carpentariae and T. grandiflora) for the ability to inhibit B. anthracis growth.

Plant source and extraction
The Terminalia carpentariae leaf and Terminalia grandiflora fruit and nut (seed) plant materials used in this study were a kind gift from David Boehme of Northern Territory Wild Harvest.Voucher samples of all plant specimens have been stored at the School of Natural Sciences, Griffith University, Brisbane (Australia).The plant materials were comprehensively desiccated in a Sunbeam food dehydrator and dried materials stored at -30 o C for later use.Prior to usage, the materials were thawed and ground into a coarse powder.Individual 1 g quantities of the materials were weighed into individual tubes and 50 mL of deionised water, methanol, hexane, chloroform or ethyl acetate were added.All solvents were obtained from Ajax Australia and were AR grade.The deionised water was sterilised prior to use.The ground plant materials were individually extracted in each solvent for 24 h at 4 o C through gentle shaking.The extracts were then filtered through filter paper (Whatman No. 54) under vacuum, followed by drying by rotary evaporation in an Eppendorf concentrator 5301.The resultant extract was weighed and redissolved in 10 mL deionised water (containing 1% DMSO).

Antibacterial screening
Environmental Bacillus anthracis strain An environmental strain of Bacillus anthracis was isolated as previously described. 31All growth studies were performed using a modified peptone/yeast extract (PYE) agar: 1 g/L peptone, 1.5 g/L yeast extract, 7.5 g/L NaCl, 1 g/L ammonium persulfate, 2.4 g/L HEPES buffer (pH 7.5) and 16 g/L bacteriological agar when required.Incubation was at 30 o C and the stock culture was subcultured and maintained in PYE media at 4 o C. The media nutrient components were supplied by Oxoid Ltd, Australia.The GenBank accession number for the 16S rRNA gene sequence for the isolate is KR003287.

Evaluation of antimicrobial activity
Antimicrobial activity of all plant extracts was determined using a modified disc diffusion assay. 32,33Briefly, 100 µL of established Bacillus anthracis culture was grown in 10 mL of fresh PYE liquid media until it reached a count of ~10 8 cells/mL.A 100 µL volume of bacterial suspension was spread onto PYE agar plates.The extracts were tested for antibacterial activity using 5 mm sterilised filter paper discs.Discs were subsequently impregnated with 10 µL of the test extract, allowed to dry and placed onto inoculated plates.The plates were allowed to stand at 4 o C for 2 h before incubation at 30 o C for 24 h.The diameters of the zones of inhibition were measured in millimetres and all measurements were to rounded to the closest whole millimetre.Each assay was performed in triplicate.Mean values (± SEM) are reported in this study.Standard discs of ampicillin (10 µg) and penicillin (2 µg) were obtained from Oxoid Ltd, Australia and served as positive controls for antibacterial activity.Filter discs impregnated with 10 µL of distilled water were used as a negative control.

Minimum inhibitory concentration (MIC) determination
The minimum inhibitory concentration (MIC) of each extract was determined as previously described. 34Briefly, the plant extracts were diluted in deionised water and tested across a range of concentrations.Discs were impregnated with 10 µL of the test dilution, allowed to dry and placed onto inoculated plates.The assay was performed as described above and graphs of inhibition zones versus concentration were plotted for each extract.Linear regression was used to determine 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 was serially diluted in artificial seawater for use in the Artemia franciscana nauplii bioassay.

Artemia franciscana nauplii toxicity screening
6][37] Briefly, 400 µL of seawater containing ~43 (mean 43.2, n=155, SD 14.5) A. franciscana nauplii were added to wells of a 48 well plate and used for bioassay.A volume of 400 µL of each diluted plant extract or the reference toxin were transferred to the wells and incubated at 25 ± 1 o 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.Nauplii were considered dead if no movement of the appendages was observed within 10 sec.After 24 h all nauplii were sacrificed and counted to determine the total % mortality per well.The LC 50 with 95% confidence limits for each treatment was determined using probit analysis.

Non-targeted GC-MS head space analysis
Separation and quantification were performed using a Shimadzu GC-2010 plus (USA) linked to a Shimadzu MS TQ8040 (USA) mass selective detector system as previously described. 23The 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×0.25 mm id×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 fora 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.

