Article pubs.acs.org/JAFC
Antiprotozoal Effects of the Tomato Tetrasaccharide Glycoalkaloid Tomatine and the Aglycone Tomatidine on Mucosal Trichomonads Jenny Liu,† Sierra Kanetake,† Yun-Hsuan Wu,† Christina Tam,‡ Luisa W. Cheng,‡ Kirkwood M. Land,† and Mendel Friedman*,§ †
Department of Biological Sciences, University of the Pacific, Stockton, California 95211, United States Foodborne Toxin Detection and Prevention, Agricultural Research Service, United States Department of Agriculture, Albany, California 94556, United States § Healthy Processed Foods Research, Agricultural Research Service, United States Department of Agriculture, Albany, California 94556, United States ‡
ABSTRACT: The present study investigated the inhibitory effects of the commercial tetrasaccharide tomato glycoalkaloid tomatine and the aglycone tomatidine on three mucosal pathogenic protozoa that are reported to infect humans, cattle, and cats, respectively: Trichomonas vaginalis strain G3, Tritrichomonas foetus strain D1, and Tritrichomonas foetus strain C1. A preliminary screen showed that tomatine at 100 μM concentration completely inhibited the growth of all three trichomonads. In contrast, the inhibition of all three pathogens by tomatidine was much lower, suggesting the involvement of the lycotetraose carbohydrate side chain in the mechanism of inhibition. Midpoints of concentration−response sigmoid plots of tomatine on the three strains correspond to IC50 values, the concentration that inhibits 50% of growth of the pathogenic protozoa. The concentration data were used to calculate the IC50 values for G3, D1, and C1 of 7.9, 1.9, and 2.2 μM, respectively. The results show an approximately 4-fold variation from the lowest to the highest value (lowest activity). Although the inhibition by tomatine was not as effective as that of the medicinal drug metronidazole, the relatively low IC50 values for both T. vaginalis and T. foetus indicated tomatine as a possible natural alternative therapeutic for trichomoniasis in humans and food-producing (cattle and pigs) and domestic (cats) animals. Because tomatine has the potential to serve as a new antiprotozoan functional (medical) food, the distribution of this glycoalkaloid in tomatoes and suggestions for further research are discussed. KEYWORDS: Trichomonas vaginalis, Tritrichomonas foetus, tomatoes, tomatine, tomatidine, growth inhibition, infections, trichomoniasis prevention
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producing (cattle and pigs) and domestic (cats) animals.8−11 Although the two protozoans are closely related, normal treatment with metronidazole on T. foetus is ineffective. Untreated trichomonosis has the potential to adversely affect livestock fertility and yield and the health of felines. Tomatoes, a major food source for humans, accumulate a variety of secondary metabolites, including tetrasaccharide glycoalkaloids α-tomatine and dehydrotomatine that are reported to be involved in host-plant resistance that involves protection against phytopathogenic bacteria, fungi, viruses, worms,12 and protozoa.13 The biosynthesis of tomatine and dehydrotomatine takes place in all parts of the tomato plant14 as well as in numerous wild-type, uncultivated potato cultivars.15 Because tomato leaves are rich in tomatine, leaves are the most likely source of commercial tomatine, an approximately 9:1 mixture of tomatine and structurally similar dehydrotomatine (Figure 1). We previously reported that commercial tomatine inhibits cancer cells,16,17 multi-organ cancers in rainbow trout,18 and the growth of transplanted colon tumors in mice19,20 and that
INTRODUCTION Trichomoniasis is a sexually transmitted infection with the protozoan organism Trichomonas vaginalis and is usually treated with a single 2 g dose of the antibiotic drug metronidazole. Side effects of the treatment include leucopenia (decreases in the number of white blood cells), adverse reaction to alcohol, and pathogenic Candida fungal superinfections.1 Trichomoniasis is reported to be the most common non-viral sexually transmitted infection (STI) in the world, an important source of reproductive morbidity, and a facilitator of human immunodeficiency virus (HIV) transmission and acquisition.2 The organism is reported to infect an estimated 3.7 million