Isomers of the Tomato Glycoalkaloids α-Tomatine and Dehydrotomatine: Relationship to Health Benefits.

High-performance liquid chromatography (HPLC) analysis of three commercial tomatine samples and another isolated from green tomatoes revealed the presence of two small peaks in addition to those associated with the glycoalkaloids dehydrotomatine and α-tomatine. The present study investigated the possible structures of the compounds associated with the two small peaks using HPLC-mass spectrophotometric (MS) methods. Although the two peaks elute much earlier on chromatographic columns than the elution times of the known tomato glycoalkaloids dehydrotomatine and α-tomatine, isolation of the two compounds by preparative chromatography and subsequent analysis by MS shows the two compounds have identical molecular weights, tetrasaccharide side chains, and MS and MS/MS fragmentation patterns to dehydrotomatine and α-tomatine. We suggest the two isolated compounds are isomeric forms of dehydrotomatine and α-tomatine. The analytical data indicate that widely used commercial tomatine preparations and those extracted from green tomatoes and tomato leaves consist of a mixture of α-tomatine, dehydrotomatine, an α-tomatine isomer, and a dehydrotomatine isomer in an approximate ratio of 81:15:4:1, respectively. The significance of the reported health benefits of tomatine and tomatidine is mentioned.

α-Tomatine degradation to tomatidine by food-related Aspergillus species belonging to the section Nigri.

α-Tomatine is a steroidal glycoalkaloid in tomato plants and degrades with ripening. The aglycone form, tomatidine, is reported to have beneficial effects. In this study, the ability of food-related microorganisms to produce tomatidine from α-tomatine was evaluated. A total of 11 strains of Aspergillus species belonging to the section Nigri exhibited tomatinase activity, and Aspergillus luchuensis JCM 22302 was selected for optimization due to its high activity in its mycelia, conidia, and non-mycotoxin-producing property. Next, using A. luchuensis JCM22302 conidia, the highest yield was obtained in a 24-h reaction with 50 m m of acetic acid-sodium acetate buffer (pH 5.5) at 37 °C. Similar to the tomato pathogen Fusarium oxysporum f. lyceopersici, the time course analysis suggested that A. luchuensis JCM 22302 removed the entire sugar moiety in a single step. Future research will focus on utilizing conidia for large-scale tomatidine production because of their high tolerance and manageability.

Concise Large-Scale Synthesis of Tomatidine, A Potent Antibiotic Natural Product.

Tomatidine has recently generated a lot of interest amongst the pharmacology, medicine, and biology fields of study, especially for its newfound activity as an antibiotic agent capable of targeting multiple strains of bacteria. In the light of its low natural abundance and high cost, an efficient and scalable multi-gram synthesis of tomatidine has been developed. This synthesis uses a Suzuki-Miyaura-type coupling reaction as a key step to graft an enantiopure F-ring side chain to the steroidal scaffold of the natural product, which was accessible from low-cost and commercially available diosgenin. A Lewis acid-mediated spiroketal opening followed by an azide substitution and reduction sequence is employed to generate the spiroaminoketal motif of the natural product. Overall, this synthesis produced 5.2 g in a single pass in 15 total steps and 15.2% yield using a methodology that is atom economical, scalable, and requires no flash chromatography purifications.

The steroidal alkaloids α-tomatine and tomatidine: Panorama of their mode of action and pharmacological properties.

