FIELD OF THE INVENTION

The invention relates to plant based pharmacological compositions, in particular but not exclusively to a method and use of an extract of genus Terminalia (particularly, T. ferclinancliana ) in the preparation of a medicament which inhibits and/or blocks the growth of gastrointestinal parasites of the genus Giardia (particularly, G. duodenalis) in humans, animals and as a treatment for potable water.

BACKGROUND OF THE INVENTION

Prior laboratory studies have indicated that pharmacologically active extracts of the fruit of the plant, Terminalia ferdinandiana , commonly also known as the Kakadu plum may be useful in the treatment of cancers and as a bactericide. The most recent and only study to date suggesting compositions of T. ferdinandiana may be used to treat protozoal conditions is contained in a scientific paper by the inventor published in Rayan, et.al. Parasitol.Res. 2015, April 16.

The present invention is a reduction to practice based on further research conducted solely by the inventor which is directed to the preparation of a medicament which inhibits and/or blocks growth of parasites of the genus, Giardia.

Giardiasis, is a disease caused by gastrointestinal protozoal parasites of the genus Giardia (particularly Giardia lamblia, Giardia duodenalis, and less frequently by Giardia intestinalis), which causes debilitating diarrhea in large numbers of people annually (Muhsen & Levine, 2012). A recent report estimated that 280 million people per year were diagnosed with giardiasis internationally (Esch & Petersen, 2013). Whilst the disease is most prevalent in developing countries and communities with untreated water supplies, inadequate sanitation and poor dietary status, it is also a significant problem in developed nations. A recent study estimated that approximately 20,000 cases are reported annually in the USA (Esch & Petersen, 2013). It is noteworthy that these figures are estimates of the reported cases only. As a large percentage of individuals with giardiasis are likely to be self-diagnosed/treated, this figure may significantly under estimate the impact of this disease.

The current drug of choice for the treatment of giardiasis is metronidazole (refer Figure 1) due to its efficacy, bioavailability and cost (Upcroft & Upcroft, 2001). However, metronidazole treatment is associated with a number of unpleasant side effects (including nausea, diarrhea, weight loss, abdominal pain and dizziness) and toxicities. It is best considered as a short term treatment only. A recent National Toxicology Program report (2011) by the US Department of Health and Human Services also listed metronidazole as 'reasonably anticipated to be a human carcinogen'. A further worrying trend is the emergence of metronidazole resistant strains of Giardia spp. (Upcroft et al., 2006). In a recent review (Watkins & Eckmann, 2014), the authors highlighted the importance of developing new giardiasis treatments: "Therefore, because of the prevalence of giardiasis and limited treatment options, the development of new agents is a high priority."

Recent studies have examined the anti-Giardial activity of conventional antimicrobials such as benzimidazoles (Hanevik et al., 2008) and novel synthetic compounds such as alpha-aminophosphonates (Staake et al., 2010). Unfortunately, whilst targeting giardiasis with purified antibiotics may initially appear a viable method of treatment, it may also be problematic as prolonged antibiotic treatment/exposure may result in the production of antibiotic resistant bacterial strains. Recent studies have begun to re-examine the use of complementary and alternative therapies including plant extracts used in traditional healing systems (Li et al, 2012), essential oils (Machado et al., 2011; Machado et al., 2010), and functional foods such as saturated fatty acid rich coconut milk (Rayan et al., 2005) to treat giardiasis.

A re-examination of traditional herbal medicines for the treatment of giardiasis is an attractive prospect as the antiseptic qualities of medicinal plants have been long recognized and recorded. There has recently been a revival of interest in herbal medications due to a perception that there is a lower incidence of adverse reactions to plant preparations compared to synthetic pharmaceuticals. Furthermore, the use of complex mixtures such as whole foods or extracts would minimize the risk of developing antibiotic resistant strains of enteric bacteria. Whilst ongoing prophylactic treatment with a single antibiotic would certainly result in resistant bacterial strains, the use of functional foods or plant extracts with potent antibacterial activity would be expected to contain several antibiotic compounds which would be likely to function via several different mechanisms. There is a growing trend in the animal husbandry industry to switch to the usage of crude plant extracts rather than using pure antibiotic compounds for this reason. A literature search has been unable to find any reports of any bacterial species developing resistance to crude plant extracts. Terminalia ferdinandiana (Kakadu plum) is an endemic Australia plant which has received recent interest due to its reported high antioxidant content (Konczak et al., 2010; Netzel et al.,