Liquid extraction yields and qualitative phytochemical screening
Extraction of 1 g of the various dried Terminalia materials with the solvents yielded dried plant extracts ranging from 16 mg (T.grandiflora nut ethyl acetate extract) to 348 mg (T.carpentariae leaf methanolic extract) (Table 1).The leaf extracts generally gave relatively high yields of dried extracted material compared to the fruit and nut 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 that methanol and water extracted the greatest amount and widest range of phytochemicals (Table 1).These solvents extracted high levels of water soluble phenolics, moderate to high levels of tannins, as well as low levels of flavonoids and anthraquinones for all Terminalia samples tested.Saponins were also generally present in the methanolic and aqueous extracts, although the levels of this class of compound were more variable.The ethyl acetate extracts generally extracted similar but lower phytochemical profiles as the methanolic and aqueous extracts.In contrast, the chloroform and hexane extracts were devoid of detectable levels of all classes of phytochemical screened for.

Antimicrobial activity
To determine the ability of the crude plant extracts to inhibit the growth of B. anthracis, aliquots (10 µL) of each extract were screened using a disc diffusion assay.The bacterial growth was inhibited by 7 of the 14 extracts screened (50%) (Figure 1).T. carpentariae methanolic leaf extract was the most potent inhibitor of B. anthracis growth (as judged by zone of inhibition), with inhibition zones of 13 ± 0.6 mm.This compares favourably with the penicillin and ampicillin controls, with zones of inhibition of 8.3 ± 0.6 and 10.0 ± 0.7 respectively.The T. grandiflora methanolic nut and T. carpentariae methanolic leaf extracts both displayed good inhibition of B. anthracis growth, with ≥ 10 mm zones of inhibition.In general, the methanolic extracts were more potent inhibitors of B. anthracis growth than were their counterparts.The antimicrobial efficacy was further quantified by determining the MIC values (Table 2).Several of the extracts were effective at inhibiting microbial growth, with MIC values against B. anthracis substantially <1000 µg/mL (<10 µg impregnated in the disc).The methanolic T. grandiflora nut and T. carpentariae leaf extracts extracts were particularly potent, with MIC values of 155 and 74 µg/mL respectively (approximately 1.6 and 0.7 µg impregnated in the disc respectively).The T. carpentariae leaf ethyl acetate extract was also a potent inhibitor of B. anthracis growth (MIC value 340 µg/mL; 3.4 µg impregnated in the disc).The T. grandiflora fruit methanolic extract also had moderate growth inhibitory activity (MIC 3872 µg/mL).All other extracts were either unable to inhibit B. anthracis growth, or only displayed low inhibitory efficacy (MIC values >5000 µg/mL).

Quantification of toxicity
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.Potassium dichromate was rapid in its onset of mortality, inducing nauplii death within the first 3 h of exposure and 100% mortality was evident following 4-5 h (results not shown).Most of the extracts displayed >75 % mortality at 24 h.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.No LC 50 values are reported for the T. grandiflora nut chloroform, hexane and ethyl acetate extracts, nor for the T. carpentariae leaf hexane extract as <50 % mortality was seen for all concentrations tested.Significant toxicity was noted for the T. grandiflora nut, fruit and leaf methanolic extracts, with LC 50 values substantially <1000 µg/mL.All other extracts were determined to be nontoxic, with LC 50 values substantially greater than 1000 µg/mL following 24 h exposure.Extracts with an LC 50 of greater than 1000 µg/mL towards Artemia nauplii have been defined as being nontoxic. 38

Non-targeted GC-MS headspace analysis Australian Terminalia extracts
As the methanolic T. carpentariae leaf extract and the methanolic T. grandiflora nut extract had the most potent B. anthracis growth inhibitory efficacy (as determined by MIC; Table 2), they were deemed the most promising extracts for further phytochemical analysis.Optimised GC-MS parameters were developed and used to examine the phytochemical composition of these extracts.The resultant gas chromatograms for the methanolic T. carpentariae leaf extract and the methanolic T. grandiflora nut extract are presented in Figures 3 and Figure 4 respectively.Major peaks were evident in the methanolic T. carpentariae leaf extract at approximately 11.1, 12.9, 14.4, 17.0, 18.2 and 19.5 min (Figure 3).Several smaller peaks were also evident throughout all stages of the chromatograms.In total, 55 unique mass signals were noted for the methanolic T. carpentariae leaf extract (Table 3).Putative empirical formulas and identifications were achieved for 21 (38%) of these compounds by comparison with the database.The methanolic T. grandiflora nut extract was also a potent inhibitor of B. anthracis growth (as determined by MIC; Table 2) and was therefore also analysed by headspace GC-MS with comparison to a GC-MS spectral database.The resultant gas chromatogramis presented in Figure 4. Several major peaks were present at times which also corresponded to peaks in the T. carpentariae leaf extract chromatogram (11.1, 12.9, 14.4 and 19.5 min).Several smaller peaks were also evident throughout all stages of the chromatograms.In total, 28 unique mass signals were noted for the methanolic T. grandiflora nutextract (Table 4).Putative empirical formulas and identifications were achieved for 11 (39%) of these compounds.