women and men in the United States annually.3 About 348 million new infections are estimated to occur annually worldwide.4,5 Although the infection is prevalent, the only treatments available for the sexual transmitted disease (STD) are the above-mentioned metronidazole and another antibiotic, tinidazole. Also, the number of untreatable cases of trichomoniasis is increasing as a result of growing strains of drug-resistant T. vaginalis. Because high rates of positive retests after a single-dose treatment are likely due to clinical resistance and drug allergy rather than re-infection, a need exists for new approaches to complement the available therapies.6,7 Strains of the related protozoan Tritrichomonas foetus are reported to cause pathogenesis of trichomonosis in food© XXXX American Chemical Society
Received: September 8, 2016 Revised: October 20, 2016 Accepted: November 4, 2016
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DOI: 10.1021/acs.jafc.6b04030 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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MATERIALS AND METHODS
Materials. Tomatine and tomatidine were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Metronidazole was obtained from Sigma (St. Louis, MO). BDL-sensi-discs were obtained from BD Diagnostics (Sparks, MD). TYM Diamond medium was obtained from Hardy Diagnostics (Santa Maria, CA). T. vaginalis strain G3 was obtained from Patricia Johnson, University of California, Los Angeles (UCLA). T. foetus D1 was obtained from Lynette Corbeil, School of Medicine, University of California, San Diego (UCSD). The feline T. foetus-like organism was obtained from Stanley Marks, School of Veterinary Medicine, University of California, Davis (UC Davis). All of the normal flora bacteria were obtained from the American Type Culture Collection (ATCC), Manassas, VA. Inhibition of the Parasites by Tomatine and Tomatidine Hydrochloride. Cultures of T. vaginalis G3 strain were grown in 10 mL of completed TYM Diamond medium of pH 6.2 in a 37 °C incubator for 24 h. Measurements of liquids were made with 5 and 10 mL serological pipettes and 2.5, 20, 200, and 1000 μL micropipettes. Cultures of T. foetus strains D1 and C1 were grown in 10 mL of completed TYM Diamond medium at pH 6.8 in a 37 °C incubator for 24 h. The cells are passed daily by placing 1000 μL of cells into a new tube of 10 mL of media to maintain cultures. Tomatine and tomatidine HCl were dissolved in dimethyl sulfoxide (DMSO) to make the 100 mM stock solution for screening. In all of the trials, a consistent number of cells is inoculated into the 5 mL of completed TYM Diamond medium as determined by counting 10 μL of cells using a light microscope, hemocytometer, and coverslips. Cells (4000) were divided by the number of cells, resulting in the microliters of cell culture added to 5 mL of TYM Diamond medium. Two positive controls were used to help determine percent inhibition by the two test compounds. The cell count for the DMSO control (cells inoculated with the highest amount of DMSO used for the trial) should match that of a wild-type control (untreated cells) count to ensure that the cells and media were not defective and that DMSO did not inhibit microbial growth. Percent inhibition was calculated by subtracting the experimental cell count from the DMSO cell count, dividing the result by the DMSO cell count, and then multiplying the result by 100%. Tomatine and tomatidine HCl were screened at 100 μM to determine potency on the G3 strain of T. vaginalis and D1 and C1 strains of T. foetus. Percent inhibition was calculated by determining the experimental cell count 24 h after inoculation and comparing it to the number of cells in a DMSO control. Further testing was performed to determine IC50 values when percent inhibition for a preliminary screen at 100 μM was higher than 90%. Additional testing was then performed with serial dilutions on the T. vaginalis and T. foetus strains to determine the IC50 values. The inhibitory activity at different concentrations for each strain was then used to generate dose− response curves on GraphPad to determine the IC50 values. The theoretical IC50 values were tested to determine if they were in fact the concentration of compound needed for 50% inhibition; i.e., IC50 values were