2007). The extremely high levels of ascorbic acid in Kakadu plum are particularly noteworthy, with levels reported as high as 6% of the recorded wet weight. This is approximately 900 times higher (g/g) than the ascorbic acid content in blueberries (which were used as a standard). As a further comparison, oranges and grapefruit (which are considered good sources of ascorbic acid) only contain approximately 0.007% wet weight (0.5% dry weight) (Johnson, 2003). Due to its high vitamin C levels, the primary use of T. ferdinandiana fruit is currently for production of vitamin C in health food, cosmetic, and pharmaceutical industries. However, T. ferdinandiana fruit also contains many other compounds which also contribute to its high antioxidant activity (Konczak et al., 2010; Netzel et al., 2007). While many of these compounds are yet to be identified, T. ferdinandiana fruit is known to contain benzoic acids, flavanols, or flavanones (Konczak et al., 2010). T. ferdinandiana fruit is a good source of gallic acid and ellagic acid (Cunningham et al., 2009; Cherikoff and Kowalski, 2008), which demonstrate strong antioxidant activity in vitro (Yilmaz & Toledo, 2004). Lipophilic T. ferdinandiana fruit extracts are also rich in lutein (a carotenoid antioxidant compound associated with eye health) and with vitamin E and vitamin E analogs (Konczak et al., 2010). Hesperitin, as well as the glycosides kaempferol, luteolin, and quercetin are some of the other antioxidants present in T. ferdinandiana fruit (Konczak et al., 2010). It has been postulated that the exceptionally high antioxidant content of T. ferdinandiana fruit may provide therapeutic effects for this plant (Mohanty & Cock, 2012). Indeed, studies within our laboratory have reported potent inhibition of bacterial growth by Kakadu plum fruit (Cock & Mohanty, 2011) and leaf extracts (Courtney et al., in press). However, despite the documented ability of Kakadu plum to inhibit prokaryotic cell growth, similar studies against eukaryotic infective agents are lacking.

It is therefore an object of the present invention which is to seek to eliminate or ameliorate the disadvantages and limitations of the prior art by providing an inventive and previously unknown use of one or more biologically active extracts of Terminalia genus to inhibit or block the growth of gastrointestinal protozoal parasites responsible for Giardiasis in humans and animals.

SUM MARY OF THE INVENTION

In one aspect, the invention resides in the use of a compound from an extract of plant genus Terminalia in the preparation of a medicament which inhibits and/or blocks the growth of protozoal gastrointestinal parasites of genus Giardia.

In another aspect, the invention resides in a method of treating Giardiasis by administering an effective dosage of the compound to humans or animals or supplies of potable water.

The compound preferably includes at least 50% of Gemfibrozil M l (5-(4-hydroxy-2,5-dimethylphenoxy)- 2,2-dimethyl-Pentanoic acid/C15 H22 04) and at least 50% of ascorbic acid.

The compound preferably also includes at least 50% of Gallic acid (C7H605) and at least 50% of ascorbic acid. The compound preferably also includes at least 50% of purine (C5 H4 N4) and at least 50% of ascorbic acid.

(The chemical compounds for this study were obtained from Banksia Chemicals Australia)

In one example, the plant used is Terminalia ferdinandiana , commonly known as the Kakadu plum and the extract is derived from the pulp of the fruit.

While the parasites of the present study are of the genus, Giardia and include particularly, Giardia duodenalis, Giardia duodenalis and Giardia intestinalis, it is possible that conditions caused by protozoal parasites of other genus may also be controlled by plant based extracts of Terminalia and subject to further investigation.

Where the extract is preferably administered by an oral route, it would be obvious that it could be administered intravenously via a drip in a sterile preparation.

While the extract could be added to a drinking container or supply of potable water, it could also be administered as a dosage by volume to control infection or proliferation of the microorganism in a communal water supply or reservoir.

The compound could also be administered as a pharmaceutical or a nutraceutical preparation in powder, tablet, pill or capsule form.

BRIEF DESCRIPTION OF THE DIAGRAMS.

In order that the invention is understood, reference is made to the accompanying figures and tables: FIG. 1 shows the structure of metronidazole

(https://en.wikipedia.Org/wiki/Metronidazole#/media/File:Met ronidazole.svg)

Table 1 shows the mass of extracted and antioxidant content of T. ferdinandiana fruit extracts. FIG. 2 shows inhibitory chemical compounds with their structures that have been identified previously from the fruit extracts of T. ferclinancliana targeting Giardia duodenalis according to the invention

Referring to Table 1 which shows putative identification of compounds in the T. ferdinondiono fruit extracts according to Name, Formula, Mass, RT, and Structure. Only compounds detected in the all of the methanolic, aqueous, ethyl acetate and chloroform extracts by high accuracy QTOF LC-MS are shown. Compounds also detected in the hexane extract are not listed.