The steroidal glycoalkaloid α-tomatine (αTM) and its aglycone tomatidine (TD) are abundant in the skin of unripe green tomato and present in tomato leaves and flowers. They mainly serve as defensive agents to protect the plant against infections by insects, bacteria, parasites, viruses, and fungi. In addition, the two products display a range of pharmacological properties potentially useful to treat various human diseases. We have analyzed all known pharmacological activities of αTM and TD, and the corresponding molecular targets and pathways impacted by these two steroidal alkaloids. In experimental models, αTM displays anticancer effects, particularly strong against androgen-independent prostate cancer, as well as robust antifungal effects. αTM is a potent cholesterol binder, useful as a vaccine adjuvant to improve delivery of protein antigens or therapeutic oligonucleotides. TD is a much less cytotoxic compound, able to restrict the spread of certain viruses (such as dengue, chikungunya and porcine epidemic diarrhea viruses) and to provide cardio and neuro-protective effects toward human cells. Both αTM and TD exhibit marked anti-inflammatory activities. They proceed through multiple signaling pathways and protein targets, including the sterol C24 methyltransferase Erg6 and vitamin D receptor, both directly targeted by TD. αTM is a powerful regulator of the NFkB/ERK signaling pathway implicated in various diseases. Collectively, the analysis shed light on the multitargeted action of αTM/TD and their usefulness as chemo-preventive or chemotherapeutic agents. A novel medicinal application for αTM is proposed.

Tomatidine inhibits porcine epidemic diarrhea virus replication by targeting 3CL protease.

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. There is an urgent need to find new therapeutic methods to prevent and control PEDV. Not only is there a shortage of commercial anti-PEDV drugs, but available commercial vaccines fail to protect against highly virulent PEDV variants. We screened an FDA-approved library of 911 natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of PEDV replication in Vero and IPEC-J2 cells in vitro. Molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of PEDV 3CL protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (ITC). The inhibiting effect of tomatidine on 3CL protease was determined using cleavage visualization and FRET assay. Tomatidine-mediated blocking of 3CL protease activity in PEDV-infected cells was examined by western blot detection of the viral polyprotein in PEDV-infected cells. It indicates that tomatidine inhibits PEDV replication mainly by targeting 3CL protease. In addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), encephalo myocarditis virus (EMCV) and seneca virus A (SVA) in vitro. These results may be helpful in developing a new prophylactic and therapeutic strategy against PEDV and other swine disease infections.

Exploring membrane permeability of Tomatidine to enhance lipid mediated nucleic acid transfections.

Intracellular delivery of nucleic acids is one of the critical steps in the transfections. Prior findings demonstrated various strategies including membrane fusion, endosomal escape for the efficient cytoplasmic delivery. In our continuing efforts to improve the nucleic acids transfections, we harnessed cell permeable properties of Tomatidine (T), a steroidal alkaloid abundantly found in green tomatoes for maximizing intracellular delivery of lipoplexes. We doped Tomatidine into liposomes of cationic lipid with amide linker (A) from our lipid library. Six liposomal formulations (AT) of Lipid A (1 mM) with varying concentrations of Tomatidine (0-1 mM) were prepared and evaluated for their transfection efficacies. Owing to its signature characteristic of cell membrane permeability, Tomatidine modulated endocytosis process, enhanced the intracellular delivery of the lipoplexes, and in turn increased the transfection efficacy of cationic liposomes. Our findings provide 'proof of concept' for enhancing transfections in gene delivery applications with Tomatidine in cationic liposomal formulations. These findings can be further applied in lipid mediated gene therapy and drug delivery applications.

Ecological Relevance of the Major Allelochemicals in Lycopersicon esculentum Roots and Exudates.

Stigmasterol, bergapten, and α-tomatine were isolated from tomato roots. The preliminary phytotoxic activities of stigmasterol and α-tomatine were evaluated in a wheat-coleoptile bioassay, and α-tomatine was more active than stigmasterol. To confirm its phytotoxic activity, α-tomatine was tested on Lactuca sativa and two weeds ( Lolium perenne and Echinochloa crus-galli), and it was active in all cases. The stimulatory activities of α-tomatine and stigmasterol on parasitic-plant germination were also evaluated, and α-tomatine was found to be active on Phelipanche ramosa, a parasitic plant of tomato. α-Tomatine was identified in root exudates by LC-MS/MS. This confirms that α-tomatine is exuded by roots into the environment, where it could act as both an allelochemical and a stimulator of P. ramosa, a parasitic plant of tomato.

The Effect of Tomatine on Gene Expression and Cell Monolayer Integrity in Caco-2.