DISCUSSION
Many Terminalia spp.have a history of therapeutic usage to treat microbial infections and numerous recent investigations have reported on their  0.12 0.21 The % area and % height is expressed as a % of the total area under all chromatographic peaks or % of the total height of all peaks respectively.Pharmacognosy Journal, Vol 8, Issue 3, May-Jun, 2016 The % area and % height is expressed as a % of the total area under all chromatographic peaks or % of the total height of all peaks respectively.antibacterial properties. 39Of the Australian species, T. ferdinandiana has been the most extensively studied.Several studies have reported it to be a potent antibacterial agent, with growth inhibitory activity reported against a broad panel of bacterial pathogens, 26 as well as against some bacterial triggers of rheumatoid arthritis 23,25 and multiple sclerosis. 24,25urthermore, T. ferdinandiana has also recently been reported to inhibit the proliferation of the gastrointestinal protozoan parasite Giardia duodenalis 40 indicating its therapeutic potential against both prokaryotic and eukaryotic pathogens.Interestingly, whilst inhibition of B. anthracis growth was not evaluated in any of the previous studies, one recent study reported potent growth inhibition of the related bacterial species B. cereus, with MIC values as low as approximately 100 µg/mL. 26B. cereus is very closely related to B. anthracis with >99% 16S rRNA gene sequence homology 41 and some bacterial taxonomonists believe that they should be classified as a single species under current standards (>97% 16S rRNA sequence homology).In contrast, other native Australian Terminalia spp.are less well studied.The methanolic T. carpentariae leaf and T. grandiflora nutextracts displayed the most potent B. anthracis growth inhibitory activity (MIC values of 74 and 155 µg/mL respectively) and were therefore analysed by qualitative GC-MS.A number of interesting compounds were identified in each of these extracts.Analysis of the methanolic T. carpentariae leaf extract putatively identified methyl N-hydroxybenzenecarboximidoate (Figure 5a), 1-octen-3-ol (Figure 5b), 5-hepten-2-one, 6-methyl-(Figure 5c), 2-tert-butoxyethanol (Figure 5d), 2-ethyl-1-hexanol (Figure 5e), dimethyl succinate (Figure 5f), isophorone (Figure 5g), α-citronellol (Figure 5h), nonanal (Figure 5i), 4-oxoisophorone (Figure 5j), ethyl benzoate (Figure 5k), methyl benzeneacetate (Figure 5l), α-terpineol (Figure 5m), 2-isopropylidene-3-methylhexa-3,5-dienal (Figure 5n), lauraldehyde (Figure 5o), 2,4-dimethyl-benzaldehyde (Figure 5p), 1,3-pentanediol, 2,2,4-trimethyl-, 1-isobutyrate (Figure 5q), 2,4-di-tert-butylphenol (Figure 5r), ethyl para-ethoxybenzoate (Figure 5s) and2,2,4-trimethyl-1,3pentanediol diisobutyrate (Figure 5t).The presence of the monoterpenoids α-citronellol and α-terpineol are particular interesting as many monoterpenoids have potent broad spectrum antibacterial activity 42 and therefore may contribute to the B. anthracis growth inhibition reported in our study.Interestingly, several monoterpenes have also been reported to suppress NF-κB signalling (the major regulator of inflammatory diseases). 43,44Thus, the terpene components may have a pleuripotent mechanism in blocking anthrax, by inhibiting the growth of the causative bacterium, as well as relieving the downstream inflammatory symptoms evident with the most common (cutaneous) form of the disease.Many of the same compounds detected in the methanolic T. carpentariae leaf extract were also putatively identified in the methanolic T. grandi-Pharmacognosy Journal, Vol 8, Issue 3, May-Jun, 2016 in the methanolic T. carpentariae leaf and T. grandiflora nut extract for a more complete understanding of the complete plant metabolome.Of note, the methanolic T. carpentariae leaf extract was determined to be nontoxic towards Artemia franciscana nauplii, with LC 50 values >1000 µg/mL.Extracts with LC 50 values >1000 µg/mL towards Artemia nauplii are defined as being nontoxic. 38This indicates that this extract may be safe for use for all forms of the disease (cutaneous, inhalation or gastrointestinal).In contrast, the methanolic T. grandiflora nut extract (which also was a potent inhibitor of B. anthracis growth) displayed toxicity towards Artemia nauplii, with an LC 50 value of 740 µg/mL.This represents low to moderate toxicity and indicates that this using human cell lines are required to further evaluate the safety of these extracts.Furthermore, whilst the results of our study are promising, it must be noted that the growth inhibitory studies screened against vegetative cells.As Bacillus spp.are spore formers, further studies are required to determine whether extracts with B. anthracis growth inhibitory activity also affect bacterial growth from the spores.