Figure 5 shows chemical structures of T. ferdinondiono fruit compounds corresponding to Table 3 detected in the methanolic, aqueous, ethyl acetate and chloroform extracts, but not in the hexane extract including: (a) purine; (b) gallic acid; (c) methoxycarbonyloxymethyl methylcarbonate; (d)ribonolactone; (e) apionic acid; (f) (IS,5R)-4-0xo-6,8-dioxabicyclo[3.2.l]oct- 2-ene-2-carboxylic acid; (g) ascorbic acid; (h) gluconolactone; (i) quinic acid, (D glucuronic acid, (k) glucohepatonic acid-l,4-lactone; (1) eujavonic acid; (m) 5-(4-hydroxy-2,5-dimethylphenoxy)- 2,2-dimethyl-Pentanoic acid (Gemfibrozil Ml); (n) p-hydroxytiaprofenic acid; (0) 2,3- Dihydroxyphenyl B-D-glucopyranosiduronic acid; (p) ferulic acid dehydrodimer ; (q) chebulic acid.

FIG. 3 shows lethality of T. ferdinandiana fruit extracts and control towards Artemia franciscana nauplii.

FIG. 4 shows positive ion chromatograms of various extracts of T. ferdi

BACKGROUND DETAILED DESCRIPTION FROM THE PREVIOUS JOURNAL PUBLICATION

1. PRIOR ART TREATMENT FOR GIARDIA.

1.1 Metronidazole.

FIG. 1 shows the structure of metronidazole, the prior art 'gold standard' drug for the treatment of giardiasis which, as previously discussed, may be problematic wherein prolonged usage may result in the production of antibiotic resistant bacterial strains. Metronidazole treatment is associated with a number of unpleasant side effects (including nausea, diarrhoea, weight loss, abdominal pain and dizziness) and toxicity. It is best considered as a short term treatment only. A recent National Toxicology Program report (2011) by the US Department of Health and Human Services listed metronidazole as 'reasonably anticipated to be a human carcinogen'. A further worrying trend is the emergence of metronidazole resistant strains of Giardia spp. (Upcroft et al., 2006).

2. Materials and methods of extraction of anti- Giardia compounds of T. ferdinandiana

2.1. T. ferdinandiana fruit pulp samples from the Northern Territory, Australia were initially frozen for transport and stored at -10°C until processed.

2.2. Preparation of the extracts.

T. ferdinandiana fruit pulp was thawed at room temperature and dried in a food dehydrator. The dried pulp material was subsequently ground to a coarse powder. A mass of 1 g of ground dried pulp was extracted extensively in 50 ml of methanol, deionized water, ethyl acetate, chloroform or hexane for 24 hours at 4 °c with gentle shaking. The extracts were filtered through filter paper (Whatman No. 54). The solvent extracts were air dried at room temperature. The aqueous extract was lyophylised by rotary evaporation in an Eppendorf concentrator. The resultant pellets were dissolved in 10 ml deionised water. The extract was passed through 0.22 micron filter (Sarstedt) and stored at 4°C. 2.3. Qualitative phytochemical studies.

Phytochemical analysis of the T. ferdinandiana extracts for the presence of saponins, phenolic compounds, flavonoids, polysteroids, triterpenoids, cardiac glycosides, anthraquinones,tannins and alkaloids was conducted by previously described assays (Arkhipov et al., 2014;Kalt & Cock, 2014).

2.4. Antioxidant capacity.

The antioxidant capacity of each sample was assessed using the DPPH free radical scavenging method (Jamieson et al., 2014; Winnett et al., 2014) with modifications. Briefly, DPPH solution was prepared fresh each day as a 400 micron solution by dissolving DPPH (Sigma) in AR grade methanol (Ajax, Australia). The initial absorbance of the DPPH solution was measured at 515 nm using a Molecular Devices, Spectra Max M3 plate reader and did not change significantly throughout the assay period. A 2 ml aliquot of each extract was evaporated and the residue re- suspended in 2 ml of methanol. Each extract was added to a 96-well plate in amounts of 5, 10, 25, 50, 75 microliters in triplicate. Methanol was added to each well to give a volume of 225 microliters. A volume of 75 microliters of the fresh DPPH solution was added to each well for a total reaction volume of 300 microliters. A blank of each extract concentration, methanol solvent, and DPPH was also performed in triplicate. Ascorbic acid was prepared fresh and examined across the range 0-25 micrograms per well as a reference and the absorbance were recorded at 515 nm. All tests were performed in triplicate and triplicate controls were included on each plate. The antioxidant capacity based on DPPH free radical scavenging ability was determined for each extract and expressed as microgram ascorbic acid equivalents per gram of original plant material extracted.

2.5. Inhibitory bioactivity against Giardia duodenalis trophozoites. 2.5.1. Parasite culture.

The Giardia duodenalis S-2 (sheep strain 2) trophozoite strain used were maintained and sub- cultured anaerobically at 37°C in TYI-S-33 growth media supplemented with 1% bovine bile (Sigma), 10 % Serum Supreme (Cambrex Bioproducts) and 200 lU/ml penicillin/200 micrograms/ml streptomycin (Invitrogen, USA). Confluent mid log phase cultures were passaged every 2 days by chilling the cultures on ice for a minimum of 10 min, followed by vortexing to dislodge the adherent trophozoites from the walls of the culture vessel. Fresh culture media (5 ml) was seeded with approximately 1 x 10(5) trophozoites for each passage.