More understanding of the risk-benefit effect of the glycoalkaloid tomatine is required to be able to estimate the role it might play in our diet. In this work, we focused on effects towards intestinal epithelial cells based on a Caco-2 model in order to analyze the influence on the cell monolayer integrity and on the expression levels of genes involved in cholesterol/sterol biosynthesis (LDLR), lipid metabolism (NR2F2), glucose and amino acid uptake (SGLT1, PAT1), cell cycle (PCNA, CDKN1A), apoptosis (CASP-3, BMF, KLF6), tight junctions (CLDN4, OCLN2) and cytokine-mediated signaling (IL-8, IL1ß, TSLP, TNF-α). Furthermore, since the bioactivity of the compound might vary in the presence of a food matrix and following digestion, the influence of both pure tomatine and in vitro digested tomatine with and without tomato fruit matrix was studied. The obtained results suggested that concentrations <20 µg/mL of tomatine, either undigested or in vitro digested, do not compromise the viability of Caco-2 cells and stimulate cytokine expression. This effect of tomatine, in vitro digested tomatine or in vitro digested tomatine with tomato matrix differs slightly, probably due to variations of bioactivity or bioavailability of the tomatine. The results lead to the hypothesis that tomatine acts as hormetic compound that can induce beneficial or risk toxic effects whether used in low or high dose.

Sarcopenia, Aging and Prospective Interventional Strategies.

Sarcopenia, or age-related muscle decline, occurs in most organisms and burdens both human health and the healthcare system. As our population ages, additional options for treating sarcopenia are needed. Mitochondrial dysfunction is implicated in the onset of sarcopenia, so therapies directed at improving mitochondrial function in muscle should be considered. Many naturally-occurring compounds, derived from commonly consumed foods, possess anti-sarcopenic effects, such asnicotinamide riboside, tomatidine, and Urolithin A. These naturally-occurring compounds can improve mitochondrial health and efficiency by modulating mitochondrial biogenesis, cellular stress resistance, or mitophagy. Further research should assess whether compounds that improve mitochondrial health can attenuate sarcopenia in humans.

A tomatinase-like enzyme acts as a virulence factor in the wheat pathogen Fusarium graminearum.

During their interactions with plants, fungal pathogens employ large numbers of pathogenesis-associated molecules including secreted effectors and enzymes that can degrade various defence compounds. However, in many cases, in planta targets of pathogen-produced enzymes remain unknown. We identified a gene in the wheat pathogen Fusarium graminearum, encoding a putative enzyme that shows 84% sequence identity to FoTom1, a tomatinase produced by the tomato pathogen Fusarium oxysporum f. sp. lycopersici. In F. oxysporum f. sp. lycopersici, FoTom1 is a virulence factor involved in the degradation of tomato defence compound tomatine, a saponin compound. Given that wheat is unknown to produce tomatine, we tested the ability of F. graminearum to degrade tomatine and found that F. graminearum was unable to degrade tomatine in culture. However, FgTom1 degraded tomatine in vitro when heterologously expressed. To determine the possible function of FgTom1 in pathogen virulence, we generated FgTom1 knockout mutants (ΔTom1). ΔTom1 mutants were not different from wild type when grown in culture but showed significant reduction in pathogen virulence in root rot and head blight assays. In an attempt to identify possible in planta targets of FgTom1, the metabolomes of wheat heads infected with wildtype pathogen and ΔTom1 were compared and several peaks differentially abundant between treatments identified. Although the exact identity of these peaks is currently unknown, this result suggested that FgTom1 may have in planta targets in wheat, possibly tomatine-like saponin compounds. Overall, our results presented here show that FgTom1 is a new virulence factor in F. graminearum.

Antiprotozoal Effects of the Tomato Tetrasaccharide Glycoalkaloid Tomatine and the Aglycone Tomatidine on Mucosal Trichomonads.

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.

Saponin Interactions with Model Membrane Systems - Langmuir Monolayer Studies, Hemolysis and Formation of ISCOMs.

