Patent Publication Number: US-2021169959-A1

Title: Terminalia ferdinandiana extract and products containing extract of terminalia ferdinandiana for antimicrobial or antibacterial applications

Description:
FIELD OF THE INVENTION 
     The present invention relates to natural extracts and/or derivatives of  Terminalia ferdinandiana  ( T. ferdinandiana ). 
     The present invention particularly, though not solely, utilises extracts and/or derivatives of  Terminalia ferdinandiana  leaf. 
     The present invention finds application in antibacterial or antimicrobial products or uses, such as for use in treating infections in humans and animals. 
     BACKGROUND TO THE INVENTION 
     Hereinafter,  Terminalia ferdinandiana  may be referred to as  T. ferdinandiana  for ease of reference. 
       T. ferdinandiana  is a small, deciduous tree which grows wild extensively throughout the subtropical woodlands of northern tracts of Australia, typically in the Northern Territory and Western Australia. 
       T. ferdinandiana  bears an abundant crop of small plum-like fruits. The fruit is known to have very high vitamin C content, and is a source of antioxidants, folic acid and iron. The fruit and extracts of the fruit are used in foods, dietary supplements and pharmaceuticals. 
     The commonest use of  T. ferdinandiana  fruits is for gourmet jams, sauces, juices, ice-cream, cosmetics, flavours and pharmaceuticals. 
     Examples of cosmetic vehicles for the  T. ferdinandiana  fruit extract have been proposed in European patent document EP 1581513. Another patent document U.S. Pat. No. 7,175,862 discloses a method of producing a powder containing ascorbic acid (vitamin C), antioxidants and phytochemicals from the fruit of the  T. ferdinandiana  plant. U.S. Pat. No. 7,175,862 mentions use of the powdered  T. ferdinandiana  fruit for the reduction of free radicals in the human body. 
       T. ferdinandiana  fruit is also known for having antimicrobial properties. As a native fruit of northern Australia, the fruit has a long history of use by indigenous Australians as a food and a medicinal agent. The fruit was eaten during long hunting trips by indigenous Australians as a source of high nutrition food. The medicinal properties of  T. ferdinandiana  have not been well understood or fully evaluated. 
     A study by I. E. Cock and S. Mohanty reporting on an evaluation of the antimicrobial properties of  T. ferdinandiana  fruit pulp was published in the Pharmacgnosy Journal 2011 [vol 3 I issue 20]. That study focussed on the bacterial growth inhibitory potential of  T. ferdinandiana  fruit pulp and recognised that further studies were needed to examine other medicinally important bioactivities of  T. ferdinandiana  fruit. 
     Despite reported growth inhibitory activity of fruit, numerous pathogens are yet to be evaluated for the ability to inhibit their growth. 
     In particular, the antibacterial properties of leaf extracts of  T. ferdinandiana  remain unrealised. 
     Many bacteria can infect humans and animals. Some bacteria, such as  Clostridium perfringens , are anaerobically active. Other bacteria, such as  Bacillus anthracis , are aerobically or anaerobically active. 
       Clostridium perfringens  causes myonecrosis, a condition of necrotic damage, specific to muscle tissue. It is often seen in infections with  C. perfringens  or any of myriad soil-borne anaerobic bacteria. Bacteria cause myonecrosis by specific exotoxins. These microorganisms are opportunistic and, in general, enter the body through significant skin breakage. Gangrenous infection by soil-borne bacteria was common in the combat injuries of soldiers well into the 20th century, because of non-sterile field surgery and the basic nature of care for severe projectile wounds. 
       Clostridium perfringens  ( C. perfringens ) is an endospore-forming, gram-positive bacterium and the etiological agent of various diseases, including clostridial myonecrosis and enteritis necroticans. 
     The  C. perfringens  bacterium grows strictly anaerobically (although it is aero-tolerant) and is found ubiquitously in the environment as part of the natural microbial flora. The bacterium is often also present in the digestive tract of humans and other vertebrates. 
     Under stresses, such as harsh environmental surroundings or when deprived of necessary nutrients,  C. perfringens  can produce endospores that place it in a metabolically dormant state as a defence mechanism until conditions are once again favourable for cellular proliferation. 
     The environmental robustness of the  C. perfringens  bacterium has significant clinical implications and under anoxic conditions is responsible for a wide variety of diseases, some of which are highly fatal. 
       Clostridial myonecrosis  (or gas gangrene) is a rapidly progressive, highly lethal infection of the skeletal muscle caused by several exotoxin-producing  Clostridium  species. Though it is caused by a number of species within the  Clostridium  genus (including  C. septicum, C. histolyticum  or  C. novyi ), the predominant cause of gas gangrene is through  C. perfringens,  which is estimated to be the causative agent in the greatest proportion of documented cases (said to be between 80% and 90% of such cases). 
     The  C. perfringens  bacterium is reliant on anaerobic conditions and thus infection occurs primarily in deep tissues, either as a result trauma or post-surgery. Associated exotoxins are subsequently produced and these necrotize the surrounding tissue, resulting in muscular degradation. Unless prompt treatment is administered, later symptoms may include acute renal failure, shock, coma and ultimately death. 
     Current strategies in the treatment of  C. perfringens  induced gas gangrene involve a combination of both antibiotic therapy and aggressive surgical debridement. 
     Without prompt treatment, gas gangrene is highly fatal and thus the removal of necrotized tissues is often necessary to reduce the chance of host death. In recent times there has been an emphasis on producing an effective vaccine, however this is viewed more as a preventative measure than as a curative therapy and thus has no use once infection has initiated. Furthermore, the sporadic, opportunistic nature of the pathogen results in difficulty in predicting who should receive the vaccination. 
     Thus, the primary method of treatment for gas gangrene currently involves the administration of a combination of penicillin and clindamycin as soon as the infection is detected. Although the bacterium has remained relatively susceptible to antibiotics, reports of antibiotic resistant  C. perfringens  have emerged and thus there is an ever-increasing need to discover and develop alternative chemotherapeutic options for the treatment of gas gangrene. 
     Zoonotic infections are diseases that can be transmitted indirectly or directly between humans and animals and are a significant burden from both health and economic standpoints. Such diseases can be spread to humans from both domesticated and wild animals and can be transferred through direct contact, the contamination of drinking water by animal secretions, or the consumption of contaminated meat products. These diseases pose an exceptional set of problems in the control and treatment of infections, as the traditionally effective strategies of herd immunity and isolation of infected individuals are not feasible. 
     Furthermore, unlike humans who can verbalise otherwise indistinguishable symptoms, infected animals may go unnoticed and further contribute to the spread of disease. From 1940 to 2004, it is thought that approximately 60% of all emerging infectious diseases were of a zoonotic nature with the majority originating in wildlife. Therefore, the development of cross species treatments plays a key role in the effective control and eradication of zoonotic diseases. 
     By way of example,  Bacillus anthracis  ( B. anthracis ), the etiological agent of anthrax, is a sporulating gram-positive bacterium found predominately in soils. Similar to other organisms within the  Bacillus  genus,  B. anthracis  is capable of producing endospores that can remain dormant for several years until conditions are again favourable for growth. These spores are metabolically inactive and are capable of surviving environmental conditions that would kill vegetative cells, including temperature, desiccation and enzymatic destruction. 
     Four forms of human anthrax disease are recognized based on their portal of entry to the human body: 1. Cutaneous, the most common form (95%), causes a localized, inflammatory, black, necrotic lesion (eschar); 2. Inhalation, a rare but highly fatal form, is characterized by flu like symptoms, chest discomfort, diaphoresis, and body aches; 3. Gastrointestinal, a rare but also fatal (causes death to 25%) type, results from ingestion of anthrax spores. Symptoms include: fever and chills, swelling of neck, painful swallowing, hoarseness, nausea and vomiting (especially bloody vomiting), diarrhea, flushing and red eyes, and swelling of abdomen; 4. Injection, symptoms are similar to those of cutaneous anthrax, but injection anthrax can spread throughout the body faster and can be harder to recognize and treat compared to cutaneous anthrax. 
     Although the vegetative  B. anthracis  cells produce the toxins associated with the disease, infection is generally initiated when spores are introduced into a host through inhalation, ingestion or via direct contact with open wounds. Once internalised, the spores revert to viable cells, proliferate and begin producing the deadly anthrax toxins. 
     The disease has been controlled to varying degrees internationally through careful monitoring and strong eradication measures. However, anthrax is endemic worldwide and is often fatal if infection occurs. 
     Current strategies in the treatment of anthrax typically involve a combination of antibiotic therapies to fight infection, as well as supportive care to manage associated symptoms. 
     The administration of intravenous or oral antibiotics are generally effective in the management of anthrax, however there is always an inherent risk that the bacteria may develop drug resistance. As such, the discovery of new drugs is of significant importance, either through the design and synthesis of new compounds, or through the investigation of antimicrobials within pre-existing natural assets. 
     Giardiasis is a major cause of infectious diarrhoea in humans and livestock worldwide. Giardiasis is caused by gastrointestinal infections of protozoal parasites of the genus  Giardia.  There is a limited range of drugs available for chemotherapeutic treatment of this disease, and they are only used after clinical diagnosis and not for prophylaxis. 
     The majority of these drugs are ineffective against some life stages of the  Giardia protozoa,  are toxic, have unpleasant side effects and may have limited availability in developing countries. Treatment failure and parasite resistance highlight the importance to develop new chemotherapeutic treatments for giardiasis with greater efficacy and less severe side effects. 
     Treatment of giardiasis using natural plant derived compounds is an attractive prospect as the medicinal qualities of plants can be very efficacious. 
     The antimicrobial effects of medicinal plants have long been recognised by many cultures and phytochemical analysis to identify the active compounds offers promise in the development of new antimicrobial agents, such as for treatment of giardiasis and anti- B. anthracis  agents. 
     Thus, the development of natural assets provides great potential in the discovery of compounds effective in managing disease causing microbes, such as bacteria causing anthrax, giardiasis or clostridium. 
     It is with such aforementioned bacteria and bacterial infections in mind that the present invention has been developed. 
     SUMMARY OF THE INVENTION 
     According to one or more forms of the present invention and methods/tests assessing  T. ferdinandiana  fruit and leaf extracts, it has been realised that products containing  T. ferdinandiana  leaf extract are efficacious in inhibiting the growth of microbes, such as bacteria, e.g. the gram-positive anaerobic bacterium  Clostridium perfringens  ( C. perfringens ) or  Bacillus anthracis  ( B. anthracis ) or the genus  Giardia,  such as  Giardia duodenalis.    
     An aspect of the present invention provides an extract of  Terminalia ferdinandiana  ( T. ferdinandiana ) for use in a medicament for treatment of microbial or bacterial infection in humans or animals. 
     A further aspect of the present invention provides a medicament including extract of  Terminalia ferdinadiana  ( T. ferdinandiana ). 
     Preferably, the extract includes extract of  T. ferdinandiana  leaf. 
     A composition for use in a medicament for use in treating microbial or bacterial infection in humans or animals, the medicament containing an extract derived from  Terminalia ferdinandiana  ( T. ferdinandiana ) leaf as an antibacterial agent. 
     The medicament or composition may be provided for use as one or more pills, tablets, capsules or in liquid form. 
     The medicament may include an extract of  T. ferdinandiana  fruit in addition to the extract of  T. ferdinandiana  leaf. 
     Preferably the  T. ferdinandiana  leaf extract includes one or more of a methanolic/ethanolic extract, aqueous extract, ethyl acetate extract, chloroform extract or hexane extract. 
     The  T. ferdinandiana  leaf extract may include a proportion of at least one antioxidant. 
     The at least one antioxidant may include one or more of an ellagic acid or trimethyl ellagic acid. 
     The extract, composition or medicament provided as an antimicrobial agent for use n treating bacterial infection in humans or animals. 
     Preferably the extract, medicament or composition is provided in pill form, capsule form, or as a liquid, including the extract of  T. ferdinandiana  leaf. 
     The extract, medicament or composition may include at least one tannin and/or at least one flavone. 
     The extract, medicament or composition may include one of or a combination of two or more of, chebulic acid, corilagen, chebulinic acid and chebulagic acid. 
     The extract, medicament or composition may include at least one flavone or flavinoid. 
     The extract, medicament or composition may include one or more antioxidants. The at least one antioxidant may include an ellagic acid. The ellagic may include ellagic acid dehydrate and/or trimethyl ellagic acid. 
     A further aspect of the present invention provides an antimicrobial composition containing an extract derived from  Terminalia ferdinandiana  ( T. ferdinandiana ) leaf. 
     Preferably, the extract or medicament is provided in a medicament for use in treating  B. anthracis  or  C. perfringens  or  Giardia  infection in humans or animals. 
     A further aspect of the present invention provides for use of an extract of  Terminalia ferdinandiana  ( T. ferdinandiana ) in the preparation of a medicament or composition for use in treating microbial or bacterial infection in humans or animals. The use may include the extract including  T. ferdinandiana  leaf extract. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       One or more embodiments of the present invention will hereinafter be described with reference to the accompanying Figures and Tables, in which: 
         FIG. 1  shows a chart of growth inhibitory activity of  T. ferdinandiana  fruit and leaf plant extracts against the  C. perfringens  environmental isolate measured as zones of inhibition (mm). 
         FIG. 2  shows a chart of the lethality of the Australian plant extracts (2000 μg/mL) and the potassium dichromate control (1000 μg/mL) towards  Artemia franciscana  nauplii after 24 h exposure. 
         FIG. 3 a    shows positive and  FIG. 3 b    negative ion RP-HPLC total compound chromatograms (TCC) of 2 μl injections of  T. ferdinandiana  leaf methanolic extract. 
         FIG. 4 a    shows positive and  FIG. 4 b    negative ion RP-HPLC total compound chromatograms (TCC) of 2 μl injections of  T. ferdinandiana  leaf ethyl acetate extract. 
         FIG. 5  shows chemical structures of  T. ferdinandiana  leaf tannin compounds detected in the methanolic and/or ethyl acetate extracts: (a) chebulic acid; (b) protocatechuic acid; (c) ellagic acid dihydrate; (d) punicalagin; (e) ellagic acid; (f) chebulagic acid; (g) castalagin; (h) corilagin; (i) punicalin; (j) chebulinic acid; (k) punicalin; (l-m) trimethylellagic acid isomers. 
         FIG. 6  shows a chart of growth inhibitory activity of  T. ferdinandiana  plant extracts against the  B. anthracis  environmental isolate measured as zones of inhibition (mm). FW=aqueous  T. ferdinandiana  fruit extract; FM=methanolic  T. ferdinandiana  fruit extract; FC=chloroform  T. ferdinandiana  fruit extract; FH=hexane  T. ferdinandiana  fruit extract; FE=ethyl acetate  T. ferdinandiana  fruit extract; LW=aqueous  T. ferdinandiana  leaf extract; LM=methanolic  T. ferdinandiana  leaf extract; LC=chloroform  T. ferdinandiana  leaf extract; LH=hexane  T. ferdinandiana  leaf extract; LE=ethyl acetate  T. ferdinandiana  leaf extract; PC=penicillin (2 μg); AMP=ampicillin (10 μg). Results are expressed as mean zones of inhibition±SEM. 
         FIG. 7  shows a chart of the lethality of the  T. ferdinandiana  fruit and leaf plant extracts (2000 pg/mL) and the potassium dichromate control (1000 μg/mL) towards  Artemia franciscana nauplii  after 24 hour exposure. FW=aqueous  T. ferdinandiana  fruit extract; FM=methanolic  T. ferdinandiana  fruit extract; FC=chloroform  T. ferdinandiana  fruit extract; FH=hexane  T. ferdinandiana  fruit extract; FE=ethyl acetate  T. ferdinandiana  fruit extract; LW=aqueous  T. ferdinandiana  leaf extract; LM=methanolic  T. ferdinandiana  leaf extract; LC=chloroform  T. ferdinandiana  leaf extract; LH=hexane  T. ferdinandiana  leaf extract; LE=ethyl acetate  T. ferdinandiana  leaf extract; PC=potassium dichromate control; SW=seawater control. Results are expressed as mean % mortality±SEM. 
         FIG. 8  shows a chart representing a head space gas chromatogram of 0.5 μL injections of  T. ferdinandiana  ethyl acetate fruit extract. The extract were dried and resuspended in methanol for analysis. 
         FIG. 9  shows a chart representing a head space gas chromatogram of 0.5 μL injections of methanolic  T. ferdinandiana  leaf extract. The extract were dried and resuspended in methanol for analysis. 
         FIGS. 10 a  to 10 n    show examples of compounds present in the leaf and fruit extracts, such as one or more furans and/or tannins. 
         FIGS. 11 a  to 11 k    show examples of compounds present in  T. ferdinandiana  leaf with properties consistent with anti-giardial activity according to at least one embodiment of the present invention. 
         FIG. 12  shows inhibitory activity of the  T. ferdinandiana  extracts and pure compounds against three strains of  Giardia duodenalis  trophozoites measured as a percentage the untreated control. 
         FIGS. 13 a  to 13 c    show Isobolograms for combinations of gallic acid and ascorbic acid tested at various ratios against (a) the sheep S2, (b) reference metronidazole sensitive (ATCC203333) and (c) reference metronidazole resistant (ATCC PRA-251)  G. duodenalis  strains. 
         FIGS. 14 a  to 14 c    show isobologramss of the association between the growth inhibitory activity and the DPGA axis. 
     