2.5.2. Evaluation of ant\-Giardia) activity by direct parasite enumeration.

Anti-Giardial activity of the extracts was assessed by direct enumeration of parasite numbers in the presence or absence of extracts (Hart et al., 2014). For each test, aliquots of the trophozoite suspension (70 microliters) containing approximately 1 x 10(5) trophozoites were added to the wells of a 96 well plate. A volume of 30 microliters of the test extracts or the vehicle solvent or culture media (for the negative controls) was added to individual wells and the plates were incubated anaerobically at 37°C for 8 hours in a humidified anaerobic atmosphere. Following the 8 h incubation, all tubes were placed on ice for a minimum of 10 min, followed by vortexing to dislodge the adherent trophozoites from the walls of the culture vessel. The suspensions were mounted onto a Neubauer haemocytometer (Weber, UK) and the total trophozoites per ml were determined. The anti-proliferative activity of the test extracts was determined and expressed as a % of the untreated control trophozoites per ml.

2.5.3. Determination of IC50 values against Giardial trophozoites.

For IC50 determinations, the plant extracts were tested by the direct enumeration method across a range of concentrations. The assays were performed as outlined above and graphs of the zone of inhibition versus concentration were plotted for each extract. Linear regression was used to calculate the IC50 values.

2.6. Toxicity screening.

2.6.1. Reference toxin for toxicity screening.

Potassium dichromate (K2Cr207) (AR grade, Chem-Supply, Australia) was prepared as a 1.6 mg/ml solution in distilled water and was serially diluted in artificial seawater for use in the Artemia franciscana nauplii bioassay.

2.6.2. Artemia franciscana nauplii toxicity screening.

Toxicity was tested using a modified Artemia franciscana nauplii lethality assay (Arkhipov et.al.

2014; Kalt & Cock, 2014). Briefly, 400 microliters of seawater containing approximately 43 (mean 43.2, n = 155, SD 14.5) A. franciscana nauplii were added to wells of a 48 well plate and immediately used for bioassay. A volume of 400 microliters of diluted plant extracts or the reference toxin were transferred to the wells and incubated at 25 ± 1°C under artificial light (1000 Lux). A negative control (400 microliters 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 72 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.

2.7. HPLC-MS/MS analysis.

Chromatographic separations were performed using 2 microliter injections of sample onto an Agilent 1290 HPLC system fitted with a Zorbax Eclipse plus C18 column (2.1 x 100 mm, 1.8 micrometer 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 utilized for the study consisted of the first 5 min run isocratically at 5% B, a gradient of (B) from 5% to 1000/0 was applied from 5 min to 30 min, followed by 3 min isocratically at 100%. Mass spectrometry analysis was performed on an Agilent 6530 quadra pole time-of-flight spectrometer fitted with a Jetstream electrospray ionisation source in both positive and negative mode.

Data was analyzed using the Masshunter Qualitative analysis software package (Agilent Technologies). Blanks using each of the solvent extraction systems were analyzed 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 analyzed 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); the Metlin metabolomics database (24,768 compounds); and the Forensic Toxicology Database by Agilent Technologies (7,509 compounds). Empirical formula for unidentified compounds was determined using the Find Formula function in the software package.

2.8. Statistical analysis.

Data are expressed as the mean ± SEM of at least three independent experiments. T test values were calculated and determined to be >0.5 which is considered to be statistically significant.

DETAILED DESCRI PTION OF CURRENT TRIAL WITH TH E ACTIVE CHEMICALS IDENTI FIED FROM TABLE 1

Parasite culture.

The Giardia duodenalis S-2 (sheep strain 2) trophozoite strain used were maintained and sub- cultured anaerobically at 37°C in TYI-S-33 growth media supplemented with 1% bovine bile (Sigma), 10 % Serum Supreme (Cambrex Bioproducts) and 200 lU/ml penicillin/200 micrograms/ml streptomycin (I nvitrogen, USA). Confluent mid log phase cultures were passaged every 2 days by chilling the cultures on ice for a minimum of 10 min, followed by vortexing to dislodge the adherent trophozoites from the walls of the culture vessel. Fresh culture media (5 ml) was seeded with approximately 1 x 10(5) trophozoites for each passage.

Evaluation of anti-Giardia) activity by direct parasite enumeration.