CONCLUSION
The B. anthracis growth inhibitory activity and low toxicity of the T. carpentariae and T. grandiflora extracts demonstrate their potential in the prevention and treatment of anthrax.Methanolic T. carpentariae leaf and T. grandiflora nut extracts were particularly potent growth inhibitors.
Further investigations aimed at the purification of the bioactive components are needed to assess the mechanisms of action of these agents.flora nut extract.In particular, methyl N-hydroxybenzenecarboximidoate, 2-(1,1-dimethylethoxy)-ethanol, 2-ethyl-1-hexanol, nonanal, ethyl benzoate, 1,3-pentanediol, 2,2,4-trimethyl-,1-isobutyrate, 2,4-di-tertbutylphenol and benzoic acid, 4-ethoxy-ethyl ester were also present in the methanolic T. grandiflora nut extract.GC-MS analysis also putatively identified 2-(1,1-dimethylethoxy)-ethanol (Figure 6a), caryophyllene (Figure 6b), 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (Figure 6c) and butyl octyl phthalate (Figure 6d) in the methanolic T. grandiflora nut extract.Previous studies have reported bacterial growth inhibitory activities for the sesquiterpenoid caryophyllene. 42It is likely that caryophyllene therefore contributes (at least in part) to the growth inhibitory activity of this extract.It is likely that other phytochemical classes also contribute to the growth inhibitory properties of these extracts.Our qualitative phytochemical screening studies indicate that polyphenolics, flavonoids, saponins, and tannins were present in the methanolic T. carpentariae leaf and T. grandiflora nut extracts.However, no compounds of these classes were identified by GC-MS headspace analysis.As GC-MS techniques generally only detect lower polarity compounds, many mid to higher polarity bioactive compounds may have been missed.Recent studies have reported the LC-MS profiles of extracts prepared from other Australian Terminalia spp. 23- 25,40Several features were common to all of these studies.In particular, all of these studies reported on the diversity of tannins in the Terminalia extracts.This is noteworthy as tannins have potent growth inhibitory activity against a broad spectrum of bacterial species. 39Recent studies have also highlighted the stilbene components in extracts prepared from different Australian Terminalia spp. 24,25Resveratrol and the glycosylated resveratrol derivative piceid, and several combretastatins were putatively identified in those studies.Stilbenes have attracted much recent interest due to their reported potent ability to of some compounds to block cancer cell progression and induce apoptosis by binding intracellular tubulin, thereby disrupting microtubule formation. 45Further studies utilising LC-MS are required to identify the mid to higher polarity compounds

Figure 1 :
Figure 1: Growth inhibitory activity of Terminalia spp.extracts against the B. anthracis environmental isolate measured as zones of inhibition (mm).N=nut; F=fruit; L=leaf; W=aqueous extract M=methanolic extract; C=chloroform extract; H=hexane extract; E=ethyl acetate extract.Results are expressed as mean zones of inhibition ± SEM.

Figure 4 :
Figure 4: GC headspace chromatograms of 0.5 µL injection of T. grandiflora nut methanolic extract.The extract was dried and resuspended in methanol for analysis.

Figure 3 :
Figure 3: GC headspace chromatograms of 0.5 µL injection of T. carpentariae leaf methanolic extract.The extract was dried and resuspended in methanol for analysis.

Table 1 : The mass of dried extracted material, the concentration after resuspension in deionised water and qualitative phytochemical screenings of the Terminalia extracts. W = aqueous extract M = methanolic extract; C = chloroform extract; H = hexane extract; E = ethyl acetate extract
+++ indicates a large response; ++ indicates a moderate response; + indicates a minor response; -indicates no response in the assay.

Table 2 : Minimum inhibitory concentration (µg/mL) of the plant extracts and LC 50 values (µg/mL) in the Artemia nauplii bioassay. W = aqueous extract M = methanolic extract; C = chloroform extract; H = hexane extract; E = ethyl acetate extract Species Part Extract MIC (µg/mL) LC50 (µg/mL)
Numbers indicate the mean MIC and LC 50 values of triplicate determinations.indicatesno bacterial growth inhibition was evident, or that an LC 50 value could not be obtained as the mortality did not reach 50 % for any dose tested.