    
    
     DESCRIPTION OF PREFERRED EMBODIMENT 
     One or more methods for obtaining extract(s) and/or derivatives of  T. ferdinandiana  for one or more embodiments of the present invention will hereinafter be described. However, it is to be understood and appreciated that the generality of the present invention is not to be limited by the specific scope of the following specific description. 
     Solvent extracts and aqueous extracts were prepared using the fruit and the leaf of T. ferdinandiana. 
       Clostridium Perfringens  ( C. Perfringens ) 
       T. ferdinandiana  fruit and leaf solvent and aqueous extracts were investigated for growth inhibitory activity by disc diffusion assay against a clinical strain of  C. perfringens.    
     Their minimum inhibitory concentration (MIC) values were determined to quantify and compare their efficacies. 
     Toxicity was determined using the  Artemia franciscana nauplii  bioassay. Active extracts were analysed by non-targeted High Performance Liquid Chromatography-Quadrupole Time-of-Flight (HPLC-QTOF) mass spectroscopy (with screening against 3 compound databases) for the identification and characterisation of individual components in the crude  T. ferdinandiana  fruit and leaf extracts. 
     Methanolic and aqueous  T. ferdinandiana  fruit and leaf extracts, as well as the leaf ethyl acetate extract, displayed growth inhibitory activity in the disc diffusion assay against  C. perfringens.    
     The leaf extracts were generally more potent growth inhibitors than the corresponding fruit extracts, although the aqueous fruit extract had substantially greater efficacy than the aqueous leaf extract. 
     The methanolic and ethyl acetate leaf extracts were particularly potent growth inhibitors, with MIC values of 206 and 117 μg/ml respectively. 
     The fruit methanolic extract also displayed good efficacy, with an MIC of 716 μg/ml. I 
     In contrast, the chloroform and hexane extracts of both fruit and leaf were completely devoid of growth inhibitory activity. 
     All  T. ferdinandiana  extracts were either nontoxic or of low toxicity in the  Artemia fransiscana  bioassay. Non-biased phytochemical analysis of the methanolic and ethyl acetate leaf extracts revealed the presence of high relative levels of a diversity of gallo- and ellagi-tannins. 
     The low toxicity of the  T. ferdinandiana  extracts and the potent growth inhibitory bioactivity of the leaf methanolic and ethyl acetate extracts against  C. perfringens  indicates their potential as medicinal agents in the treatment and prevention of clostridial myonecrosis and enteritis necroticans. Metabolomic profiling studies indicate that these extracts contained a diversity of tannins. 
     Plant source and extraction:  T. ferdinandiana  fruit, leaves and pulp were obtained. The pulp was frozen prior to transport and kept at −10° C. until processed. The leaves were extensively dehydrated in a dehydrator and the desiccated material was stored at −30° C. The fruit and leaf materials were thoroughly dried and ground into a coarse powder prior to use. A mass of 1g of ground powder was extensively extracted in 50 mL of either de-ionised water, methanol, chloroform, hexane or ethyl acetate for 24 h at 4° C. with gentle agitation. The extracts were filtered through filter paper (Whatman No. 54) and air dried at room temperature. The aqueous extract was lyophilised by rotary evaporation in a concentrator. The resultant pellets were dissolved in 10 mL deionised water (containing 0.5% DMSO). The extract was passed through a 0.22 μm filter (Sarstedt) and stored at 4° C. until used. 
     Qualitative phytochemical studies: Phytochemical analysis of the extracts for the presence of triterpenoids, tannins, saponins, phytosteroids, phenolic compounds, flavonoids, cardiac glycosides, anthraquinones and alkaloids were conducted by previously described assays. 
     Antioxidant capacity: The antioxidant capacity of each sample was assessed using a modified 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method. Ascorbic acid (0-25 μg per well) was used as a reference and the absorbance was measured and recorded at 515 nm. All tests were completed alongside controls on each plate and all were performed in triplicate. The antioxidant capacity based on DPPH free radical scavenging ability was determined for each extract and expressed as pg ascorbic acid equivalents per gram of original plant material extracted. 
     Antibacterial screening: Clinical  Clostridium perfringens  screening: A clinical strain of  C. perfringens  was obtained. 
     Evaluation of antimicrobial activity: Antimicrobial activity of all of the leaf and fruit  T. ferdinandiana  plant extracts was determined using a modified disc diffusion assay. Briefly, 100 μL of  C. perfringens  was grown in 10 mL of fresh thioglycollate media until they reached a count of ˜10 8  cells/mL. A volume of 100 μL of the bacterial suspension was spread onto nutrient agar plates and extracts were tested for antibacterial activity using 6 mm sterilised filter paper discs. Discs were impregnated with 10 μL of  T. ferdinandiana  extracts, allowed to dry and placed onto the inoculated plates. The plates were allowed to stand at 4° C. for 2 hours before incubation at 30° C. for 24 hours. The diameters of the inhibition zones were measured to the closest whole millimetre. Each assay was performed in at least triplicate. Mean values (±SEM) are reported herein. Standard discs of penicillin (2 μg) and ampicillin (10 μg) were obtained and used as positive controls to compare 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 concentrations (MIC) of the extracts was determined as previously described. Briefly, the  T. ferdinandiana  fruit and leaf plant extracts were diluted in deionised water and tested across a range of concentrations. Discs were impregnated 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 determine MIC values. 
     Toxicity screening: Reference toxin for toxicity screening: Potassium dichromate (K 2 Cr 2 O 7 ) was prepared in distilled water (4 mg/mL) 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  Artermia franciscana nauplii  lethality assay. 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 immediately used in the bioassay. Volumes of 400 μL of reference toxin or the diluted plant extracts 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 deemed dead if no movement of the appendages was detected within 10 seconds. After 24 h, all nauplii were sacrificed and counted to determine the total % mortality per well. The LC50 with 95% confidence limits for each treatment was calculated using probit analysis. 
     Non-targeted HPLC-MS QTOF analysis: For chromatographic separations, 2 μL of sample was injected onto an HPLC system fitted with a column (2.1×100 mm, 1.8 μm particle size). The mobile phases consisted of (A) ultrapure water and (B) 95:5 acetonitrile/water at a flow rate of 0.7 mL/min. Both mobile phases were modified with 0.1% (v/v) glacial acetic acid for mass spectrometry analysis in positive mode and with 5 mM ammonium acetate for analysis in negative mode. The chromatographic conditions utilised for the study consisted of the first 5 min run isocratically at 5% B, a gradient of (B) from 5% to 100% was applied from 5 min to 30 min, followed by 3 min isocratically at 100%. Mass spectrometry analysis was performed on a quadrapole time-of-flight mass spectrometer (QTOF MS) fitted with an electrospray ionisation source in both positive and negative mode. 
     Data was analysed using known qualitative analysis software. Blanks using each of the solvent extraction systems were analysed using the ‘Find by Molecular Feature’ algorithm in the software package to generate a compound list of molecules with abundances greater than 10,000 counts. This was then used as an exclusion list to eliminate background contaminant compounds from the analysis of the extracts. Each extract was then analysed using the same parameters using the ‘Find by Molecular Feature’ function to generate a putative list of compounds in the extracts. Compound lists were then screened against three accurate mass databases; a database of known plant compounds of therapeutic importance generated specifically for this study (800 compounds); a known metabolomics database (24,768 compounds); and a known forensic toxicology database (7,509 compounds). Empirical formula for unidentified compounds was determined using the Find Formula function in the software package. 
     Statistical analysis: Data is expressed as the mean±SEM of at least three independent experiments. 
     Liquid extraction yields and qualitative phytochemical screening 
       T. ferdinandiana  plant extractions (1 g) with various solvents yielded dried plant extracts ranging from 18 mg to 483 mg (fruit extracts) and 58 mg to 471 mg (leaf extracts) (see Table 1). 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Table 1: The mass of dried extracted material, the concentration after re-suspension in deionised water, 
               
               
                 qualitative phytochemical screenings and antioxidant capacities of the  T. ferdinandiana  extracts: 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Concentration 
                 Antioxidant Capacity 
                   
                   
                   
                   
                   
               
               
                   
                 Mass of Dried 
                 of Resuspended 
                 (mg Ascorbic Acid 
                 Total 
                 Water Soluble 
                 Water Insoluble 
                 Cardiac 
               
               
                 Extract 
                 Extract (mg) 
                 Extract (mg/mL) 
                 Equivalency) 
                 Phenolics 
                 Phenolics 
                 Phenolics 
                 Glycosides 
                 Saponins 
               
               
                   
               
               
                 KFW 
                 483 
                 48.3 
                 264 
                 +++ 
                 +++ 
                 +++ 
                 − 
                 + 
               
               
                 KFM 
                 359 
                 35.9 
                 660 
                 +++ 
                 +++ 
                 +++ 
                 − 
                 ++ 
               
               
                 KFC 
                 62 
                 6.2 
                 7 
                 + 
                 − 
                 − 
                 − 
                 − 
               
               
                 KFH 
                 18 
                 1.8 
                 1 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                 KFE 
                 30 
                 3 
                 39 
                 ++ 
                 ++ 
                 + 
                 − 
                 + 
               
               
                 KLW 
                 471 
                 47.1 
                 340 
                 +++ 
                 +++ 
                 +++ 
                 ++ 
                 +++ 
               