confirmed by retesting the predicted values on parasite cultures. Disc Diffusion Antibiotic Susceptibility Assay on Normal Flora Bacteria. Single colonies of non-pathogenic strains of Escherichia coli K-12 MG 1655, Lactobacillus acidophilus (ATCC 43560), Lactobacillus rhamnosus (ATCC 53103), and Lactobacillus reuteri (ATCC 23272) were grown in either Luria Broth (LB) or Lactobacilli MRS under anaerobic conditions. Sterile wooden cotton swabs were dipped into the overnight cultures and streaked to form a continuous lawn of bacterial growth. Stock compound solutions (100 mM) in DMSO were diluted into media to a final concentration of 100 mM, along with a vehicle control of DMSO. Blank BDL-sensi-discs (6 mm) were incubated with the test compounds (100 mM) or vehicle control at room temperature for 20 min. BDL-antibiotic-laden discs containing levofloxacin (5 mg), gentamicin (10 mg), and gentamicin (120 mg) were placed onto the bacterial-streaked agar plates and incubated overnight at 37 °C. Zones of inhibition representing antibiotic and/or compound sensitivity were measured in millimeters for each of the antibiotics, test compounds, and vehicle controls.
Figure 1. Structures of α-tomatine, dehydrotomatine, tomatidine, and metronidazole. α-Tomatine (molecular weight of 1034.20) consists of tomatidine (molecular weight of 415.66) with an attached tetrasaccharide (lycotetraose) side chain consisting of two glucose, one xylose, and one galactose molecule. The structure of dehydrotomatine is identical to that of α-tomatine, except for the presence of a double bond in ring B of the tomatidine (steroidal) part of the compound.
dietary tomatine and high-tomatine green tomatoes decreased plasma low-density (bad) cholesterol in hamsters fed a high saturated fat, high cholesterol diet.21,22 These results imply that tomatine might also inhibit other pathogenic cell lines, including disease-causing trichomonad protozoa. The effect of synthetically derived compounds on trichomonad protozoa has been investigated. Land and collaborators evaluated in cell assays the inhibition of T. vaginalis and other parasitic protozoan growth by a series of structurally different synthetic compounds.23−28 Although the cited studies indicate that the synthetic formulations seem to be highly active against pathogenic protozoans, their adoption for human and animal use would require demonstration of both efficacy and safety. Because this might not be the case for natural food-compatible plant-derived products that are considered to be generally recognized as safe (GRAS), we are exploring the potential of plant extracts and some of their bioactive compounds to inhibit the growth of (inactivate) pathogenic trichomonad species. The objective of the present study was to determine in cell assays the potential of commercial tomatine and the aglycone tomatidine, which lacks the carbohydrate side chain, to inhibit the growth of the following three parasitic protozoa that are reported to infect humans, cattle, and cats: human T. vaginalis, bovine T. foetus, and a feline T. foetus-like pathogen. As part of this effort, we also determined, using an in vitro assay, the effect of tomatine on a number of different members of the normal flora microbiome. B
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RESULTS AND DISCUSSION Biological Evaluations. In Vitro Parasite Cell Inhibition Assays. As mentioned, the increasing number of drug resistance cases of T. vaginalis and the lack of treatment for infection of T. foetus in animals has given rise to the need for developing alternative treatments of infection.7,29 We therefore performed a structure−activity analysis for both T. vaginalis and T. foetus on glycoalkaloids and alkaloids found in tomatoes, tomatine and the aglycone tomatidine, to investigate their potential as alternative therapies. We found through general screenings at 100 μM that tomatine completely inhibited T. vaginalis strain G3 and T. foetus strains D1 and C1 growth at 100% (Table 1). Further
Table 2. Calculated IC50 Values for the Inactivation of Three Protozoan Parasites by Tomatine Compared to the Medicinal Drug Metronidazole IC50 (μM) T. vaginalis G3 T. foetus D1 T. foetus C1
inhibition (%) T. vaginalis G3 T. foetus D1 T. foetus C1
tomatidine HCl
100 100 100
3.2 22.86 10.21