Anti-Giardial activity of the extracts was assessed by direct enumeration of parasite numbers in the presence or absence of extracts (Hart et al., 2014). For each test, aliquots of the trophozoite suspension (70 microliters) containing approximately 1 x 10(5) trophozoites were added to the wells of a 96 well plate. A volume of 30 microliters of the test extracts or the vehicle solvent or culture media (for the negative controls) was added to individual wells and the plates were incubated anaerobically at 37°C for 8 hours in a humidified anaerobic atmosphere. Following the 8 h incubation, all tubes were placed on ice for a minimum of 10 min, followed by vortexing to dislodge the adherent trophozoites from the walls of the culture vessel. The suspensions were mounted onto a Neubauer haemocytometer (Weber, UK) and the total trophozoites per ml were determined. The anti-proliferative activity of the test extracts was determined and expressed as a % of the untreated control trophozoites per ml.

2.5.3. Determination of IC50 values against Giardial trophozoites. For IC50 determinations, the chemical compounds were tested individually followed by various combinations at various ratio by the direct enumeration method across a range of concentrations. The assays were performed as outlined above and graphs of the zone of inhibition versus concentration were plotted for each extract. Linear regression was used to calculate the IC50 values.

3. RESULTS.

For this experiment commercially available known chemicals were purchased from Banksia chemicals Australia.

3.1. Liquid extraction from the previous study yields and qualitative phytochemical screening were the used as the basis of this study where the chemicals were commercially obtained and chosen from the list in table 1 and figure 2 that showed putative identification of compounds in the T. Ferdinandiana fruit extracts according to Name, Formula, Mass, RT, and Structure. Only compounds detected in the all of the methanolic, aqueous, ethyl acetate and chloroform extracts by high accuracy QTOF LC-MS are shown. Compounds also detected in the hexane extract are not listed.

Table 2 refers to the mass of dried extracted material, the concentration after re-suspension in deionized water, qualitative phytochemical screenings and antioxidant contents of T. ferdinandiana fruit extracts.

Extraction of 1 g of dried J. ferdinondiono fruit with various solvents yielded dried plant extracts ranging from 30 mg (ethyl acetate extract) to 483 mg (water extract) (Table 1). Deionized water and methanol gave relatively high yields of dried extracted material, whilst all other solvents extracted lower masses. The dried extracts were re-suspended in 10 ml of deionized water resulting in the extract concentrations shown in Table 1. Qualitative phytochemical studies (Table 1) showed that methanol and water extracted the widest range of phytochemicals. Both showed high levels of phenolic (both water soluble and insoluble phenolic) and flavonoids, as well as moderate to high levels of tannins. Saponins were also present in low to moderate levels. Triterpenes and alkaloids were also present in low levels in the methanol extract. The ethyl acetate extract also had moderate levels of phenolics, flavonoids and triterpenes as well as low levels of saponins. Low levels of phenolics were detected in the chloroform extract whilst no phytochemical class was present in detectable levels in the hexane extract.

3.2. Antioxidant capacity.

Antioxidant capacity (expressed as ascorbic acid equivalence) for the T. ferdinandiana fruit extracts are shown in Table 1. The antioxidant capacity ranged from a low of 1 mg ascorbic acid equivalence per gram of dried plant material extracted (hexane extract) to a high of 660 mg ascorbic acid equivalence per gram of dried plant material extracted (methanol extract).

Whilst significantly lower than the methanol extract, the aqueous extract a lso had a high antioxidant capacity with 264 mg ascorbic acid equivalence per gram of dried plant material extracted.

3.3. Inhibition of Giardia duodenalis proliferation.

T. ferdinandiana fruit extracts were screened for their ability to inhibit Giardia duodenalis growth (Figure 2). The methanol, water, ethyl acetate and chloroform extracts displayed significant inhibitory activity. The hexane extracts was completely ineffective as an inhibitor of proliferation, with no significant difference to the untreated control levels. The methanolic and aqueous extracts were particularly potent, each inhibiting 100 % of the Giardial growth (compared to the untreated control). Furthermore, both of these extracts were extremely rapid in producing their inhibitory activity, with 100 % inhibition seen in less than 5 min of exposure (unpublished results). The ethyl acetate and chloroform extracts also significantly inhibited trophozoite growth, albeit with a much lower efficacy than was evident for the methanolic and aqueous extracts (to approximately 76 and 71 % of the growth of the negative controls respectively).

Figure 3 shows inhibitory activity of T. ferdinondiono fruit extracts against Giordio duodenolis trophozoites measured as a percentage the untreated control wherein

M = methanolic extract; W = water extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract; PC = metronidazole control (50 micrograms/ml); NC = negative control Results are expressed as mean ± SEM of at least triplicate determinations. * indicates results that are significantly different to the untreated control (p<0.01).