               
                 KLM 
                 331 
                 33.1 
                 150 
                 +++ 
                 +++ 
                 +++ 
                 +++ 
                 ++ 
               
               
                 KLC 
                 59 
                 5.9 
                 5 
                 + 
                 − 
                 − 
                 − 
                 − 
               
               
                 KLH 
                 58 
                 5.8 
                 0.4 
                 + 
                 − 
                 − 
                 − 
                 − 
               
               
                 KLE 
                 59 
                 5.9 
                 22 
                 +++ 
                 +++ 
                 +++ 
                 − 
                 − 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Alkaloids 
                 Alkaloids 
                   
                   
                 Free 
                 Combined 
               
               
                   
                 Extract 
                 Triterpenes 
                 Phytosteroids 
                 (Mayer Test) 
                 (Wagner Test) 
                 Flavonoids 
                 Tannins 
                 Anthraquinones 
                 Anthraquinones 
               
               
                   
                   
               
               
                   
                 KFW 
                 − 
                 − 
                 − 
                 − 
                 +++ 
                 ++ 
                 − 
                 − 
               
               
                   
                 KFM 
                 + 
                 − 
                 + 
                 + 
                 +++ 
                 ++ 
                 − 
                 − 
               
               
                   
                 KFC 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 KFH 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 KFE 
                 ++ 
                 − 
                 − 
                 − 
                 ++ 
                 − 
                 − 
                 − 
               
               
                   
                 KLW 
                 ++ 
                 − 
                 − 
                 − 
                 ++ 
                 +++ 
                 + 
                 + 
               
               
                   
                 KLM 
                 + 
                 − 
                 + 
                 + 
                 ++ 
                 +++ 
                 + 
                 + 
               
               
                   
                 KLC 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 KLH 
                 − 
                 − 
                 − 
                 − 
                 ++ 
                 + 
                 − 
                 − 
               
               
                   
                 KLE 
                 − 
                 − 
                 − 
                 − 
                 ++ 
                 ++ 
                 − 
                 − 
               
               
                   
                   
               
            
           
         
       
     
     In Table 1 above, + + + indicates a large response; + + indicates a moderate response; + indicates a minor response; − indicates no response in the assay. KFW=aqueous  T. ferdinandiana  fruit extract; KFM=methanolic  T. ferdinandiana  fruit extract; KFC=chloroform  T. ferdinandiana  fruit extract; KFH=hexane  T. ferdinandiana  fruit extract; KFE=ethyl acetate  T. ferdinandiana  fruit extract; KLW=aqueous  T. ferdinandiana  leaf extract; KLM=methanolic  T. ferdinandiana  leaf extract; KLC=chloroform  T. ferdinandiana  leaf extract; KLH=hexane  T. ferdinandiana  leaf extract; KLE=ethyl acetate  T. ferdinandiana  leaf extract. Antioxidant capacity was determined by DPPH reduction and is expressed as mg ascorbic acid equivalence per g plant material extracted. 
     Aqueous and methanolic extracts provided significantly greater yields of extracted material relative to the chloroform, ethyl acetate and hexane counterparts, which gave low to moderate yields. The dried extracts were resuspended in 10 mL of deionised water (containing 1% DMSO), resulting in the concentrations presented in Table 1. 
     Antioxidant content: Antioxidant capacity for the plant extracts (Table 1) ranged from 0.4 mg (hexane leaf extract) to a high of 660 mg ascorbic acid equivalence per gram of dried plant material extracted (methanolic fruit extract). The aqueous and methanolic extracts generally had higher antioxidant capacities than the corresponding chloroform, hexane and ethyl acetate extracts. 
     Antimicrobial activity: To determine the ability of the fruit and leaf crude extracts to inhibit  C. perfringens  growth, 10 μL of each extract was screened using a disc diffusion assay. 
     As shown in the chart in  FIG. 1 , Bacterial growth was strongly inhibited by 5 of the 10 extracts screened (50%). 
     The methanolic leaf extract was the most potent inhibitor of growth (as judged by zone of inhibition), with inhibition zones of 16±0.6 mm. This compares favourably with the penicillin (2 μg) and ampicillin controls (10 μg), with the zones of inhibition of 12.3±0.3 and 13±1.0 mm respectively. 
     The methanolic fruit extract as well as both the aqueous and ethyl acetate leaf extracts also displayed good inhibition of  C. perfringens  growth, with ≥9 mm zones of inhibition. 
     Typically, the leaf extracts were more potent inhibitors of  C. perfringens  growth than were their corresponding fruit extract counterparts. 
       FIG. 1  shows a chart of growth inhibitory activity of  T. ferdinandiana  fruit and leaf plant extracts against the  C. perfringens  environmental isolate measured as zones of inhibition (mm). KFW=aqueous  T. ferdinandiana  fruit extract; KFM=methanolic  T. ferdinandiana  fruit extract; KFC=chloroform  T. ferdinandiana  fruit extract; KFH=hexane  T. ferdinandiana  fruit extract; KFE=ethyl acetate  T. ferdinandiana  fruit extract; KLW=aqueous  T. ferdinandiana  leaf extract; KLM=methanolic  T. ferdinandiana  leaf extract; KLC=chloroform  T. ferdinandiana  leaf extract; KLH=hexane  T. ferdinandiana  leaf extract; KLE=ethyl acetate  T. ferdinandiana  leaf extract; PC=penicillin (2 μg); AMP =ampicillin (10 μg). Results are expressed as mean zones of inhibition±SEM. 
     The antimicrobial efficacy was further quantified through the determination of MIC values against the  T. ferdinandiana  extracts (Table 2). 
     Table 2 below shows minimum inhibitory concentration (μg/mL) of the  T. ferdinandiana  fruit and leaf extracts and LC50 values (μg/mL) in the  Artemia nauplii  bioassay (Numbers indicate the mean MIC and LC50 values of triplicate determinations.—indicates no inhibition): 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Extract 
                 MIC 
                 LC 50   
               
               
                   
                   
               
             
            
               
                   
                 aqueous fruit extract 
                 1192  
                 2,080 
               
               
                   
                 methanolic fruit extract 
                 716 
                 2,115 
               
               
                   
                 chloroform fruit extract 
                 — 
                 — 
               
               
                   
                 hexane fruit extract 
                 — 
                 — 
               
               
                   
                 ethyl acetate fruit extract 
                 — 
                 — 
               
               
                   
                 aqueous leaf extract 
                 3125  
                 1,330 
               
               
                   
                 methanolic leaf extract 
                 206 
                 1,133 
               
               
                   
                 chloroform leaf extract 
                 — 
                 — 
               
               
                   
                 hexane leaf extract 
                 — 
                 — 
               
               
                   
                 ethyl acetate leaf extract 
                 117 
                   767 
               
               
                   
                   
               
            
           
         
       
     
     The aqueous and methanolic extracts (both fruit and leaf), as well as the leaf ethyl acetate extract, were effective at inhibiting  C. perfringens  growth, with MIC values generally &lt;1000 μg/ml (&lt;10 μg impregnated in the disc). 
     The methanolic and ethyl acetate leaf extracts were particularly potent, with MIC values of 206 μg/mL (approximately 2.1 μg infused into the disc) and 117 μg/mL (approximately 1.2 μg infused into the disc) respectively. 
     These results compare well with the growth inhibitory activity of the penicillin and ampicillin controls which were tested at 2 μg and 10 μg respectively. 
     The methanolic fruit extract was also a potent  C. perfringens  growth inhibitor (MIC value of 716 μg/ml). 
     Whilst less potent, the aqueous fruit extract also displayed good growth inhibitory activity (MIC values of 1192 μg/ml). 
     In contrast, both chloroform and hexane extracts, as well as the fruit ethyl acetate extract, were not active, or were of only low efficacy in the assay. 
     Quantification of toxicity: All extracts were initially screened in the assay at 2000 μg/mL (see  FIG. 2 ). 
     As a reference toxin, potassium dichromate was also tested in the bioassay. The potassium dichromate reference toxin was rapid in its onset of mortality, inducing nauplii death within the first 3 h of exposure and 100% mortality evident within 4-5 h (results omitted). 
     All aqueous and methanolic extracts as well as the ethyl acetate leaf extract showed &gt;90% mortality rates at 24 h. 
     The other extracts showed &lt;10% mortality rates at 24 h, with the exception of the chloroform leaf extract. 
     To further quantify the effects of toxin concentration on the initiation of mortality, the extracts were serially diluted in artificial seawater to test across a series of concentrations in the  Artemia franciscana nauplii  bioassay at 24 hours. The LC 50  values of the  T. ferdinandiana  extracts towards  A. franciscana  are presented in Table 2. No LC 50  values are reported in either of the hexane or chloroform extracts, nor for the ethyl acetate fruit extract, as &lt;50% mortality was seen in all tested concentrations. 
     Extracts with an LC 50  greater than 1000 μg/ml towards  Artemia nauplii  have been defined as being nontoxic in this assay. As only the ethyl acetate fruit extract had an LC 50  value of &lt;1000 μg/ml, all other extracts were considered nontoxic. Whilst the LC 50  value for the ethyl acetate leaf extract is &lt;1000 μg/ml, a value of 767 μg/ml indicates low to moderate toxicity. 
       FIG. 2  shows a chart of the lethality of the Australian plant extracts (2000 μg/mL) and the potassium dichromate control (1000 μg/mL) towards Artemia franciscana nauplii after 24 h exposure. KFW=aqueous T. ferdinandiana fruit extract; KFM=methanolic  T. ferdinandiana  fruit extract; KFC=chloroform  T. ferdinandiana  fruit extract; KFH=hexane  T. ferdinandiana  fruit extract; KFE=ethyl acetate  T. ferdinandiana  fruit extract; KLW=aqueous  T. ferdinandiana  leaf extract; KLM=methanolic  T. ferdinandiana  leaf extract; KLC=chloroform  T. ferdinandiana  leaf extract; KLH=hexane  T. ferdinandiana  leaf extract; KLE=ethyl acetate  T. ferdinandiana  leaf extract; PC=potassium dichromate control; SW=seawater control. Results are expressed as mean % mortality±SEM. 
     It will be appreciated that methanolic extracts include ethanolic extracts. 
     HPLC-MS QTOF analysis: As the methanolic and ethyl acetate leaf extracts had the greatest antibacterial efficacy (as determined by MIC), they were deemed the most promising extracts for further phytochemical analysis. Optimised HPLC-MS QTOF parameters used previously for the analysis of  T. ferdinandiana  leaf extracts were also used for the determination of the methanolic and ethyl acetate leaf extract compound profiles. The total compound chromatograms of the methanolic and ethyl acetate extracts are presented in  FIGS. 3 a , 3 b  and 4 a , 4 b    respectively. 
     The  T. ferdinandiana  methanolic extract positive ( FIG. 3 a   ) and negative ion ( FIG. 3 b   ) total compound chromatogram chromatograms revealed multiple overlapping peaks in the early stages of the chromatogram corresponding to the elution of polar compounds. 
     Most of the extract compounds had eluted within 12 minutes of the chromatogram (corresponding to approximately 32% acetonitrile). 
     However, several prominent peaks between 12 and 16 min in both chromatograms, and between 24 and 30 minutes (51-66% acetonitrile) indicates the broad spread of polarities of the compounds in this extract. 
     The leaf ethyl acetate positive ion ( FIG. 4 a   ) chromatogram had a similar elution profile to the corresponding methanolic extract, albeit with fewer peaks evident. 
     Many of the peaks in this chromatogram corresponded to peaks at similar elution volumes in the methanolic extract, indicating that many compounds were extracted by both solvents. 
     In contrast, much fewer peaks were evident in the leaf ethyl acetate negative ion chromatogram ( FIG. 4 b   ). 
     However, this chromatogram had significant background absorbance levels than the positive ion chromatogram due to ionisation of negative ions in this mode, possibly masking the signals for some peaks. 
       FIG. 4 a    shows positive and  FIG. 4 b    negative ion RP-HPLC total compound chromatograms (TCC) of 2 μl injections of  T. ferdinandiana  leaf ethyl acetate extract. 
     In total, fifty-four unique mass signals were noted for the T. ferdinandiana leaf methanolic and/or ethyl acetate extracts (Table 3). 
     All of the fifty-four unique molecular mass signals detected were putatively identified by comparison to the ‘Metlin’ metabolomics, forensic toxicology (Agilent) and phytochemicals (developed in this laboratory) databases. 
     Seventeen and eight compounds were detected only in the methanolic and ethyl acetate extracts respectively. The remaining twenty-nine compounds were present in both extracts. 
     The diversity of tannin compounds is noteworthy, with fourteen tannin compounds putatively identified across the methanolic and ethyl acetate leaf extracts. 
     In particular, chebulic acid ( FIG. 5 a   ), protocatechuic acid ( FIG. 5 b   ), ellagic acid dehydrate ( FIG. 5 c   ), punicalagin ( FIG. 5 d   ), ellagic acid ( FIG. 5 e   ), chebulagic acid ( FIG. 5 f   ), castalagin ( FIG. 5 g   ), corilagin ( FIG. 5 h   ), punicalin ( FIG. 5 i   ), chebulinic acid ( FIG. 5 j   ), punicalin ( FIG. 5 k   ), trimethylellagic acid isomers ( FIG. 5 l    and  FIG. 5 m   ) were putatively identified. 
     Table 3 shows qualitative HPLC-MS/MS analysis of the  T. ferdinandiana  leaf methanolic and ethyl acetate extracts, elucidation of empirical formulas and putative identification of the compound. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Name 
                 Formula 
                 Mass 
                 RT 
                 KLM 
                 KLE 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Chebulic acid (isomer 1) 
                 C 14  H 12  O 11   
                 356.0395 
                 0.363 
                 − 
                 +/− 
               