Saponins are used in medicine due to their pharmacological and immunological effects. To better understand interactions of saponins with model membranes and natural membranes of, for example, erythrocytes, Langmuir film balance experiments are well established. For most saponins, a strong interaction with cholesterol was demonstrated in dependence of both the aglycone part and the sugar moieties and is suggested to be correlated with a strong hemolytic activity, high toxicity, and high surface activity, as was demonstrated for the steroid saponin digitonin. In general, changes in the sugar chain or in substituents of the aglycone result in a modification of the saponin properties. A promising saponin with regard to fairly low hemolytic activity and high adjuvant effect is α-tomatine, which still shows a high affinity for cholesterol. An interaction with cholesterol and lipids has also been proven for the Quillaja saponin from the bark of Quillaja saponaria Molina. This triterpene saponin was approved in marketed vaccines as an adjuvant due to the formation of immunostimulating complexes. Immunostimulating complexes consist of a Quillaja saponin, cholesterol, phospholipids, and a corresponding antigen. Recently, another saponin from Quillaja brasiliensis was successfully tested in immunostimulating complexes, too. Based on the results of interaction studies, the formation of drug delivery systems such as immunostimulating complexes or similar self-assembled colloids is postulated for a variety of saponins.

Enzyme-driven metabolomic screening: a proof-of-principle method for discovery of plant defence compounds targeted by pathogens.

Plants produce a variety of secondary metabolites to defend themselves from pathogen attack, while pathogens have evolved to overcome plant defences by producing enzymes that degrade or modify these defence compounds. However, many compounds targeted by pathogen enzymes currently remain enigmatic. Identifying host compounds targeted by pathogen enzymes would enable us to understand the potential importance of such compounds in plant defence and modify them to make them insensitive to pathogen enzymes. Here, a proof of concept metabolomics-based method was developed to discover plant defence compounds modified by pathogens using two pathogen enzymes with known targets in wheat and tomato. Plant extracts treated with purified pathogen enzymes were subjected to LC-MS, and the relative abundance of metabolites before and after treatment were comparatively analysed. Using two enzymes from different pathogens the in planta targets could be found by combining relatively simple enzymology with the power of untargeted metabolomics. Key to the method is dataset simplification based on natural isotope occurrence and statistical filtering, which can be scripted. The method presented here will aid in our understanding of plant-pathogen interactions and may lead to the development of new plant protection strategies.

Therapeutic Potential of Steroidal Alkaloids in Cancer and Other Diseases.

Steroidal alkaloids are a class of secondary metabolites isolated from plants, amphibians, and marine invertebrates. Evidence accumulated in the recent two decades demonstrates that steroidal alkaloids have a wide range of bioactivities including anticancer, antimicrobial, anti-inflammatory, antinociceptive, etc., suggesting their great potential for application. It is therefore necessary to comprehensively summarize the bioactivities, especially anticancer activities and mechanisms of steroidal alkaloids. Here we systematically highlight the anticancer profiles both in vitro and in vivo of steroidal alkaloids such as dendrogenin, solanidine, solasodine, tomatidine, cyclopamine, and their derivatives. Furthermore, other bioactivities of steroidal alkaloids are also discussed. The integrated molecular mechanisms in this review can increase our understanding on the utilization of steroidal alkaloids and contribute to the development of new drug candidates. Although the therapeutic potentials of steroidal alkaloids look promising in the preclinical and clinical studies, further pharmacokinetic and clinical studies are mandated to define their efficacy and safety in cancer and other diseases.

Elucidation of the mass fragmentation pathways of tomatidine and ß1-hydroxytomatine using orbitrap mass spectrometry.

Tomatoes, members of the Solanaceae plant family, produce biologically active secondary metabolites, including glycoalkaloids, which may have both adverse and beneficial biological effects. Using the linear ion trap (LIT) mass spectrometry, multi-stage collision induced dissociation (CID) experiments (MSn) were performed to elucidate characteristic fragmentation pathways of the glycoalkaloid, tomatidine and of ß1-hydroxytomatine. High resolution with high accuracy mass analysis using an Orbitrap fourier transform MS with higher-energy collisional induced dissociation (HCD) was used to produce mass spectra data across a wide spectral range for confirmation of proposed ion structures and formulae.

Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.

The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.

α-Tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells.

α­Tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α­tomatine on human myeloid leukemia HL­60 cells were investigated. Treatment of HL­60 cells with α­tomatine resulted in growth inhibition and apoptosis in a concentration­dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL­60 cells. Growth inhibition and apoptosis induced by α­tomatine in HL­60 cells was partially abrogated by addition of cholesterol indicating that interactions between α­tomatine and cell membrane­associated cholesterol may be important in mediating the effect of α­tomatine. Activation of nuclear factor­κB by the phorbol ester, 12­O­tetradecanoylphorbol­13­acetate failed to prevent apoptosis in HL­60 cells treated with α­tomatine. In animal experiments, it was found that treatment of mice with α­tomatine inhibited the growth of HL­60 xenografts in vivo. Results from the present study indicated that α­tomatine may have useful anti­leukemia activities.

Biosynthesis of steroidal alkaloids in Solanaceae plants: incorporation of 3ß-hydroxycholest-5-en-26-al into tomatine with tomato seedlings.

The C-26 amino group of tomatine, a representative Solanaceae steroidal alkaloid, is introduced in an early step of its biosynthesis from cholesterol. We recently proposed a transamination mechanism for the C-26 amination as opposed to the previously proposed mechanism involving a nitrogen nucleophilic displacement. In the present study, a deuterium labeled C-26 aldehyde, (24,24,27,27,27-(2)H5)-3ß-hydroxycholest-5-en-26-al, was synthesized and fed to a tomato (Solanum lycopersicum) seedling. LC-MS analysis of the biosynthesized tomatine indicated that the labeled aldehyde was incorporated into tomatine. The finding strongly supports the intermediacy of the aldehyde and the transamination mechanism during C-26 amination.

Unraveling the structure-activity relationship of tomatidine, a steroid alkaloid with unique antibiotic properties against persistent forms of Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is responsible for difficult-to-treat and relapsing infections and constitutes one of the most problematic pathogens due to its multiple resistances to clinically available antibiotics. Additionally, the ability of S. aureus to develop small-colony variants is associated with a reduced susceptibility to aminoglycoside antibiotics and in vivo persistence. We have recently demonstrated that tomatidine, a steroid alkaloid isolated from tomato plants, possesses anti-virulence activity against normal strains of S. aureus as well as the ability to potentiate the effect of aminoglycoside antibiotics. In addition, tomatidine has shown antibiotic activity against small-colony variants of S. aureus. We herein report the first study of the structure-activity relationship of tomatidine against S. aureus.

Cholesterol aided etching of tomatine gold nanoparticles: a non-enzymatic blood cholesterol monitor.

Colloidal gold is extensively used for molecular sensing because of the wide flexibilities it offers in terms of modifications of the gold nanoparticles (GNPs) surface with a variety of functional groups. We describe a simple, enzyme free assay for the detection of cholesterol, and demonstrate its applicability by estimating cholesterol in human serum samples. To enable cholesterol detection, we functionalized GNPs with tomatine, a glycoalkaloid found in the leaves and stem of tomato plants. The binding of cholesterol onto tomatine functionalized gold nanoparticles (TGNPs) was characterized by a blue shift in the plasmon absorption spectra (SPR) followed by reduction in the particle size. The TGNPs have been core etched with increasing concentration of cholesterol and with 800 ng/mL of cholesterol particles in the size range of 10-12 nm have been obtained. This behavior was attributed to the enhanced hydrophobicity of the surface acquired by cholesterol binding resulting in the folding or shrinkage of molecule in turn leading to core etching. The method was successfully applied for the detection of cholesterol in real samples and agrees well with values obtained from the conventional method. Because of its significant plasmonic shift and simplicity, this biosensor could be used for cholesterol detection as it does not demand either any hazardous and costly chemicals or any complex synthetic routes.