The inhibitory T. ferdinondiono extracts were further tested over a range of concentrations to determine the IC50 -values (Table 2) for each extract against G. duodenolis. Inhibition of trophozoite growth was dose-dependent, with the level of inhibitory activity decreasing at lower concentrations. The water extract was a particularly good inhibitor of G. duodenolis proliferation, with an IC50 of 143 micrograms/ml. The methanol extract, whilst less potent, also displayed good anti-Giardial activity (at approximately 704 micrograms/ml respectively). We were unable to determine IC50 values for the ethyl acetate and chloroform extracts as the levels of inhibition did not exceed 50 % at any concentration tested. However, it is noteworthy that the ethyl acetate extract was at a low concentration (3 mg/ml, which equates to 900 micrograms/ml tested in the assay). Table 3 shows the T. ferdinondiono fruit extract concentrations which inhibit 50 % of G. duodenalis growth (IC50) (micrograms/ml) or induce 50 % mortality in the Artemia nauplii bioassay (LC50 values) (micrograms/ml).

3.4. Quantification of toxicity.

T. ferdinandiana fruit extracts were initially screened at 2000 micrograms/ml in the assay (Figure 2). For comparison, the reference toxin potassium dichromate (1000 micrograms/ml) was also tested in the bioassay. The potassium dichromate reference toxin was rapid in its onset of mortality, inducing mortality within the first 3 hours of exposure and 100 % mortality was evident following 4-5 hours (unpublished results). The methanol and water extracts also induced significant mortality following 24 h exposure, indicating that they were toxic at the concentration tested. The ethyl acetate, chloroform and hexane extracts did not induce mortality significantly different to the seawater control and were therefore deemed to be nontoxic.

Figure 3 shows the lethality of T. ferdinandiana fruit extracts (1000 micrograms/ml) and potassium dichromate control (1 000 ~g/ml) towards Artemia franciscana nauplii after 24 hours exposure wherein M = methanolic extract; W = water extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract; PC = potassium dichromate control (1000 micrograms/ml); NC = negative (seawater) control. Results are expressed as mean ± SEM of at least triplicate determinations.

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 2 shows the LC50 values of the T. ferdinandiana fruit extracts towards A. franciscana. No LC50 values are reported for the ethyl acetate, chloroform and hexane extracts as less than 50 % mortality was seen for all concentrations tested. Extracts with an LC50 greater than 1000 micrograms/ml towards Artemia nauplii have been defined as being nontoxic in this assay (Cock & Ruebhart, 2009). As none of the extracts had a LC50 <1000 micrograms/ml, all were considered nontoxic.

3.5. HPLC-MS/MS analysis.

Figure 4 shows positive ion RP-HPLC total compound chromatograms (TCC) of 2 microliter injections of T. ferdinandiana fruit (a) methanolic extract; (b) aqueous extract; (c) ethyl acetate extract; (d) chloroform extract; (e) hexane extract.

As the methanolic, aqueous, ethyl acetate and chloroform extracts all displayed anti-Giardial activity, yet the hexane extract did not, they were further examined by high accuracy HPLCMS QTOF (Arkhipov et al., 2014). The individual extract compound profiles were subsequently compared to identify any compounds that were common across these extracts, yet not present in the hexane extract (which did not display any anti-Giardial activity) as this has previously been shown to be an effective method of narrowing the focus of compounds responsible for a bioactivity (Cock & Matthews, in press). The resultant total compound positive ion chromatograms are presented in Figure 4a-e.

The J. ferdinandiana methanolic (Figure 4a) and aqueous extract (Figure 4b) positive ion base peak chromatogram revealed multiple overlapping peaks, particularly in the early stages of the chromatogram corresponding to the elution of polar compounds. Most of the extracted compounds had eluted in the first 10 minutes, corresponding to 5-25 % acetonitrile. Indeed, much of the peaks eluted in the first 5 minutes during the isocratic stage of the chromatogram (5 % acetonitrile). However, the presence of several prominent peaks between 10 and 16 min indicates the broad spread of polarities of the compounds in this extract. The ethyl acetate, chloroform and hexane extracts are characterized by much fewer peaks than evident in the methanolic and aqueous extracts, and a shift towards elution at high acetonitrile percent. In particular, a prominent peak is present in the ethyl acetate, chloroform and hexane extracts (as well as the methanolic extract) at approximately 15.5-16 min.

3.6. Qualitative mass spectral analysis of . ferdinandiana fruit extracts.

In total, 1116 unique mass signals were noted across the five J. ferdinandiana fruit extracts (results not shown). Of these, only 19 mass signals were present in all of the methanolic, aqueous, ethyl acetate and chloroform extracts, but not in the hexane extract. Putative empirical formulas were achieved for all of these compounds. Of the 19 unique molecular mass signals detected across these extracts, 17 compounds (89 %) were putatively identified by com parison to the Metlin metabolomics, forensic toxicology (Agilent) and phytochemicals (developed in this laboratory) databases (Table 3). Their structures are shown in Figure 2.