               
                 Shikimic acid 
                 C 7  H 10  O 5   
                 174.0542 
                 0.403 
                 − 
                 − 
               
               
                 Theophylline 
                 C 7  H 8  N 4  O 2   
                 180.0649 
                 0.424 
                 − 
                 − 
               
               
                 (1S,5R)-4-Oxo-6,8- 
                 C 7  H 6  O 5   
                 170.0221 
                 0.484 
                 − 
               
               
                 dioxabicyclo[3.2.1]oct- 
               
               
                 2-ene-2-carboxylic acid 
               
               
                 Mannitol 
                 C 6  H 14  O 6   
                 182.0793 
                 0.505 
                   
                 + 
               
               
                 Diprophylline 
                 C 10  H 14  N 4  O 4   
                 254.1012 
                 0.512 
                   
                 + 
               
               
                 Protocatechuic acid 
                 C 7  H 6  O 4   
                 154.0272 
                 0.522 
                   
                 − 
               
               
                 Propionylglycine methyl ester 
                 C 6  H 11  N O 3   
                 145.0744 
                 0.529 
                 + 
               
               
                 Naphtho [2″,3″: 4′,5′] 
                 C 19  H 10  N 4  S 
                 326.0645 
                 0.621 
                 − 
               
               
                 imidazo [2′,1′: 2,3] 
               
               
                 [1,3] thiazolo [4,5-b] quinoxaline 
               
               
                 Vanilpyruvic acid 
                 C 10  H 10  O 5   
                 210.0529 
                 0.632 
                   
                 − 
               
               
                 Valdipromide 
                 C 11  H 23 N O 
                 185.1785 
                 0.91 
                   
                 + 
               
               
                 Ellagic acid dihydrate 
                 C 14  H 10  O 10   
                 338.0285 
                 1.067 
                 +/− 
                 + 
               
               
                 Punicalagin 
                 C 48  H 28  O 30   
                 1084.065 
                 1.157 
                 − 
               
               
                 Chebulic acid (isomer 2) 
                 C 14  H 12  O 11   
                 356.0388 
                 1.533 
                 +/− 
                 + 
               
               
                 Phloroglucinol 
                 C 6  H 6  O 3   
                 126.0322 
                 1.605 
                 + 
               
               
                 Phosphoribosylaminoimidazole 
                 C 13  H 19  N 4  O 12  P 
                 454.0751 
                 2.489 
                 − 
               
               
                 succinocarboxamide (SAICAR) 
               
               
                 2-Cyclohexylpiperidine oxalate 
                 C 13  H 23  N O 4   
                 257.163 
                 3.268 
                   
                 + 
               
               
                 (2-Methyl-4-oxo-4H-pyran-3-yloxy)- 
                 C 8  H 8  O 5   
                 184.0377 
                 3.745 
                 − 
               
               
                 acetic acid 
               
               
                 Ellagic acid 
                 C 14  H 6  O 8   
                 302.0073 
                 4.372 
                 +/− 
                 + 
               
               
                 (2-Methyl-4-oxo-4H-pyran-3-yloxy)- 
                 C 8  H 8  O 5   
                 184.0374 
                 4.788 
               
               
                 acetic acid 
               
               
                 1α,25-Dihydroxy-26,27-dimethyl- 
                 C 29  H 44  O 3   
                 440.3262 
                 6.929 
                   
                 + 
               
               
                 22,22,23,23-tetradehydrovitamin D3 
               
               
                 Chebulagic acid (isomer 1) 
                 C 41  H 30  O 27   
                 954.0979 
                 7.629 
                 − 
                 − 
               
               
                 Castalagin (isomer 1) 
                 C 41  H 26  O 26   
                 934.0719 
                 7.671 
                 − 
                 − 
               
               
                 Corilagin 
                 C 27  H 22  O 18   
                 634.0815 
                 7.773 
                 +/− 
                 +/− 
               
               
                 8-Epiiridotrial glucoside 
                 C 16  H 24  O 8   
                 344.1475 
                 8.42 
                 − 
               
               
                 Punicalin 
                 C 34  H 22  O 22   
                 782.0619 
                 8.498 
                 + 
                 + 
               
               
                 Chebulinic acid (isomer 1) 
                 C 41  H 32  O 27   
                 956.1131 
                 8.602 
                 − 
                 − 
               
               
                 Luteolin (isomer 1) 
                 C 21  H 20  O 11   
                 448.1018 
                 8.726 
                 +/− 
                 +/− 
               
               
                 Castalagin (isomer 2) 
                 C 41  H 26  O 26   
                 934.0705 
                 8.767 
                 − 
                 − 
               
               
                 Vitexin 
                 C 21  H 20  O 10   
                 432.1067 
                 9.201 
                 +/− 
                 +/− 
               
               
                 Exifone 
                 C 13  H 10  O 7   
                 278.0431 
                 9.314 
                 +/− 
                 +/− 
               
               
                 Rutin 
                 C 27  H 30  O 16   
                 610.1542 
                 9.34 
                 − 
               
               
                 Punicalin 
                 C 34  H 22  O 22   
                 782.0619 
                 9.368 
                 + 
                 + 
               
               
                 Luteolin (isomer 2) 
                 C 21  H 20  O 11   
                 448.1011 
                 9.779 
                 +/− 
                 +/− 
               
               
                 Chebulagic acid (isomer 2) 
                 C 41  H 30  O 27   
                 954.0978 
                 9.847 
                 − 
                 − 
               
               
                 Casuarenin 
                 C 41  H 28  O 26   
                 936.0868 
                 9.852 
                 − 
                 − 
               
               
                 Norstictic acid pentaacetate 
                 C 28  H 24  O 15   
                 600.1117 
                 9.996 
                 +/− 
                 + 
               
               
                 9,12,13-Trihydroxy-10,15- 
                 C 18  H 32  O 5   
                 328.2253 
                 11.398 
                 − 
               
               
                 octadecadienoic acid 
               
               
                 Punicalin 
                 C 34  H 22  O 22   
                 782.0619 
                 11.448 
                 + 
               
               
                 Jasmonic acid 
                 C 12  H 18  O3 
                 210.1257 
                 11.536 
                   
                 + 
               
               
                 Luteolin (isomer 3) 
                 C 15  H 10  O 6   
                 286.0484 
                 11.864 
                 +/− 
                 +/− 
               
               
                 Quercetin 
                 C 15  H 10  O 7   
                 302.0434 
                 11.91 
                 − 
               
               
                 4,12-Dihydroxy-hexadecanoic acid 
                 C 16  H 32  O 4   
                 288.2306 
                 11.913 
                 − 
               
               
                 Methyl-p-coumarate 
                 C 10  H 10  O 3   
                 178.0632 
                 12.005 
                 − 
               
               
                 9,12,13-Trihydroxy-10-octadecenoic 
                 C 18  H 34  O 5   
                 330.2412 
                 12.491 
                 − 
               
               
                 acid 
               
               
                 Trimethyl ellagic acid (isomer 1) 
                 C 17  H 12  O 8   
                 344.0543 
                 12.574 
                 +/− 
                 +/− 
               
               
                 methyl 9,12-dihydroxy- 
                 C 19  H 34  O 5   
                 342.2412 
                 12.664 
                 − 
               
               
                 13-oxo-10-octadecenoate 
               
               
                 Gingerol 
                 C 17  H 26  O 4   
                 294.1837 
                 13.015 
                 − 
                 − 
               
               
                 Trimethyl ellagic acid (isomer 2) 
                 C 17  H 12  O 8   
                 344.0545 
                 14.238 
                 +/− 
                 +/− 
               
               
                 Trimethyl ellagic acid (isomer 3) 
                 C 17  H 12  O 8   
                 344.0545 
                 15.116 
                 +/− 
                 +/− 
               
               
                 16-Hydroperoxy-9Z,12,14E- 
                 C 18  H 30  O 4   
                 310.2145 
                 15.251 
                 − 
               
               
                 octadecatrienoic acid 
               
               
                 9,13-Dihydroxy-11-octadecenoic acid 
                 C 18  H 34  O 4   
                 314.246 
                 20.265 
                 − 
                 − 
               
               
                 Heptyl heptanoate 
                 C 14  H 28  O 2   
                 228.2091 
                 20.996 
                 − 
                 − 
               
               
                 Palmitic acid 
                 C 16  H 32  O 2   
                 256.2413 
                 23.869 
                 − 
                 − 
               
               
                   
               
            
           
         
       
     
     In Table 3 above, + and − refers to the relevant ionisation mode in which the compound was detected. KLM= T. ferdinandiana  leaf methanolic extract; KLE= T. ferdinandiana  ethyl acetate extract. 
     The diversity of ellagitannins in the methanolic and ethyl acetate  T. ferdianadiana  leaf extracts was particularly noteworthy. 
     As well as from ellagic acid and the dehydrated and trimethylated derivatives, the more complex, higher molecular weight compounds (j) chebulinic acid and punicalin were also putatively identified and are likely to contribute to the  C. perfringens  growth inhibitory activity of these extracts. 
     Ellagitannins are considered to be identified as potent inhibitors of the growth of a broad panel of bacteria, with MIC values as low as 62.5 μg/ml. 
     The  T. ferdinandiana  extracts are shown to display low toxicity towards  Artemia franciscana.  Indeed, with the exception of the leaf ethyl acetate extract (MIC 767 μg/mL), the LC 50  values for all extracts were well in excess of 1000 μg/mL and are therefore nontoxic. 
       Bacillus Anthracis  ( B. Anthracis ) 
     The ability to inhibit the growth of  B. anthracis  was investigated using a disc diffusion assay. 
     The minimum inhibitory concentration (MIC) values of the fruit and the leaf extracts were determined in order to quantify and compare their efficacies. 
     Toxicity was determined using an  Artemia franciscana nauplii  bioassay. 
     The most potent  T. ferdinandiana  fruit and leaf extracts were investigated using known non-targeted gas chromatography/mass spectrometry—GC-MS headspace analysis (with screening against a compound database) for the identification and characterisation of individual components in the crude  T. ferdinandiana  extracts. 
     Results: Solvent extractions of  T. ferdinandiana  fruit and leaf displayed good growth inhibitory activity in the disc diffusion assay against  B. anthracis.    
     Fruit ethyl acetate and methanolic  T. ferdinandiana  leaf extracts were particularly potent growth inhibitors, with MIC values of 451 and 377 μg/mL respectively. 
     The fruit methanolic and chloroform extracts, as well as the aqueous leaf extracts, also were good inhibitors of  B. anthracis  growth (MIC values of 1800 and 1414 μg/mL respectively). 
     The aqueous fruit extract and leaf chloroform extracts had only low inhibitory activity. 
     All other extracts were completely devoid of growth inhibitory activity. 
     Furthermore, all of the extracts with growth inhibitory activity were nontoxic in the  Artemia fransiscana  bioassay, with LC50 values &gt;1000 μg/mL. Non-biased GC-MS phytochemical analysis of the most active extracts (fruit ethyl acetate and methanolic leaf) putatively identified and highlighted several compounds that may contribute to the ability of these extracts to inhibit the growth of  B. anthracis.    
     The low toxicity of the  T. ferdinandiana  fruit ethyl acetate and methanolic leaf extracts, as well as their potent growth inhibitory bioactivity against  B. anthracis,  indicates their previously unrealised suitability as medicinal agents in the treatment and prevention of anthrax. 
     Qualitative phytochemical studies: Phytochemical analysis of the extracts for the presence of alkaloids, anthraquinones, cardiac glycosides, flavonoids, phenolic compounds, phytosteroids, saponins, tannins and triterpenoids were conducted. 
     Antioxidant capacity: The antioxidant capacity of each sample was assessed using the DPPH free radical scavenging method with modifications. 
     Ascorbic acid (0-25 μg per well) was used as a reference and the absorbances were recorded at 515 nm. 
     All tests were completed alongside controls on each plate and all were performed in triplicate. The antioxidant capacity based on DPPH free radical scavenging ability was determined for each extract and expressed as μg ascorbic acid equivalents per gram of original plant material extracted. 
     Antibacterial screening: Environmental  Bacillus anthracis  screening: An environmental strain of  Bacillus anthracis  was isolated and identified. All 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 16g/L bacteriological agar when required. Incubation was at 30° C. and the stock culture was subcultured and maintained in PYE media at 4° C. 
     Evaluation of antimicrobial activity: Antimicrobial activity of all plant extracts was determined using a modified disc diffusion assay. Briefly, 100 μL of the test bacterium was grown in 10 mL of fresh nutrient broth media until they reached a count of ˜108 cells/mL. 
     A volume of 100 μL of the bacterial suspension was spread onto nutrient agar plates and extracts were tested for antibacterial activity using 5 mm sterilised filter paper discs. Discs were impregnated with 10 μL of the test sample, allowed to dry and placed onto the inoculated plates. The plates were allowed to stand at 4° C. for 2 hours before incubation at 30° C. for 24 hours. 
     The diameters of the inhibition zones were measured to the closest whole millimetre. 
     Each assay was performed in at least triplicate. Mean values (±SEM) are reported in this study. Standard discs of penicillin (2 μg) and ampicillin (10 μg) were obtained and used 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 concentrations (MIC) of the extracts was determined as previously described. 
     Briefly, the plant extracts were diluted in deionised water and tested across a range of concentrations. Discs were impregnated 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. MIC values were determined using linear regression. 
     Toxicity screening: Reference toxin for toxicity screening: Potassium dichromate (K 2 Cr 2 O 7 ) was prepared in distilled water (4 mg/mL) 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  A. franciscana nauplii  lethality assay. Briefly, 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 in the bioassay. 
     A volume of 400 μL of reference toxin or the diluted plant extracts 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 hours, all nauplii were sacrificed and counted to determine the total % mortality per well. The LC50 with 95% confidence limits for each treatment was calculated using probit analysis. 
     Non-targeted GC-MS head space analysis: Separation and quantification were performed using a mass selective detector system. Briefly, the system was equipped with an auto-sampler fitted with a solid phase micro-extraction fibre (SPME) handling system utilising a 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. 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 
     Liquid extraction yields and qualitative phytochemical screening: Extractions of the various dried  T. ferdinandiana  fruit and leaf plant materials (1 g) with various solvents yielded dried plant extracts ranging from 18 mg (hexane fruit extract) to 483 mg (aqueous fruit extract) (Table 4). 
     Methanolic and aqueous extracts gave significantly higher yields of dried extracted material compared to the chloroform, hexane and ethyl acetate counterparts, which gave low to moderate yields. The dried extracts were resuspended in 10 mL of deionised water (containing 1% DMSO), resulting in the extract concentrations shown in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Table 4: The mass of dried extracted material, the concentration after resuspension in deionised water, 
               