metronidazole
7.9 2.7 2.0
0.72 0.49 0.55
than 90% in the general screening at 100 μM on T. vaginalis and both strains of T. foetus; therefore, the aglycone was not selected for the IC50 trials. Effect of Tomatine on Normal Flora Bacteria. Because the three parasites evaluated in this study are mucosal pathogens of the urogenital and gastrointestinal tracts that live within complex microbial ecosystems, it is of interest to know if the antiparasitic test compounds affect (inhibit) the normal flora microbiome. To address this possibility, we determined the effect of tomatine on a culture of standard non-pathogenic Lactobacillus and E. coli usually found in these environments. The results of the in vitro study show that, unlike the antibiotics, tomatine did not inhibit the growth of the normal non-pathogenic Lactobacillus and E. coli present in the gut and/ or urogenital tract, suggesting that the action of tomatine in vivo might not be affected by concurrent inactivation of bacteria associated with the microbiome. Tomatine Content of Tomatoes. Because consumption of tomatine-containing tomatoes and tomato products has the potential to mitigate trichomoniasis in humans and animals, we will briefly mention results of reported studies on the content of α-tomatine in tomatoes. α-Tomatine is found in high amounts in immature (green) tomatoes in wild tomato fruit, in a high-tomatine red tomato variety grown in the Andes Mountains of Peru, in uncultivated potato accessions, but in low amounts in red tomatoes.30−35 The tomatine content of fresh tomatoes ranged from about 4 to 42 mg/kg on a freezedried dry weight basis, and the levels of commercially available pickled green and fried green tomatoes are 50−100 times higher than those of the standard red varieties. The α-tomatine content from organically and conventionally grown red tomatoes harvested over a 10-year period ranged from 4.3 to 111.8 μg/g of dry weight, with the average value for organic tomatoes twice that for conventional tomatoes.36 Structure−Activity Relationships. Although the IC50 values of the medicinal drug metronidazole are lower than those of tomatine, the relatively low IC50 values for both T. vaginalis and T. foetus strains indicate tomatine as a possible therapeutic agent for trichomoniasis. The positive findings at micromolar concentrations of tomatine suggest that the glycoalkaloid should be investigated further for developing alternative treatments for trichomoniasis patients and those who have metronidazole-resistant strains of the parasitic protozoan. The therapeutic potential of tomatine against T. foetus strains that infect food-producing cattle and pigs as well as cats also merits study. These data also suggest that tomatine could serve as a chemical scaffold for new compound synthesis in an effort to improve its inhibitory effects. The cited observations imply that carbohydrate side chains of both potato and tomato glycoalkaloids seem to largely govern anticancer and antiviral as well as antiprotozoal activities, as indicated by the fact that in the present study tomatine was much more active than tomatidine and that sequential removal
Table 1. General Screening Data for Tomatine and Tomatidine HCl on T. vaginalis Strain G3 and T. foetus Strains D1 and C1 tomatine
tomatine
testing of tomatine on G3, D1, and C1 at various concentrations (see the sigmoid concentration−inhibitory activity plots shown in Figure 2) resulted in calculated IC50 values of 7.887, 1.886, and 2.225 μM, respectively (Table 2). The IC50 value indicates the 50% inhibition of either T. vaginalis or T. foetus growth inhibition. Commercial tomatidine hydrochloride, however, had percent inhibition values of less
Figure 2. Concentration response of commercial tomatine on human T. vaginalis strain G3, bovine T. foetus strain D1, and feline T. foetuslike strain C1. C
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biomarkers, and complexation with cholesterol (for which tomatine has a strong affinity) that are associated with disruption of cell membranes also operate during the destruction of Trichomonas cells. The cited previous results and the results of the present study suggest that tomatine has the potential for both cancer and trichomoniasis prevention and treatment in animals and humans.