3.7 Bioassay of efficacy of individual compounds against Giardia duodenalis (- Not active; N/A not available)

3.8a Bioassay of efficacy of compounds in combination with a possible synergistic composition of compounds against Giardia duodenalis (- Not active; N/A not available; * significant activity observed)

3.8b Bioassay of efficacy of compounds with ascorbic acid in a possible synergistic composition compounds against Giardia duodenalis

( - Not active; * significant activity observed)

4. Discussion

Giardiasis is a major cause of infectious diarrhoea in humans and livestock worldwide. There are currently a limited range of drugs available for chemotherapeutic treatment of this disease, with the majority of these used only following clinical diagnoses and generally not for prophylaxis. These drugs are ineffective against some of the life stages of the pathogenic protozoa, have unpleasant and unwanted side effects and may have limited availability in developing countries. Frequent reports of drug toxicity, treatment failure and parasite resistance also highlight the importance to develop new chemotherapeutic treatments with greater efficacy and less severe side effects. Recent studies have highlighted the potential of plant medicines and have demonstrated that some plant components are very effective inhibitors of G. duodenalis growth, with similar potency to the gold standard drug metronidazole (Rayan et al. 2005). Our studies demonstrate that 1: ferdinandiana fruit extracts also possess significant G. duodenalis growth inhibitory activity. IC50 values of approximately 700 and 140 micrograms/ml are reported for the methanolic and aqueous fruit extracts respectively. Also noteworthy was the rapid action of these extracts, with both the methanolic and aqueous extracts blocking 100 % of G. duodenalis growth within 5 minutes of exposure. Furthermore, all T ferdinandiana fruit extracts were nontoxic in the Artemia nauplii bioassay, further demonstrating their suitability for chemotherapeutic treatment and prophylactic prevention of giardiasis. The phytochemical composition of all T ferdinandiana fruit extracts was determined and compared to identify compounds common between extracts with G. duodenalis growth inhibitory activity, but not present in extracts lacking this activity. Similar metabolomics comparison studies have previously been used very successfully to narrow the focus of phytochemicals and allow for the identification of bioactive components in extracts from other plant species.

In a recent study examining the anti-viral activity of Scaevola spinescens, a comparison of the metabolomic profiles of solvents of varying polarities was able to highlight 2 compounds from 239 detected mass signals as possibly contributing to this activity (Cock & Matthews, in press). Of these 2 compounds, 1 had been previously been reported to have anti-viral activity, validating this approach.

High accuracy QTOF HPLC-MS was used to examine the metabolomics profiles of the various T. ferdinandiana fruit extracts in our study. Liquid chromatography - mass spectroscopy (LC-MS) is a good choice for the analysis of compounds of a wide variety of compounds, particularly those of medium and high polarity. Coupling LC with high mass accuracy spectroscopy techniques using both mild ionization and electrospray ionization (ESI) can generate large amounts of useful information for compound identification and metabolomic analysis. Using these methods, molecular ions can be detected and their empirical formulas accurately determined and compared to databases. Furthermore, coupling this with ESI analysis also allows for the detection and characterization of characteristic fragments, allowing for rapid identification of unknown compounds in a crude extract. A number of interesting compounds were identified in all extracts displaying anti-Giardial activity, that were also absent in the inactive hexane extract. Purine (Figure 5a) was putatively identified in all of the inhibitory extracts. Numerous studies have reported that Giardia duodenalis are unable to synthesize their own purine or pyrimidine nucleotides (Baum et al., 1989; Jarroll et al., 1989). Instead, G. duodenalis are reliant on salvage pathways to supply them with nucleotides for nucleic acid synthesis. These studies also reported that G. duodenalis are incapable of inter conversion between purine nucleotides. Furthermore, purine analogues have been reported to inhibit the growth of G. duodenalis and have been proposed as potential chemotherapeutic agents to treat Giardiasis (Berens & Marr, 1986). It is therefore possible that the G. duodenalis examined in our studies may incorporate the purine analogue identified in the inhibitory extracts into their nucleic acids during replication, causing DNA mismatches and blocking Giardial proliferation.

The inhibitory extracts also contained a relative abundance of the gallotannin components gallic acid (Figure 5b) and chebulic acid (Figure 5q). Gallotannins have been reported to inhibit the growth of a broad spectrum of microbial species (Buzzini et al., 2008) through a variety of mechanisms including binding cell surface molecules including lipotoichoic acid and proline-rich cell surface proteins (Wolinsky & Sato, 1984; Hogg & Embery, 1982), and by inhibiting glucosyltransferase enzymes (Wu-Yan et al., 1988). Whilst we were unable to find similar studies reporting inhibitory effects of tannins on Giardia spp. growth, a number of studies have reported high tannin contents in a variety of plants used in traditional medicine to treat giardiasis (Tapia- Perez et al., 2003). Furthermore, tannins extracted from other plant species inhibit the growth of other protozoa including Schistosoma mansoni (the parasite responsible for schistosoma) (Abozeid et al., 2012).