               
                 qualitative phytochemical screenings and antioxidant capacities of the  T ferdinandiana  extracts: 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Concentration 
                 Antioxidant Capacity 
                   
                   
                   
                   
                   
               
               
                   
                 Mass of Dried 
                 of Resuspended 
                 (mg Ascorbic Acid 
                 Total 
                 Water Soluble 
                 Water Insoluble 
                 Cardiac 
               
               
                 Extract 
                 Extract (mg) 
                 Extract (mg/mL) 
                 Equivalency) 
                 Phenolics 
                 Phenolics 
                 Phenolics 
                 Glycosides 
                 Saponins 
               
               
                   
               
               
                 FW 
                 483 
                 48.3 
                 264 
                 +++ 
                 +++ 
                 +++ 
                 − 
                 + 
               
               
                 FM 
                 359 
                 35.9 
                 660 
                 +++ 
                 +++ 
                 +++ 
                 − 
                 ++ 
               
               
                 FC 
                 62 
                 6.2 
                 7 
                 + 
                 − 
                 − 
                 − 
                 − 
               
               
                 FH 
                 18 
                 1.8 
                 1 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                 FE 
                 30 
                 3 
                 39 
                 ++ 
                 ++ 
                 + 
                 − 
                 + 
               
               
                 LW 
                 471 
                 47.1 
                 340 
                 +++ 
                 +++ 
                 +++ 
                 ++ 
                 +++ 
               
               
                 LM 
                 331 
                 33.1 
                 150 
                 +++ 
                 +++ 
                 +++ 
                 +++ 
                 ++ 
               
               
                 LC 
                 59 
                 5.9 
                 5 
                 + 
                 − 
                 − 
                 − 
                 − 
               
               
                 LH 
                 58 
                 5.8 
                 0.4 
                 + 
                 − 
                 − 
                 − 
                 − 
               
               
                 LE 
                 59 
                 5.9 
                 22 
                 +++ 
                 +++ 
                 +++ 
                 − 
                 − 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Alkaloids 
                 Alkaloids 
                   
                   
                 Free 
                 Combined 
               
               
                   
                 Extract 
                 Triterpenes 
                 Phytosteroids 
                 (Mayer Test) 
                 (Wagner Test) 
                 Flavonoids 
                 Tannins 
                 Anthraquinones 
                 Anthraquinones 
               
               
                   
                   
               
               
                   
                 FW 
                 − 
                 − 
                 − 
                 − 
                 +++ 
                 ++ 
                 − 
                 − 
               
               
                   
                 FM 
                 + 
                 − 
                 + 
                 + 
                 +++ 
                 ++ 
                 − 
                 − 
               
               
                   
                 FC 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 FH 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 FE 
                 ++ 
                 − 
                 − 
                 − 
                 ++ 
                 − 
                 − 
                 − 
               
               
                   
                 LW 
                 ++ 
                 − 
                 − 
                 − 
                 ++ 
                 +++ 
                 + 
                 + 
               
               
                   
                 LM 
                 + 
                 − 
                 + 
                 + 
                 ++ 
                 +++ 
                 + 
                 + 
               
               
                   
                 LC 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 LH 
                 − 
                 − 
                 − 
                 − 
                 ++ 
                 + 
                 − 
                 − 
               
               
                   
                 LE 
                 − 
                 − 
                 − 
                 − 
                 ++ 
                 ++ 
                 − 
                 − 
               
               
                   
                   
               
            
           
         
       
     
     In Table 4 above, + + + indicates a large response; + + indicates a moderate response; + indicates a minor response; − indicates no response in the assay. FW=aqueous  T. ferdinandiana  fruit extract; FM=methanolic  T. ferdinandiana  fruit extract; FC=chloroform  T. ferdinandiana  fruit extract; FH=hexane  T. ferdinandiana  fruit extract; FE=ethyl acetate  T. ferdinandiana  fruit extract; LW=aqueous  T. ferdinandiana  leaf extract; LM=methanolic  T. ferdinandiana  leaf extract; LC=chloroform  T. ferdinandiana  leaf extract; LH=hexane  T. ferdinandiana  leaf extract; LE=ethyl acetate  T. ferdinandiana  leaf extract. Antioxidant capacity was determined by DPPH reduction and is expressed as mg ascorbic acid equivalence per g plant material extracted. 
     Antimicrobial activity: To determine the ability of the  T. ferdinandiana  fruit and leaf 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 strongly inhibited by 7 of the 10 extracts screened (70%) ( FIG. 6 ). 
     The methanolic leaf extract was the most potent inhibitor of  B. anthracis  growth (as judged by zone of inhibition), with inhibition zones of 15.3±0.6 mm. This compares favourably with the penicillin (2 μg) and ampicillin controls (10 μg), with zones of inhibition of 8.3±0.6 and 10.0±0.7 respectively. 
     The methanolic fruit extract as well as the ethyl acetate and aqueous leaf extracts also displayed good inhibition of B. anthracis growth, with 8 mm zones of inhibition. 
     In general, the leaf extracts were more potent inhibitors of  B. anthracis  growth than were their fruit extract counterparts. 
     The antimicrobial efficacy was further quantified through the determination of MIC values against the  T. ferdinandiana  extracts (Table 5). 
     Most of the extracts were effective at inhibiting  B. anthracis  growth, with MIC values &lt;1000 μg/ml for several extracts (&lt;10 μg impregnated in the disc). 
     The ethyl acetate fruit extract and the methanolic leaf extract were particularly potent, with MIC values of 451 μg/mL (approximately 4.5 μg infused into the disc) and 377 μg/mL (approximately 3.8 μg infused into the disc) respectively. 
     These results compare well with the growth inhibitory activity of the penicillin and ampicillin controls which were tested at 2 μg and 10 μg respectively. 
     The methanolic fruit extract was also a potent  B. anthracis  growth inhibitor (MIC value of 877 μg/ml). 
     Whilst less potent, the fruit chloroform and aqueous leaf extracts also had good growth inhibitory activity (MIC values of 1800 and 1414 μg/ml respectively). 
     In contrast, the aqueous fruit and hexane extracts, as well as the leaf chloroform hexane and ethyl acetate extracts, were not active, or were of only low efficacy in the assay. 
     Table 5 below shows minimum inhibitory concentration (μg/mL) of the  T. ferdinandiana  fruit and leaf extracts and LC50 values (μg/mL) in the  Artemia nauplii  bioassay. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 Extract 
                 MIC 
                 LC50 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 aqueous fruit extract 
                 &gt;10,000 
                 2,080 
               
               
                   
                 methanolic fruit extract 
                 877 
                 2,115 
               
               
                   
                 chloroform fruit extract 
                 1800 
                 — 
               
               
                   
                 hexane fruit extract 
                 — 
                 — 
               
               
                   
                 ethyl acetate fruit extract 
                 451 
                 — 
               
               
                   
                 aqueous leaf extract 
                 1414 
                 1,330 
               
               
                   
                 methanolic leaf extract 
                 377 
                 1,133 
               
               
                   
                 chloroform leaf extract 
                 5,000 
                 — 
               
               
                   
                 hexane leaf extract 
                 — 
                   767 
               
               
                   
                 ethyl acetate leaf extract 
               
               
                   
                   
               
               
                   
                 Numbers indicate the mean MIC and LC50 values of triplicate determinations. 
               
               
                   
                 — indicates no inhibition. 
               
            
           
         
       
     
     Quantification of toxicity: All extracts were initially screened at 2000 μg/mL in the assay (see  FIG. 7 ). 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 of mortality, inducing nauplii death within the first 3 hours of exposure and 100% mortality was evident following 4-5 hours (results not shown). 
     All methanolic and aqueous extracts showed &gt;90% mortality rates at 24 hour, as did the ethyl acetate leaf extract. The remainder of the extracts showed &lt;10% mortality rates at 24 hour, with the exception of the chloroform leaf extract. 
     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 at 24 hours. 
     Table 5 shows the LC50 values of the  T. ferdinandiana  extracts towards  A. franciscana.  No LC50 values are reported for either of the chloroform and hexane extracts, nor for the ethyl acetate fruit extract, as less than 50% mortality was seen for all concentrations tested. 
     Extracts with an LC50 greater than 1000 μg/ml towards  Artemia nauplii  have been defined as being nontoxic in this assay. 
     As only the ethyl acetate fruit extract had a LC50 &lt;1000 μg/ml, all other extracts were considered nontoxic. Whilst the LC50 value for leaf ethyl acetate extract is below 1000 pg/ml, the value of 767 μg/ml indicates low to moderate toxicity. 
     Non-targeted GC-MS headspace analysis of  T. ferdinandiana  fruit and leaf extracts: As the fruit ethyl acetate and methanolic leaf extracts had the greatest growth inhibitory efficacy against  B. anthracis  (as determined by MIC; see Table 5), 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 fruit ethyl acetate and methanolic leaf extracts are presented in  FIGS. 8 and 9  respectively. Several major peaks were noted in the fruit ethyl acetate extract at approximately 15.1 (3, 3-dimethyl-hexane, 7.1% relative abundance), 19.7 (2-methyl-2-phenyl-oxirane, 14.6% relative abundance), 20.9 (m-di-tert-butylbenzene, 22% relative abundance) and 28.9 min (3,5-bis(1,1-dimethylethyl)-phenol, 19.4% relative abundance). Numerous overlapping peaks were also evident in the middle stages of the chromatogram from 10-25 min. In total, 42 unique mass signals were noted for the  T. ferdinandiana  fruit ethyl acetate extract (Table 6). Putative empirical formulas and identifications were achieved for all of these compounds. 
     Table 6 below shows qualitative GC-MS analysis of the  T. ferdinandiana  fruit ethyl acetate extract, elucidation of empirical formulas and putative identification of each compound: 
                                                         Retention   Relative           Molecular   Empirical   Time   Abundance       Putative Identification   Mass   Formula   (min)   (% Area)                                                    Ethanone, 1-(2-furanyl)-   110   C 6  H 6  O 2     10.202   0.9       2-Heptanone, 4-methyl-   128   C 8  H 16  O   11.184   3.8       Propanoic acid, 2-   144   C 5  H 8  N 2  O 3     11.8   1       acetylhydrazono-       Tridecane   184   C 13  H 28     12.087   0.6       Acetic acid, heptyl ester   158   C 9  H 18  O 2     12.263   0.2       3-Pentanol, 2,2-dimethyl-   116   C 7  H 16  O   12.525   2.8       2-Heptanone, 4,6-dimethyl-   142   C 9  H 18  O   12.866   3.2       3-Heptanol, 2-methyl-   130   C 8  H 18  O   13.063   0.2       1-Octanol, 2-butyl-   186   C 12  H 26  O   13.233   0.8       Heptane, 3,3,5-trimethyl-   142   C 10  H 22     13.531   0.3       Undecane   156   C 11  H 24     13.69   1.5       Octane, 3,3-dimethyl-   142   C 10  H 22     13.96   2.5       1-Hexanol, 2-ethyl-   130   C 8  H 18  O   14.105   3.8       1,3-Propanediamine, N,N-   102   C 5  H 14  N 2     14.3   0.3       dimethyl-       Hexane, 3,3-dimethyl-   114   C 8  H 18     15.079   7.1       1 -Octanol   130   C 8  H 18  O   15.422   0.3       Propanoic acid, anhydride   130   C 6  H 10  O 3     15.625   0.2       1,3-Benzenediol, 4-ethyl-   138   C 8  H 10  O 2     16.034   0.4       1-Undecene, 4-methyl-   168   C 12  H 24     16.461   1.9       Carbonic acid, nonyl prop-1-   228   C 13  H 24  O 3     17.241   0.2       en-2-yl ester       Octanoic acid   144   C 8  H 16  O 2     18.481   0.8       Oxalic acid, 6-ethyloct-3-yl   314   C 18  H 34  O 4     18.665   0.4       isohexyl ester       2-Octanol, 2,6-dimethyl-   158   C 10  H 22  O   18.914   0.1       Undecane, 4,7-dimethyl-   184   C 13  H 28     19.387   0.8       Oxirane, 2-methyl-2-phenyl-   134   C 9  H 10  O   19.699   14.6       Nonane, 2,6-dimethyl-   156   C 11  H 24     19.803   0.6       Dodecane, 4-methyl-   184   C 13  H 28     20.035   0.3       Undecane, 2,4-dimethyl-   184   C 13  H 28     20.678   0.3       4-tert-Butylcyclohexyl methyl   262   C 13  H 27  O 3  P   20.777   0.3       ethylphosphonate       m-Di-tert-butylbenzene   190   C 14  H 22     20.931   22       Hexane, 3,3-dimethyl-   114   C 8  H 18     21.08   0.2       Nonadecane   268   C 19  H 40     21.703   1       Butyl 2-butoxyacetate   188   C 10  H 20  O 3     21.841   0.7       Octane, 2,3,6,7-tetramethyl-   170   C 12  H 26     22.087   0.1       Hexane, 3,3-dimethyl-   114   C 8  H 18     22.959   0.2       1,1,6,6-   180   C 13  H 24     23.348   0.6       Tetramethylspiro[4.4]nonane       2,2,4-Trimethyl-1,3-   286   C 16  H 30  O 4     23.611   0.4       pentanediol diisobuty       n-Decanoic acid   172   C 10  H 20  O 2     23.862   0.1       Propanoic acid, 2-methyl-,   216   C 12  H 24  O 3     24.169   0.6       3-hydroxy-2,2       1-Dodecanol   186   C 12  H 26  O   27.352   0.1       Phenol, 3,5-bis(1,1-   206   C 14  H 22  O   28.856   19.4       dimethylethyl)-       2,2,4-Trimethyl-1,3-   286   C 16  H 30  O 4     31.769   0.5       pentanediol diisobutyrate                    
The relative abundance expressed in this table 6 is a measure of the area under the peak expressed as a % of the total area under all chromatographic peaks
 
