Repurposing Common Food Additives (Benzo Derivatives) As New Anti-parasitic Agents

This study examined the anti-protozoal effects of selected benzo derivatives, namely ten gallic acid (GA) alkyl esters (viz., benzoic acid analogs) and twenty-three benzaldehyde analogs, against six different anaerobic human protozoal pathogensTrichomonas vaginalis, Tritrichomonas foetus, Tritrichomonas foetus-like, Giardia lamblia, Entamoeba histolytica, and Naegleria fowleri. The efficacy of benzaldehyde and gallate (3,4,5-trihydroxybenzoic acid) analogs were investigated in two respects: (1) changing types of side chains and their positions on the benzaldehyde ring [structure–activity relationships (SAR)]; and, (2) changing lengths of alkyl chains esterified with the carboxyl group on gallate. Results of parasite growth inhibition assays indicated that benzo derivatives could be further developed as potent anti-protozoal drug candidates/leads, where GA having longer alkyl chains exhibited higher anti-protozoal activity than compounds with shorter alkyl chains or all benzaldehyde analogs tested. The chemical libraries were also screened against common human microbiome bacteria and no detectable inhibition was observed. Structure-activity relationships and their implications for new drug discovery against these sexually-transmitted, water-borne, and foodborne parasites are discussed.


Introduction
Gallic acid (GA; 3,4,5-trihydroxybenzoic acid) is a redox-active polyphenol, viz., possessing both antioxidant and prooxidant potential, which is abundantly found in many plants (Badhani et al., 2015). Redox-active phenolics can serve as potent redox-cyclers in pathogens, hence function as effective antimicrobial agents by disrupting redox homeostasis and/or redox-sensitive structures in the cell (Guillen and Evans, 1994;Jacob, 2006). In addition to its free acid form, plant hydrolyzable tannins are important source of GA, which could also function as a natural anti-mycotoxigenic agent (Mahoney and Molyneux, 2004). Since oxidative stress triggers aflatoxin production in Aspergillus sp. (Jayashree and Subramanyam, 2000), the anti-aflatoxigenic effect of GA could result from the antioxidant potential of the chemical (Kim et al., 2005). Besides, GA has also been documented for its pharmacological effects, such as anticancer (Kosuru et al., 2018), antiinflammatory (Kroes et al., 1992) and anti-diabetic complication activity (Huang et al., 2018).
While GA has long been studied for control of bacterial pathogens (Kubo et al., 2002a;Wang et al., 2017), pathogen-inhibitory effect of GA has also been investigated in lower eukaryotes, such as yeast or filamentous fungal pathogens. For instance, administration of GA prevented the growth of Candida albicans (yeast) or Trichophyton rubrum (filamentous fungus), where GA disrupted ergosterol biosynthesis (thus interfering with the integrity of cellular membrane) by lowering the activity of sterol 14α-demethylase P450 (CYP51) and squalene epoxidase in the membrane (Li et al., 2017).

Introduction (Continued)
Of note, methods have been developed for the enhancement of bioavailability or biologically relevant application of GA. For example, to improve GA solubility for treating C. albicans biofilms, antifungal efficacy of GA/hydroxypropyl-β-cyclodextrin complex (HPβCD) has been examined (Teodoro et al., 2017). Results showed that GA/HPβCD complex was active against C. albicans biofilms, where the complex preserved the antifungal potency of the pure GA while it also exerted anti-inflammatory activity.
However, despite its potential as an effective antimicrobial agent, the level of antifungal activity of GA is still not comparable to conventional drugs or fungicides. One of the strategies for potency improvement of lead compounds is a structural modification of chemical backbones. To enhance the antifungal efficacy of GA, the effect of incorporating alkyl esters into GA backbone has been studied by changing the length of alkyl esters, as follows: (1) GA alkyl esters, such as octyl gallate (OG), reduced fluidity of the fungal membrane and/or inhibited the activity of H(+)-ATPase, thus prevented the growth of fungal growth with enhanced GA potency (Fujita and Kubo, 2002a), (2) Tests using mutants of Saccharomyces cerevisiae (model yeast) or Aspergillus fumigatus (causative agent of human invasive aspergillosis) indicated that GA alkyl derivatives also negatively affected fungal antioxidant systems, where S. cerevisiae mitochondrial superoxide dismutase mutant or A. fumigatus antioxidant mitogen-activated protein kinase (MAPK) mutant were highly susceptible to alkyl gallates (Kim et al., 2010a(Kim et al., & 2010b, (3) In addition to inhibiting Introduction (Continued) antioxidant system, OG also negatively affected cell wall-integrity system of fungi (Kim et al., 2014), which was determined via the inhibition of S. cerevisiae cell wall-integrity MAPK pathway mutants, (4) Antimicrobial activity of alkyl gallates has been quantified against Aspergillus brasiliensis (mold) and also Pseudomonas aeruginosa (Gram negative bacterium) with a hazard assessment (Buckley et al., 2017). OG was determined to possess better antimicrobial activity with lower hazards, when compared to other alkyl derivatives or conventional preservatives (Buckley et al., 2017), (5) Meanwhile, in a separate study, dodecyl gallate (DG) was determined as the most effective alkyl gallate for control of the yeast pathogen C. albicans (Singulani et al., 2017), where GA and DG were the least toxic compounds comparing to other derivatives (Zebrafish embryo model).
Similar to the benzoic acid (-COOH) analogs, as described above, certain benzaldehyde (-CHO) analogs could also be developed as redox-active antimicrobial agents. Benzaldehyde is also a natural product, which can be isolated from crops such as almond (Geng et al., 2016).