Of the remaining compounds putatively identified in all inhibitory extracts, the majority of these contain lactone moieties. These include ribonolactone (Figure 5d), (IS,5R)-4-0xo-6,8dioxabicyclo[ 3.2.1]oct-2-ene-2-carboxylic acid (Figure 5f), ascorbic acid (Figure 5g), gluconolactone (Figure 5h), glucuronic acid (Figure 5j), glucohepatonic acid-l,4-lactone (Figure 5k), p-hydroxytiaprofenic acid (Figure 5n) and 2,3-dihydroxyphenyl B-D-glucopyranosiduronic acid (Figure 50). This is a noteworthy finding as many of the current chemotherapeutic drugs used to treat giardiasis are lactone containing compounds, particularly lactone substituted nitroimidazoles (eg. metronidazole, secnidazole, tinidazole, ornidazole and albendazole). It has been suggested that compounds containing a lactone moiety may block the Giardial-lipid deacylation/reacylation pathways (Das et al. 2001). As

Giardia spp. are unable to synthesize lipids by de novo pathways, they use host gastrointestinal precursor lipids for the synthesis of membrane and cellular lipids by deacylation/reacylation reactions (Das et al. 2001). Thus, it is likely that lactone containing compounds may contribute to the inhibition of G. duodenalis growth by the blockage of lipid synthesis and metabolism pathways.

Quinic acid (Figure 5i) was also identified in all anti-Giardial T. ferdinandiana fruit extracts.

Recent studies have reported that substituted quinic acid compounds block leucyl-tRNA synthase activity in G. duodenalis (Zhang et al., 2012). Aminoacyl-tRNA synthases are essential for translation of the genetic code by attaching the correct amino acid to each tRNA. Thus, blockage of leucyl-tRNA synthase activity would result in ineffective Leu-tRNA production and thus the inhibition of protein synthesis. We were unable to find reports of non-substituted quinic acid having the same activity. However, if subsequent testing confirms this activity, it is possible that quinic acid may also contribute to the anti- Giardial activity of the T. ferdinandiana fruit extracts. We were unable to find reports of Giardia spp. growth inhibitory activity for any of the other compounds putatively identified in the inhibitory T. ferdinandiana fruit extracts. However, it is possible that-these may also contribute to the anti-Giardial activity reported here.

FINDINGS OF THIS RESEARCH:

Individually, and collectively, none of the chemicals had any effect on the protozoan parasite Giardia duodenalis (refer table 3.7). Except, in particular combinations of known concentrations, of the following compounds which proved to inhibit the parasite (refer table 3.8a). The three potential chemicals, Gemfibrozil Ml (5-(4-hydroxy-2,5-dimethylphenoxy)-2,2-dimethyl- Pentanoic acid/C15 H22 04); Gallic acid (C7H605) and purine (C5 H4 N4) worked in conjunction with ascorbic acid (C6 H8 06) at equal concentrations(refer table 3.8b).

At their highest concentrations in an equal ratio of ascorbic acid; gemfibrozil had an inhibition ratio of 71.5%, for Gallic acid it was 54.3% and for purine 65.3% refer table 3.8 At increasing concentration there was a linear decrease in parasite numbers, for the combination of ascorbic acid with Gallic acid or purine the inhibitory concentration required to inhibit 50% of the parasite i.e. IC50 is approx. 0.5mg/ml

Table 4.1 Structure of the active chemical compounds that demonstrated anti giardial activity in this trial.

But in the case of gemfibrozil further research is required as there appears to have an extremely complex synergistic activity, as at the highest concentration there was extreme activity but any change in its concentration resulted in an absence of activity. The ascorbic acid within these equations kept the compounds of choice in a reduced state which enabled them to have an increased antiparasitic activity. Further research is required to elaborate on the quantifying ratios and concentrations of gemfibrozil and its relationship with ascorbic acid as an antiparasitic agent.

This research is the first of its kind to test these potential chemical compounds against the protozoan parasite Giardia duodenalis.

5. CONCLUSION

The lack of toxicity and potent G. duodenalis growth inhibitory activity of the T. ferdinandiana fruit extracts demonstrate their potential as therapeutic agents for the treatment of Giardiasis.

At their highest concentrations the potential chemical compounds within an equal ratio of ascorbic acid; gemfibrozil had an inhibition ratio of 71.5%, for Gallic acid it was 54.3% and for purine 65.3%. The chemical compounds also demonstrated an IC50 of 500ug/ml (refer table 3.8.) which is within the therapeutic ratio of potential chemotherapeutic agents for antigiardial activity.

It will of course be realized that while the foregoing has been given by way of illustrative example of this invention, all such and other modifications and variations thereto as would be apparent to persons skilled in the art are deemed to fall within the broad scope and ambit of this invention as is herein set forth.

In the specification the terms "comprising" and "containing" shall be understood to have a broad meaning similar to the term "including" and will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the terms "comprising" and "containing" such as "comprise", "comprises", "contain" and "contains".

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