     The gas chromatogram for the methanolic leaf extract ( FIG. 9 ) had substantially fewer peaks evident than the fruit ethyl acetate extract ( FIG. 8 ). In total, nineteen unique mass signals were noted in the methanolic leaf extract chromatogram. 
     Several major peaks were present at approximately 11.3 (methoxy-phenyl-oxime, 22.7% relative abundance), 13.7 (1-octen-3-ol, 2.4% relative abundance), 14.4 (2-(1,1-dimethylethoxy)-ethanol, 27.7% relative abundance), 19.5 (2-methyl-2-phenyl-oxirane, 11.4% relative abundance) and 21.5 min (3,5-dimethyl-benzaldehyde, 15.6% relative abundance). 
     Several small peaks were also evident throughout the chromatogram. Of the nineteen unique mass signals, putative empirical formulas and identifications were achieved for sixteen of these compounds. 
     Table 7 below shows a qualitative GC-MS analysis of the methanolic  T. ferdinandiana  leaf extract, elucidation of empirical formulas and putative identification of each compound. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                   
                 Retention 
                 Relative 
               
               
                 Putative 
                 Molecular 
                 Empirical 
                 Time 
                 Abundance 
               
               
                 Identification 
                 Mass 
                 Formula 
                 (mins) 
                 (% Area) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Oxime-, methoxy- 
                 151 
                 C 8  H 9  NO 2   
                 11.266 
                 22.7 
               
               
                 phenyl- —   
               
               
                 1-Octen-3-ol 
                 128 
                 C 8  H 16  O 
                 13.727 
                 2.4 
               
               
                 Ethanol, 2-(1,1- 
                 118 
                 C 6  H 14  O 2   
                 14.418 
                 27.7 
               
               
                 dimethylethoxy)- 
               
               
                 1-Hexanol, 2-ethyl- 
                 130 
                 C 8  H 18  O 
                 15.373 
                 1.1 
               
               
                 Cineole 
                 154 
                 C 10  H 18  O 
                 15.499 
                 1.8 
               
               
                 Ethyl 2-(5-methyl-5- 
                 242 
                 C 13  H 22  O 4   
                 16.879 
                 1 
               
               
                 vinyltetrahydrofuran- 
               
               
                 2-yl carbonate 
               
               
                 Nonanal 
                 142 
                 C 9  H 18  O 
                 17.04 
                 1.3 
               
               
                   
                   
                   
                 17.873 
                 2.1 
               
               
                   
                   
                   
                 18.231 
                 0.6 
               
               
                 Oxirane, 2-methyl-2- 
                 134 
                 C 9  H 10  O 
                 19.56 
                 11.4 
               
               
                 phenyl- 
               
               
                 Ethyl benzoate 
                 150 
                 C 9  H 10  O 2   
                 20.06 
                 0.8 
               
               
                 2-lsopropylidene-3- 
               
               
                 methylhexa-3,5- 
                 150 
                 C 10  H 14  O 
                 21.031 
                 0.5 
               
               
                 dienal 
               
               
                 Decanal 
                 156 
                 C 10  H 20  O 
                 21.11 
                 0.3 
               
               
                 Benzaldehyde, 3,5- 
                 134 
                 C 9  H 10  O 
                 21.527 
                 15.6 
               
               
                 dimethyl- 
                   
                   
                 24.786 
                 0.9 
               
               
                 Propanoic acid, 2- 
                 216 
                 C 12  H 24  O 3   
                 26.499 
                 2.2 
               
               
                 methyl-, 3-hydroxy- 
               
               
                 2,2,4 
               
               
                 2,4-Di-tert- 
                 206 
                 C 14  H 22  O 
                 31.641 
                 1 
               
               
                 butylphenol 
               
               
                 Ethyl para- 
                 194 
                 C 11  H 14  O 3   
                 32.054 
                 5.9 
               
               
                 ethoxybenzoate 
               
               
                 2,2,4-Trimethyl-1,3- 
                 286 
                 C 16  H 30  O 4   
                 33.822 
                 0.7 
               
               
                 pentanediol 
               
               
                 diisobutyrate 
               
               
                   
               
            
           
         
       
     
     The relative abundance expressed in table 7 is a measure of the area under the peak expressed as a % of the total area under all chromatographic peaks. 
     Qualitative GC-MS headspace analysis of the most potent  B. anthracis  growth inhibitory  T. ferdinandiana  extracts (fruit ethyl acetate and methanolic leaf extracts) identified a number of interesting compounds. 
     The presence of the furan compounds 1-(2-furanyl)-ethanone ( FIG. 10 a   ) and ethyl 2-(5-methyl-5-vinyltetrahydrofuran-2-yl) carbonate ( FIG. 10 b   ) are noteworthy. The nitro furans have particularly well studied antimicrobial mechanisms, acting via the inhibition of nucleic acid synthesis. 
     Similarly, synthetic furan derivatives (modified by the addition of a rhodanine moiety) are known to be potent inhibitors of the growth of a panel of multidrug resistant bacteria, with MIC values as low as 2 μg/mL against some species. 
     Reports of anti-bacterial activity for the two furan derivatives present in the  T. ferdinandiana  extracts are not known, and it is pertinent that the two furan derivatives are likely to contribute to the effectiveness of the present extracts. 
     It is likely that other phytochemical classes also contribute to the growth inhibitory properties of these extracts. Phytochemical screening indicates that polyphenolics, flavonoids, saponins, and terpenes were present in the  T. ferdinandiana  extracts. 
     Gallic ( FIG. 5 c   ) and ellagic acids ( FIG. 5 d   ) and their methylated derivatives, chebulic acid ( FIG. 5 e   ), galloyl pyrogallol ( FIG. 5 f   ), corilagen ( FIG. 5 g   ), punicalin ( FIG. 5 h   ), castalagin ( FIG. 5 i   ) and chebulagic acid ( FIG. 5 j   ) were detected in  T. ferdinandiana  extracts in each of those studies. These tannins have potent, broad spectrum growth inhibitory activity against a variety of bacterial species. 
     Gallotannins have particularly well reported inhibitory properties. They function via multiple mechanisms including interacting with both cell surface proteins and through interactions with intracellular enzymes. 
     Ellagitatannins also interact with cellular proteins and induce disruptions in bacterial cell walls. 
     Resveratrol ( FIG. 10 k   ) and the glycosylated resveratrol derivative piceid ( FIG. 10 l   ), diethylstilbestrol monosulfate ( FIG. 10 m   ) and combretastatin A1 ( FIG. 10 n   ) were putatively identified. Identification of combretastatin A1 was particularly interesting as combretastatins have attracted much recent interest due to their potent ability to block cancer cell progression and induce apoptosis by binding intracellular tubulin, thereby disrupting microtubule formation. 
       FIG. 10  ( 10   a - n ) show respective chemical structures of (a) 1-(2-furanyl)-ethanone, (b) ethyl 2-(5-methyl-5-vinyltetrahydrofuran-2-yl)carbonate, (c) gallic acid, (d) ellagic acid; (e) chebulic acid, (f) galloyl pyrogallol, (g) corilagen, (h) punicalin, (i) castalagin, (j) chebulagic acid, (k) resveratrol, (l) piceid, (m) diethylstilbestrol monosulfate, (n) combretastatin A1. 
     Several important terpenoids have also been identified in  T. ferdinandiana  extracts. 
     With the exception of the  T. ferdinandiana  ethyl acetate leaf extract, the findings reported here demonstrate that the  T. ferdinandiana  extracts were nontoxic towards  Artemia franciscana nauplii,  with LC50 values substantially &gt;1000 μg/mL. 
     Extracts with LC50 values &gt;1000 μg/mL towards Artemia nauplii are defined as being nontoxic. Even the ethyl acetate leaf extract which induced significant mortality was deemed low to moderate toxicity due to its moderate LC50 value. 
     Whilst toxicity investigations indicate that these extracts may be safe for use as  B. anthracis  growth inhibitors, studies using human cell lines are required to further evaluate the safety of these extracts. 
       T. ferdinandiana  Extracts as Inhibitors of  Giardia  Proliferation and/or Control of Giardiasis. 
     By way of particular, though non-limiting, examples, inhibition of  Giardia duodenalis  proliferation by  T. ferdinandiana  extracts and pure compounds will hereinafter be described. It is to be understood that one or more forms of the present invention are not to be limited to control or inhibition of only  Giardia duodenalis  (aka  Giardia lamblis  and  Giardia intestinalis ) but of other  Giardia  and microbial/bacterial strains. 
     Inhibitory activity of  T. ferdinandiana  extracts and pure compounds have been tested, such as against three strains of  Giardia duodenalis  trophozoites measured as a percentage the untreated control, as shown by way of example in the test results shown in  FIG. 12 . 
     A panel of 11 compounds identified in  T. ferdinandiana  fruit extracts with potent  G. duodenalis  growth inhibitory activity have been investigated for the ability to inhibit  G. duodenalis  proliferation. 
     Eight of the 11 compounds inhibited the growth of all three  G. duodenalis  strains. 
     DPGA was the most potent antigiardial compound, with IC50 values as low as 126 μM (38 mg/mL). Notably, DPGA inhibited a metronidazole resistant  G. duodenalis  strain with similar potency as determined for the metronidazole sensitive strains. 
     Furthermore, the potency of DPGA was greatly potentiated when it was tested in combination with ascorbic acid, to approximately 17 μM (5 mg/mL) for the metronidazole sensitive  G. duodenalis  strains and 40 μM (12 mg/mL) for the resistant strain. 
       T. ferdinandiana  tannins (gallic acid and chebulic acid) were also found to be inhibitors of  G. duodenalis  growth, with enhanced levels of activity when tested in combination with ascorbic acid. 
     All of the tested compounds (and their combinations with ascorbic acid) displayed low toxic and all compounds conformed to Lipinski&#39;s rules of 5 with few violations, indicating their potential as drug leads and chemotherapies for the treatment and prevention of giardiasis. 
     A purine analogue ( FIG. 11 a   ) was identified in all extracts with growth inhibitory activity. 
     Interestingly, numerous studies have reported that  Giardia duodenalis  are unable to synthesise their own purine or pyrimidine nucleotides and are reliant on salvage pathways to supply them with nucleotides for nucleic acid synthesis. 
     Furthermore,  G. duodenalis  are incapable of interconversion between purine nucleotides and therefore require the correct purine nucleotides for replication. Indeed, purine analogues inhibit the growth of  G. duodenalis  and have been highlighted as potential chemotherapeutic agents for giardiasis. 
       FIGS. 11 a  to 11 k    show respective chemical structures of the compounds reported in anti-proliferative methanolic and aqueous  T. ferdinandiana  fruit extracts: (a) purine; (b) gallic acid; (c) chebulic acid; (d) ribonolactone; (e) ascorbic acid; (f) gluconolactone; (g) glucohepatonic acid-1,4-lactone; (h) quinic acid, (i) eujavonic acid; (j) 5-(4-hydroxy-2,5-dimethylphenoxy)-2,2-dimethyl-pentanoic acid (HMDP); (k) 2,3-dihydroxyphenyl B-D-glucopyranosiduronic acid (DPGA). 
     An abundance of naturally occurring tannins in the bioactive  T. ferdinandiana  extracts is noted, with particularly high levels of gallic acid ( FIG. 11 b   ) and chebulic acid ( FIG. 11 c   ). 
     Gallotannins inhibit the growth of multiple microbial species via binding cell surface lipotoichoic acid and proline-rich membrane proteins, and by inhibiting glucosyltransferase enzymes. 
     Ribonolactone ( FIG. 11 d   ), ascorbic acid ( FIG. 11 e   ), gluconolactone ( FIG. 11 f   ) and glucohepatonic acid-1,4-lactone ( FIG. 11 g   ) present in extracts of T .ferdinandiana as lactone moieties is of value as many of the current anti-giardial chemotherapeutic drugs used are lactone containing compounds, particularly lactone substituted nitroimidazoles (e.g. metronidazole, secnidazole, tinidazole, ornidazole and albendazole). 
     Compounds containing a lactone moiety are understood to block the giardial lipid deacylation/reacylation pathways, thereby inhibiting proliferation. As  Giardia  spp. are unable to synthesise lipids by de novo pathways, they must use host gastrointestinal precursor lipids for the synthesis of membrane and cellular lipids by deacylation/reacylation reactions. 
     Thus, the lactone containing compounds in the  T. ferdinandiana  extracts can be understood to contribute to  G. duodenalis  growth inhibition via inhibition of lipid metabolism pathways. 
     Quinic acid ( FIG. 11 h   ) in the  T. ferdinandiana  extracts has been noted. Substituted quinic acid compounds can block leucyl-tRNA synthase activity in  G. duodenalis  cells. As aminoacyl-tRNA synthases are essential for translation of the genetic code by attaching the correct amino acid to each tRNA, blockage of leucyl-tRNA synthase activity results in ineffective Leu-tRNA production and thus the inhibition of protein synthesis. Therefore, quinic acid can be understood to also contribute to the antigiradial activity of the  T. ferdianadiana  fruit extracts. 
     Eujavonic acid ( FIG. 11 i   ),  5 -( 4 -hydroxy-2,5-dimethylphenoxy)-2,2-dimethyl-pentanoic acid (HMDP) ( FIGS. 11 j   ) and  2 , 3 -dihydroxyphenyl B-D-glucopyranosiduronic acid (DPGA) ( FIG. 11 k   ) are further antigiardial compounds. 
     Furthermore, as  T. ferdinandiana  fruit have a relatively high ascorbic acid content, ascorbic acid may be efficacious in altering and/or enhancing the growth inhibitory activity of the individual components. 
     Therefore, all compounds were also assessed in combination with ascorbic acid to quantify its effects on the activity of those components. 
       T. ferdinandiana  fruit extraction yields and qualitative phytochemical screening. 
     Extraction of 1 g of dried  T. ferdinandiana  fruit with methanol and deionised water yielded relatively high masses of dried extracted material (370 μg/mL and 290 μg/mL for the methanolic and aqueous extracts respectively). The dried extracts were resuspended in 10 mL of deionised water (containing 0.5% DMSO) resulting in the extract concentrations shown in Table 8 below. 
     