Introduction (Continued)
Prior study in fungal pathogens showed that benzaldehyde derivatives, such as cinnamaldehyde, could target cellular antioxidant system (viz., MAPK pathway, antioxidant enzymes including Cu,Zn-SOD, Mn-SOD, or glutathione reductase), where the growth of fungal mutants lacking genes in antioxidant system was greatly inhibited (Kim et al., 2011). Other benzaldehyde analogs, such as 2-hydroxy-4-methoxybenzaldehyde, further inhibited the growth of cell wall integrity MAPK mutants of the model yeast S. cerevisiae (Kim et al., 2015).
Anti-protozoal effect of benzaldehyde-containing drug candidates have been investigated, where 1,3,4-thiadiazole derivatives of R-(+)-limonene benzaldehydethiosemicarbazones triggered T. cruzi death by causing cellular oxidative stress (Martins et al., 2016). Study showed these compounds increased the level of mitochondrial-derived superoxide radicals, total reactive oxygen species, nitric oxides, and lipid peroxidation, while the same treatment reduced thiol (antioxidant) levels in T. cruzi (Martins et al., 2016).
Anti-protozoal efficacy of benzaldehyde and gallate (3,4,5-trihydroxybenzoic acid) analogs were investigated in two respects: changing types of side chains and their positions on the benzaldehyde ring [structure-activity relationships (SAR)]; and, changing lengths of alkyl chains esterified with the carboxyl group on the gallate. In each modification, the effectiveness of structural analogs was tested in three different protozoa, viz., T. vaginalis trophozoites (G3, C1) and T. foetus bovine strain (D1).

Results and discussion (Continued)
growth inhibition, respectively, which is similar to that of pentyl-or ethyl gallate (Group 3 or 4, respectively). Therefore, results indicated differential susceptibility of test pathogens to these alkyl gallates. The remaining alkyl gallates achieved ≤ 30% of growth inhibition in C1 at 100 µM (Group 5).

Results and discussion (Continued)
The cutoff chain length of gallate analogs for achieving~100% growth inhibition in G3, C1 or D1 pathogens was C 8 , C 10 or C 12 , respectively. The order of susceptibility of pathogens to alkyl gallates tested was G3 > C1 > D1 (high to low), where four, two or one alkyl gallate(s) could be selected as Group 1 compound(s), respectively [Note: In the model yeast Saccharomyces cerevisiae, the cutoff for growth inhibitory and/or fungicidality was C 10 (Fujita and Kubo, 2002b)].
In contrast to that observed in G3, nonyl gallate (longer alkyl chain analog) possessed very low anti-protozoal activity in both C1 (41.2% inhibition) and D1 (35.3% inhibition) (which is also lower than that of other longer chain alkyl gallates tested in C1 and D1; See Table 1).
Except Group 1 compounds selected in G3 (where the longer the alkyl chain, the higher the activity, based on IC50 values), there was no general relationship/trend between the length of alkyl chain and the incremental increase of anti-protozoal activity of compounds in test pathogens.

Effect of benzaldehyde analogs
Effect of changing types of side chains on the benzaldehyde ring was also investigated. Twenty-three benzaldehyde analogs were examined against G3, C1 and D1, where test compounds were categorized as Group 1 to 4 depending on the level of anti-protozoal activity ( Table 2). Results showed that the level of activity of benzaldehyde analogs was much lower than that of alkyl gallates. For example, the highest growth inhibition rate, which was observed in G3 only, was 51.8% (achieved by 4-methoxy-2-methylbenzaldehyde), whereas up to 100% growth inhibition could be achieved in all protozoa tested (G3, C1, D1) by longer chain alkyl gallates (octyl-, nonyl-, decyl-or lauryl gallate; See Table 1). As observed in alkyl gallate testing, there was differential susceptibility of G3, C1 and D1 strains to benzaldehydes as follows:
It is intriguing to note that the level of anti-protozoal activity of 4-methoxy-2methylbenzaldehyde (51.8%; Group 1) was roughly equivalent to the sum of the % inhibition of both 2-methylbenzaldehyde (31.3%; Group2) and 4methoxybenzaldehyde (20.1%; Group 3; Table S1), indicating methoxyl and methyl moiety at 4-and 2-position on the benzaldehyde ring, respectively, provided an additive effect for anti-protozoal efficacy.

T. foetus D1 (Least susceptible)
In D1, the highest anti-protozoal activity (15.9% to 24.6%) was achieved only by four benzaldehydes, which belong to Group 3 ( Table 2). The remaining nineteen benzadehydes were categorized as Group 4 compounds, which achieved less than 15% of growth inhibition. In particular, 4-methoxy-2-methylbenzaldehyde or cinnamaldehyde (which were also potent anti-protozoal compounds in G3) did not achieve similar level of anti-protozoal activity to that observed in C1.
As determined in alkyl gallates (See above), there was also differential susceptibility of test strains to benzaldehyde analogs, where G3 was the most susceptible to benzaldehydes, followed by C1 and D1. Collectively, it's concluded that certain benzo derivatives (benzoic acid or benzaldehyde analogs) could be developed as potent anti-protozoal agents or leads with structural-activity relationship, where the level of activity is dependent upon types of protozoa pathogens.

Conclusions
In this study, anti-protozoal effects of selected benzo derivatives, namely ten GA alkyl esters (viz., benzoic acid analogs) and twenty-three benzaldehyde analogs, were investigated against T. vaginalis trophozoites (G3), T. foetus-like C1, and T. foetus bovine strain (D1). Results indicated that benzo derivatives could be developed as potent anti-protozoal drug candidates/leads, where GA having longer alkyl chains exhibited much higher anti-protozoal activity compared to that with shorter alkyl chains or all benzaldehyde analogs tested.