of sugar residues of the tetrasaccharide side chain of tomatine generally paralleled reduction in inhibitory activities against cancer cells.17 In summary, this study identified tomatine, a natural glycoalkaloid found in tomatoes, to have antimicrobial activity against pathogenic protozoa that infect both humans and animals. Tomatine showed great inhibitory effects on both T. vaginalis and T. foetus strains that are required for developing an alternative therapy against trichomoniasis in humans, bovines, and felines. We have demonstrated that tomatine inhibits T. vaginalis growth, with an IC50 value of ∼8 μM. In addition, we have demonstrated that tomatine inhibits the protozoal pathogen T. foetus strains, with an IC50 of ∼2 μM or about a 4-fold greater activity than against T. vaginalis. Research Needs. The results of the present study suggest that future studies should evaluate the efficacy of high-tomatine tomato diets, especially high-tomatine green tomato extracts, against trichomoniasis in susceptible humans and trichomoniasis in animals, such as cattle and cats. Another key consideration is whether combinations of low doses of tomatine with medicinal drugs, such as metronidazole, have the potential to act additively or synergistically with reduced side effects and whether they will be active against resistant strains. There is also a need to develop high-tomatine red tomatoes by suppressing the genes in the tomato plant that govern the biosynthesis of enzymes that degrade most of the tomatine during ripening of tomatoes and to evaluate the antiprotozoal potential of extracts of wild-type potatoes that contain both potato and tomato glycoalkaloids. We also do not know if bioactive phenolic and flavonoid compounds and the red pigment lycopene also present in tomatoes37,38 might enhance the antiprotozoal effects of tomatine. This aspect also merits study. Studies are also needed to determine if trisaccharide glycoalkaloids that are present in potatoes (α-chaconine and α-solanine) and in some eggplant cultivars and numerous nonfood plants (solamargine and solasonine) and their aglycones would also inhibit Trichomonas cells as they do cancer cells.19 Such studies might make it possible to define structure−activity relationships for these bioactive tomato, potato, and eggplant components. In this regard, it is relevant to note that the trisaccharide potato glycoalkaloid α-chaconine was more effective than tomatine in inhibiting the human herpes simplex virus type 1 in tissue culture, whereas the trisaccharide αsolanine was less active than tomatine.39 Because their common aglycone solanidine derived by removing the trisaccharide carbohydrate side chains from both glycoalkaloids was inactive against the pathogenic herpes virus, the authors suggested that inactivation of the herpes virus results from insertion of the carbohydrate-containing glycoalkaloids into the viral envelope, presumably initiated by the carbohydrate side chain. A recent screening of plant-derived compounds against the human pathogenic Zika virus40 leads us to postulate whether tomatine and related glycoalkaloids might also inhibit the growth of this virus. Because tomatidine also inhibited the growth of the protozoa, albeit to a lesser extent, and both tomatine and tomatidine are reported to be present in tomato extracts,37 it would be interesting to find out whether the combination of the two compounds would have additive, synergistic, or antagonistic activities. We do not know if the proposed mechanisms for the inhibition of cancer and the herpes simplex virus cells mentioned above involving signaling pathways,
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AUTHOR INFORMATION
Corresponding Author
*Telephone: 510-559-5615. E-mail: mendel.friedman@ars. usda.gov. ORCID
Mendel Friedman: 0000-0003-2582-7517 Funding
The authors thank the Department of Biological Sciences, University of the Pacific, for research support. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Carol. E. Levin for assistance with the preparation of the manuscript. ABBREVIATIONS USED ATCC, American Type Culture Collection; GRAS, generally recognized as safe; HIV, human immunodeficiency virus; IC50, half maximal inhibitory concentration
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REFERENCES
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