       
         
           
               
               
             
               
                   
                 TABLE 8 
               
             
            
               
                   
                   
               
               
                   
                 Toxicity 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Mass of 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 LC 50  in 
               
               
                   
                 extracted 
                   
                   
                   
                   
                   
                   
                   
                 Anti-giardial 
                 LC 50  in 
                 the HDF 
               
               
                   
                 material 
                   
                   
                   
                   
                   
                   
                   
                 IC 50   
                 ALA 
                 assay 
               
               
                 Extract 
                 (mg) 
                 Polyphenolics 
                 Flavonoids 
                 Phytosterols 
                 Saponins 
                 Triterpenoids 
                 Tannins 
                 Alkaloids 
                 (ug/mL) 
                 (ug/mL) 
                 (ug/mL) 
               
               
                   
               
               
                 M 
                 370 
                 +++ 
                 +++ 
                 − 
                 ++ 
                 + 
                 ++ 
                 + 
                 740 
                 1150 
                 1450 
               
               
                 W 
                 290 
                 +++ 
                 +++ 
                 − 
                 + 
                 − 
                 ++ 
                 + 
                 143 
                 1093 
                 1540 
               
               
                   
               
               
                 +++ indicates a larqe response; ++ indicated a moderate response; + indicates a low response; − indicates no response. Values indicate the mean IC 50  or LC 50  values of three experiments each with triplicate determinations. M = methanolic extract; W = aqueous extract. 
               
            
           
         
       
     
     Qualitative phytochemical studies (Table 8) showed that both extracts contained high levels of phenolics and flavonoids, as well as moderate to high levels of tannins. Saponins were also present in low to moderate levels. Triterpenes and alkaloids were present in low levels. 
     Several of the pure  T. ferdinandiana  fruit compounds also significantly inhibited  G. duodenalis  trophozoite proliferation when tested at 300 μg/mL ( FIG. 12 ). 
     DPGA was noted to be a particularly good growth inhibitor, blocking 100% of trophozoite growth. 
     DPGA was as effective against the metronidazole resistant  G. duodenalis  strain as it was against the sensitive strains, indicating that DPGA may block giardial growth by different mechanisms than metronidazole. 
     Several of the other compounds also significantly inhibited  G. duodenalis  trophozoite proliferation, albeit with lower efficacy. Gallic acid (˜50% inhibition of proliferation), chebulic acid (˜40% inhibition), quinic acid (˜30% inhibition), eujavonic acid (˜20% inhibition) and 5-(4-hydroxy-2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid (˜20% inhibition) each inhibited all three  G. duodenalis  strain, including the metronidazole resistant strain. 
     Two of the other  T. ferdinandiana  fruit compounds (ascorbic acid, ˜15% inhibition; glucohelapatonic acid lactone, ˜30% inhibition) also significantly inhibited the metronidazole sensitive  G. duodenalis  strain, yet were ineffective inhibitors of the metronidazole resistant  G. duodenalis  strain. 
     Purine, ribolactone and gluconolactone did not significantly affect the growth of any of the  G. duodenalis  strains. 
     Methanol and water  T. ferdinandiana  fruit extracts displayed potent inhibitory activity, each inhibiting 100% of the Giardial growth (compared to the untreated control). 
     The efficacy of the extracts were further evaluated by determination of the concentration required to inhibit  G. duodenalis  growth by 50% (IC50). The water extract was a particularly good inhibitor of  G. duodenalis  proliferation, with an IC50 of 143 μg/mL. The methanol extract, whilst less potent, also displayed good anti-Giardial activity (704 μg/mL). 
       FIG. 12 : Inhibitory activity of the  T. ferdinandiana  extracts and pure compounds against three strains of  Giardia duodenalis  trophozoites measured as a percentage the untreated control. NC=negative control; M=methanolic extract; W=water extract; 1=purine; 2=gallic acid; 3=chebulic acid; 4=ribolactone; 5=ascorbic acid; 6=gluconolactone; 7=glucohelapatonic acid lactone; 8=quinic acid; 9=eujavonic acid; 10=HMDP; 11=DPGA; PC=metronidazole control (50 μg/ml). Results are expressed as the mean±SEM of three independent experiments with internal triplicate determinations (n=9). *, # and ̂ indicate results that are significantly different to the untreated controls for the sheep S2, ATCC 203333 and ATCC PRA-251  G. duodenalis  strains respectively (p&lt;0.01). 
     Quantification of IC 50  for the Pure  T. Ferdinndiana  Compounds 
     The anti-proliferative activity of the pure  T. ferdinandiana  compounds was further tested over a range of concentrations to determine the IC 50  values against  G. duodenalis  trophozoites (Table 9). 
     
       
         
           
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
             
            
               
                   
                 IC 50  (μg/mL) Values and 
               
               
                   
                 Class of Combination 
               
            
           
           
               
               
               
            
               
                 Compound alone or 
                 Sheep S2 Strain 
                 ATCC203333 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 in combination 
                 Single 
                   
                 Class of 
                 Single 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 with ascorbic acid 
                 Compound 
                 Combination 
                 ΣFIC 50   
                 Interaction 
                 Compound 
                 Combination 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Gallic acid 
                 1156 
                 (6795 μM) 
                 146 
                 (858 μM) 
                 0.15 
                 Synergy 
                 1368 
                 (8041 μM) 
                 228 
                 (1340 μM) 
               
               
                 Chebulic acid 
                 1283 
                 (3602 μM) 
                 427 
                 (1199 μM) 
                 0.56 
                 Additive 
                 985 
                 (2765 μM) 
                 320 
                 (898 μM) 
               
               
                 Glucohepatonic 
                 1746 
                 (9914 μM) 
                 1330 
                 (7552 μM) 
                 1.19 
                 independent 
                 2255 
                 (12804 μM) 
                 1585 
                 (9567 μM) 
               
               
                 acid lactone 
               
               
                 Quinic acid 
                 1172 
                 (6099 μM) 
                 418 
                 (2175 μM) 
                 0.51 
                 Additive 
                 1428 
                 (7431 μM) 
                 632 
                 (3289 μM) 
               
               
                 Eujavonic acid 
                 1438 
                 (6038 μM) 
                 525 
                 (2204 μM) 
                 0.58 
                 Additive 
                 1683 
                 (7067 μM) 
                 695 
                 (2918 μM) 
               
               
                 HMDP 
                 1835 
                 (6895 μM) 
                 725 
                 (2724 μM) 
                 0.69 
                 Additive 
                 1585 
                 (5955 μM) 
                 655 
                 (2461 μM) 
               
               
                 DPGA 
                 47 
                 (156 μM) 
                 5 
                 (17 μM) 
                 0.11 
                 Synergy 
                 38 
                 (126 μM) 
                 6 
                 (20 μM) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Ascorbic acid 
                 1869 
                 (10618 μM) 
                 NA 
                 NA 
                 NA 
                 1906 
                 (10828 μM) 
                 NA 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 IC 50  (μg/mL) Values and Class of Combination 
               
            
           
           
               
               
               
               
            
               
                   
                 Compound alone or 
                 ATCC203333 
                 ATCC PRA-251 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 in combination 
                   
                 Class of 
                 Single 
                   
                   
                 Class of 
               
               
                   
                 with ascorbic acid 
                 ΣFIC 50   
                 Interaction 
                 Compound 
                 Combination 
                 ΣFIC 50   
                 Interaction 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Gallic acid 
                 0.38 
                 Synergy 
                 1255 
                 (7377 μM) 
                 276 
                 (1522 μM) 
                 0.32 
                 Synergy 
               
               
                   
                 Chebulic acid 
                 0.6 
                 Additive 
                 1220 
                 (3425 μM) 
                 446 
                 (1252 μM) 
                 0.52 
                 Additive 
               
               
                   
                 Glucohepatonic 
                 1.57 
                 Independent 
                 3536 
                 (20089 μM) 
                 3207 
                 (18209 μM) 
                 1.25 
                 Independent 
               
               
                   
                 acid lactone 
               
               
                   
                 Quinic acid 
                 0.73 
                 Additive 
                 1755 
                 (9133 μM) 
                 882 
                 (4590 μM) 
                 0.95 
                 Additive 
               
               
                   
                 Eujavonic acid 
                 0.64 
                 Additive 
                 1850 
                 (7768 μM) 
                 878 
                 (3687 μM) 
                 0.72 
                 Additive 
               
               
                   
                 HMDP 
                 0.81 
                 Additive 
                 2032 
                 (7635 μM) 
                 1058 
                 (3975 μM) 
                 1.1 
                 Independent 
               
               
                   
                 DPGA 
                 0.18 
                 Synergy 
                 72 
                 (238 μM) 
                 12 
                 (40 μM) 
                 0.18 
                 Synergy 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Ascorbic acid 
                 NA 
                 NA 
                 2850 
                 (16190 μM) 
                 NA 
                 NA 
                 NA 
               
               
                   
                   
               
               
                   
                 Restults are expressed as mean of three independent experiments with internal triplicate determinations (n = 9). Interactions classes are synergistic (ΣFIC 50  ≤ 0.5), additive (ΣFIC 50  &gt; 0.5-1.0), independent (ΣFIC 50  &gt; 1.0-4.0) or antagonistic (ΣFIC 50  &gt; 4.0). NA = results not available. 
               
            
           
         
       
     
     Most of the compounds produced only moderate to low G. duodenalis inhibitory activity, with IC 50  values &gt;1000 μg/mL. DPGA was a substantially more potent inhibitor of  G. duodenalis  proliferation than the other compounds, with IC 50  values for the different strains ranging from 38-72 μg/mL (126-238 μM). Interestingly, DPGA was a relatively minor component of the aqueous and methanolic  T. ferdinandiana  extracts, accounting for substantially less than 0.1% of the total extract&#39;s mass (results not shown) and it is therefore unlikely that DPGA alone would account for the strong activity of the crude aqueous extract (143 μg/mL). Instead, it is likely that other compounds in the extract synergise the activity of one or more of the anti-proliferative  T. ferdinandiana  compounds. 
     As  T. ferdinandiana  fruit has a very high ascorbic acid content, it is possible that ascorbic acid may have synergistic interactions with one or more of the the  T. ferdinandiana  compounds. Therefore, these compounds were further investigated in combination with ascorbic acid to identify any interactions which may occur. 
     Combinational Effects of the  T. Ferdinndiana  Compounds and Ascorbic Acid on  G. Duodenalis  Proliferation 
     A range of combinational effects were observed between  T. ferdinandiana  extract components and ascorbic acid (Table 9). 
     Of particular note, two combinations produced synergistic interactions (gallic acid+ascorbic acid; DPGA+ascorbic acid). Some combinations produced approximately 10 fold increases in activity compared to the activity of either compound alone. The increase in activity for DPGA in combination with ascorbic acid was particularly noteworthy against the sheep S2  G. duodenalis  strain, with IC 50  values decreasing from 47 μg/mL (156 M) alone, to 5 μg/mL (17 μM) in combination with ascorbic acid. A similar increase in potency was recorded against the metronidazole sensitive reference  G. duodenalis  strain (ATCC203333), with a decrease of IC 50  from 38 μg/mL (126 μM) alone, to 6 μg/mL (20 μM) in combination with ascorbic acid. Whilst slightly less potent against the metronidazole resistant  G. duodenalis  strain (ATCC PRA-251), the DPGA+ascorbic acid combination also produced clinically relevant IC 50  values of 12 μg/mL (40 μM). Substantial increases in potency were also recorded for the gallic acid+ascorbic acid combinations against all  G. duodenalis  strains. The decrease in IC 50  against the sheep S2 strain from 1150 μg/mL (6795 μM) alone, to 146 μg/mL (858 μM) in combination with ascorbic acid was notable. This combination was also synergistic against the other  G. duodenalis  strains. Interestingly, the IC 50  values were similar between both the metronidazole sensitive and resistant  G. duodenalis  strains, both with IC 50  values of approximately 250 μg/mL (1469 μM). 
     The majority of the other combinations produced additive effects. These combinations may therefore also be beneficial in the treatment of giardiasis, as they produce enhanced efficacy over either component when used separately. 
     A further combination (glucohepatonic acid lactone) was non-interactive. Whilst this combination does not provide any significant therapeutic benefit above that of either compound alone, the components also do not antagonise each other&#39;s effects and therefore it would not be detrimental if the two components were to be administered concurrently. Notably, none of the combinations produced antagonistic effects. 
     Synergistic Interactions Between Gallic Acid and Ascorbic Acid 
     As the gallic acid/ascorbic acid combination induced a synergistic interaction (Table 9), the combination was further examined using isobologram analysis across a range of gallic acid:ascorbic acid ratios to identify the ideal ratios to obtain synergy. 
     Similar susceptibility profiles were evident against all three  G. duodenalis  strains. 
     In all cases, the data correlated more closely with the gallic acid axis than with the ascorbic acid axis, indicating that the anti-proliferative activity is most reliant on the gallic acid. 
     However, whilst ratios containing between 30-60% gallic acid induced synergistic responses, the lower (20%) and higher ratios (70%) generally produced additive effects. 
     As these responses are greater than either of the individual components alone, they would therefore be beneficial for the treatment of giardiasis. 
     As synergy was determined using the ΣFIC 50  formula, synergy is defined as a response at least 4 times greater than that of the individual components alone. Thus the ratios which induce synergistic responses are far preferable as antigiardial therapies compared to the other ratios. Therefore, the ideal gallic acid/ascorbic acid ratios for the treatment of giardiasis can preferably include the combinations containing 30-60% gallic acid. 
     As shown by way of  FIGS. 13 a  to 13 c   , Isobolograms for combinations of gallic acid and ascorbic acid tested at various ratios against (a) the sheep S2, (b) reference metronidazole sensitive (ATCC203333) and (c) reference metronidazole resistant (ATCC PRA-251)  G. duodenalis  strains. GA=gallic acid; AA=ascorbic acid. Results represent mean FIC 50  values of three independent experiments, each consisting of 3 replicates (i.e. 9 data points for each ratio). Ratios lying on or underneath the 0.5/0.5 line are considered to be synergistic (Σ FIC 50 ≤0.5). Any points between the 0.5/0.5 and 1.0/1.0 lines are deemed to be additive (Σ FIC 50 &gt;0.5-1.0). 
     Synergistic Interactions Between 2,3-Dihydroxyphenyl-B-Gloucopyranosiduronic Acid (DPGA) and Ascorbic Acid 
     DPGA also induced synergistic  G. duodenalis  growth inhibition when tested in combination with ascorbic acid (Table 9). 
     The association between the growth inhibitory activity and the DPGA axis was even more pronounced than for gallic acid ( FIGS. 14 a  to 14 c   ), indicating that this compound is more important than ascorbic acid for the anti-proliferative activity of this combination. This is consistent with the IC 50  data for the compounds which reports the IC 50  of DPGA as approximately 5% of the IC 50  of ascorbic acid (Table 9). Thus, DPGA is approximately a 20 times more potent  G. duodenalis  growth inhibitor than ascorbic acid when the components were tested separately. Interestingly, all combinations containing ≤60% DPGA produced synergistic inhibition of the growth for the metronidazole sensitive  G. duodenalis  strains ( FIGS. 14 a  and 14 b   ). Therefore, ≥40% ascorbic acid is required to effectively synergise the effects of DPGA. 
     The growth inhibition isobologram against the metronidaxole resistant  G. duodenalis  strain displays a different trend ( FIG. 14 c   ). The majority of the combination ratios produced additive interactions against this strain. These ratios would still be beneficial for treating giardiasis as the growth inhibitory activity of the combination is greater than that of either component alone. However, whilst the treatment efficacy is increased for these ratios, the increase in relatively minor. In contrast, combinations containing 30-50% DPGA had substantially increased efficacy (≥4 fold increases in potency compared to the sum of the compounds tested alone). Therefore, the ideal synergistic ratio for the treatment and prevention of giardiasis against the metronidazole resistant  G. duodenalis  strain was identified to be 30-50% DPGA in combination with ascorbic acid. 
       FIGS. 14 a  to 14 c    show isobolograms for combinations of DPGA and ascorbic acid tested at various ratios against (a) the sheep S2, (b) reference metronidazole sensitive (ATCC203333) and (c) reference metronidazole resistant (ATCC PRA-251)  G. duodenalis  strains. DPGA =2,3-dihydroxyphenyl-B-gloucopyranosiduronic acid; AA=ascorbic acid. Results represent mean FIC 50  values of three independent experiments, each consisting of 3 replicates (i.e. 9 data points for each ratio). Ratios lying on or underneath the 0.5/0.5 line are considered to be synergistic (Σ FIC 50 ≤0.5). Any points between the 0.5/0.5 and 1.0/1.0 lines are deemed to be additive (Σ FIC 50 &gt;0.5-1.0). 
     Quantification of Toxicity 
     All extracts were screened across a range of concentrations using both the  Artemia nauplii  lethality assay (ALA) and a human dermal fibroblast assay (HDF) (Table 10). 
     For comparison, the reference toxin potassium dichromate (1000 μg/mL) was also tested. No LC 50  values are reported for purine, ribolactone, gluconolactone, glucohepatonic acid lactone, quinic acid, eujavonic acid, HMDP, or DPGA as less than 50% mortality was seen for all concentrations of these compounds tested in both assays. 
     All of these compounds were therefore deemed to be nontoxic. In contrast, gallic acid, chebulic acid and ascorbic acid displayed apparent toxicity in both assays following 24 hours exposure. However, it is noteworthy that the toxicity detected in our study generally correlated with acidic components. Acidic pH can suppress the rate of mitochondrial protein synthesis and potentially be fatal to the growth and development of both  Artemia nauplii  and HDF cells. Indeed, previous studies have reported that extracts high in ascorbic acid can provide fallacious toxicity determinations. Thus, this assay may have overestimated the toxicity of these compounds. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Table 10: Toxicity of the  T. ferdinandiana  compounds alone and in 
               
               
                 combination with ascorbic acid determined by  Artemia  lethality 
               
               
                 assay (ALA) and human dermal fibroblast (HDF) cytotoxicity assay. 
               
            
           
           
               
               
               
            
               
                   
                 Toxicity (μg/mL) 
                   
               
            
           
           
               
               
               
            
               
                   
                 Compound/ascorbic 
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Compound Alone 
                   
                 acid combination 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Compound 
                 ALA 
                 HDF 
                 ALA 
                 HDF 
               
               
                   
                   
               
               
                   
                 Purine 
                 CND 
                 CND 
                 NT 
                 NT 
               
               
                   
                 Gallic acid 
                 132 
                 320 
                  147 
                  336 
               
               
                   
                 Chebulic acid 
                 165 
                 380 
                  224 
                  375 
               
               
                   
                 Ribolactone 
                 CND 
                 CND 
                 NT 
                 NT 
               
               
                   
                 Ascorbic acid 
                 203 
                 358 
                 NA 
                 NT 
               
               
                   
                 Gluconolactone 
                 CND 
                 CND 
                 NT 
                 NT 
               
               
                   
                 Glucohepatonic 
                 CND 
                 CND 
                 &gt;500 
                 &gt;500 
               
               
                   
                 acid lactone 
               
               
                   
                 Quinic acid 
                 CND 
                 CND 
                 &gt;500 
                 &gt;500 
               
               
                   
                 Eujavonic acid 
                 CND 
                 CND 
                 &gt;500 
                 &gt;500 
               
               
                   
                 HMDP 
                 CND 
                 CND 
                 &gt;500 
                 &gt;500 
               
               
                   
                 DPGA 
                 CND 
                 CND 
                 &gt;500 
                 &gt;500 
               
               
                   
                 PC 
                  37 
                  42 
                 NT 
                 NT 
               
               
                   
                   
               
               
                   
                 PC = potassium dichromate; CND = could not determine as the mortality or % inhibition did not exceed 50% at any concentration tested; NT = not tested. 
               
            
           
         
       
     
     Therapeutic Index and Drug Like Properties 
     To determine the suitability of the  T. ferdinandiana  compounds as therapeutic agents, their drug-like properties were examined with reference to Lipinsky&#39;s rules of five. 
     All of the compounds had ≤10 H bond acceptors, molecular weights &lt;500 Da and octanol-water coefficients ≤5. The majority of the compounds also had ≤5 H bond donors. 
     The only compounds that violated this rule (chebulic acid and DPGA) included the compound with the greatest  G. duodenalis  anti-proliferative activity (DPGA), both alone and in combination with ascorbic acid. Both DPGA and chebulic acid have 6 H bond donors and therefore exceed Lipinsky&#39;s rules of five by one H bond donor. However, given their conformity in all other categories, these compounds were deemed to have good drug-like properties. 
     The therapeutic index (TI) was also calculated for the pure compounds and combinations. We were unable to calculate TI&#39;s for purine, ribolactone, gluconolactone, glucohepatonic acid lactone, quinic acid, eujavonic acid, HMDP and DPGA as none of these compounds displayed toxicity at any concentration tested. However, with the exception of DPGA, these compounds generally displayed only low  G. duodenalis  anti-proliferative activity and were therefore of little use therapeutically. For DPGA, this lack of apparent toxicity indicates that the compound would have a high TI and therefore be a promising drug-lead. If the dose range that DPGA was tested over was extended to test higher concentrations to determine an LC 50 , the TI would be relatively high. 
     An interesting trend was noted for the TI of gallic acid. The TI of this compound alone was relatively low (0.3) due to its apparent toxicity, indicating that it may have limited therapeutic potential. However, when the TI of gallic acid was determined in combination with ascorbic acid, it had increased substantially to 2.3. Thus, it is likely that ascorbic acid may provide dual benefits in combination with DPGA: it may synergise the anti-proliferative activity of DPGA, as well as protecting the cells against its toxicity. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Drug like properties and therapeutic index of the  T. ferdinandiana   
               
               
                 compounds alone and in combination with ascorbic acid. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 ≤5 H bond 
                 ≤10 H bond 
                 MW ≤500 
                 Octanol-water 
                 Therapeutic Index 
                 Therapeutic Index in 
               
               
                 Compound 
                 donors 
                 acceptors 
                 Da 
                 coefficient ≤5 
                 of Compound 
                 combination with AA 
               
               
                   
               
               
                 Purine 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 TBT 
                 NT 
               
               
                 Gallic acid 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 0.3 
                 2.3 
               
               
                 Chebulic acid 
                 No 
                 Yes 
                 Yes 
                 Yes 
                 0.3 
                 0.9 
               
               
                 Ribolactone 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 TBT 
                 NT 
               
               
                 Ascorbic acid 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 0.2 
                 NA 
               
               
                 Gluconolactone 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 TBT 
                 NT 
               
               
                 Glucohepatonic 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 TBT 
                 TBT 
               
               
                 acid lactone 
               
               
                 quinic acid 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 TBT 
                 TBT 
               
               
                 eujavonic acid 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 TBT 
                 TBT 
               
               
                 HMDP 
                 Yes 
                 Yes 
                 Yes 
                 Yes 
                 TBT 
                 TBT 
               
               
                 DPGA 
                 No 
                 Yes 
                 Yes 
                 Yes 
                 TBT 
                 TBT 
               
               
                   
               
               
                 TBT = the therapeutic index could not be determined as the toxicity was too low to determine an LC 50 ; NT = not tested in combination as the TI of the compound alone was inactive.