Synthesis of Demissidine Analogues from Tigogenin via Imine Intermediates.

A five-step transformation of a spiroketal side chain of tigogenin into an indolizidine system present in solanidane alkaloids such as demissidine and solanidine was elaborated. The key intermediate in the synthesis was spiroimine 3 readily obtained from tigogenin by its RuO4 oxidation to 5,6-dihydrokryptogenin followed by amination with aluminum amide generated in situ from DIBAlH and ammonium chloride. The mild reduction of spiroimine to a 26-hydroxy-dihydropyrrole derivative and subsequent mesylation resulted in the formation of 25-epidemissidinium salt or 23-sulfone depending on reaction conditions.

Solasodine, Isolated from Solanum sisymbriifolium Fruits, Has a Potent Anti-Tumor Activity Against Pancreatic Cancer.

BACKGROUND: Increasing evidences have revealed that solasodine, isolated from Solanum sisymbriifolium fruits, has multiple functions such as anti-oxidant, anti-tumor and anti-infection. However, its role in pancreatic cancer has not been well studied. METHODS: To explore the role of solasodine in pancreatic cancer, human pancreatic cell lines including SW1990 and PANC1 were treated with different concentrations of solasodine for 48 h, and cell viability was evaluated by MTT assay, cell invasion and migration were evaluated by Transwell assay. The effect of solasodine on the apoptosis of SW1990 and PANC1 cells was detected by flow cytometry. To further explore the antitumor effect of solasodine in vivo, an SW1990 tumor-bearing mouse model was constructed. The effects of solasodine on cytokines in the serum of SW1990 tumor-bearing mice were also evaluated by ELISA assay. RESULTS: Specifically, in vitro, solasodine could significantly inhibit the proliferation of pancreatic cancer cell lines SW1990 and PANC1 cells. Flow cytometric analysis indicated that solasodine could induce apoptosis of SW1990 and PANC1 cells. Western blot assay indicated that solasodine could significantly inhibit the activation of Cox-2/Akt/GSK3ß signal pathway. Meanwhile, the release of Cytochrome c from mitochondria to cytoplasm which can raise the caspases cascade (C-caspase 3 and C-caspase 9) was significantly enhanced by solasodine. In vivo, the results showed that solasodine had potent anti-tumor activities with a lower cytotoxicity. In addition, the serum TNF-α, IL-2 and IFN-γ levels in SW1990 tumor-bearing mice after the treatment of solasodine was significantly increased. CONCLUSION: Taken together, our results suggested that the solasodine could prevent the progression of pancreatic cancer by inhibiting proliferation and promoting apoptosis, as well as stimulating immunity, suggesting that solasodine might be a potential therapeutic strategy for pancreatic cancer.

Insights into the stability of carotenoids and capsaicinoids in water-based or oil-based chili systems at different processing treatments.

Ultrasonication, microwave, heat, and light treatments, as well as storage conditions, were investigated for their effects on the stability of carotenoids and capsaicinoids in water/oil chili systems. The stability of carotenoids and capsaicinoids were found to vary in response to different processing treatments. Carotenoid and capsaicinoid contents in chili juice (CJ, water system) were increased by low-power ultrasonic and microwave treatments, but decreased by high-power treatments. The thermal stability of carotenoids and capsaicinoids in hot pot bottom (HPB, oil system) were superior to those in CJ. Moreover, ultraviolet light significantly reduced the contents of carotenoids and capsaicinoids in both CJ and HPB. It was also demonstrated that low temperature conditions (4 °C) significantly delayed the degradation of carotenoids and capsaicinoids in chili-based food. In conclusion, our findings suggest that the stability of carotenoids and capsaicinoids can be tuned using different processing and storage techniques appropriate to different systems.

The synthesis and cholinesterase inhibitory activities of solasodine analogues with seven-membered F ring.

Solasodine analogues containing a seven-membered F ring with a nitrogen atom placed at position 22a were prepared from diosgenin or tigogenin in a four-step synthesis comprising of the simultaneous opening of the F-ring and introduction of cyanide in position 22α, activation of the 26-hydroxyl group as mesylate, nitrile reduction, and N-cyclization. Solasodine, six obtained 22a(N)-homo analogues, as well as four 26a-homosolasodine derivatives and their open-chain precursors (13 in total) were tested as potential inhibitors of acetyl- and butyryl-cholinesterases and showed activity at micromolar concentrations. The structure-activity relationship study revealed that activities against studied esterases are affected by the structure of E/F rings and the substitution pattern of ring A. The most potent compound 8 acted as non-competitive inhibitors and exerted IC50 = 8.51 µM and 7.05 µM for eeAChE and eqBChE, respectively. Molecular docking studies revealed the hydrogen bond interaction of 8 with S293 of AChE; further rings are stabilized via hydrophobic interaction (ring A) or interaction with Y341 and W286 (rings B and C). Biological experiments showed no neurotoxicity of differentiated SH-SY5Y cells. More importantly, results from neuroprotective assay based on glutamate-induced cytotoxicity revealed that most derivatives had the ability to increase the viability of differentiated SH-SY5Y cells in comparison to galantamine and lipoic acid assayed as standards. The newly synthesized solasodine analogues are able to inhibit and to bind cholinesterases in noncompetitive mode of inhibition and exhibited neuroprotection potential of differentiated neuroblastoma cells after Glu-induced toxicity.

Synthesis and Stereochemical Assignment of Conioidine A: DNA- and HSA-Binding Studies of the Four Diastereomers.

Conioidine A (1), isolated in 1993 with unknown relative and absolute configuration, was suggested to be a DNA-binding compound by an indirect technique. Four stereoisomers of conioidine A have been synthesized from d- and l-proline, and the natural product has been identified as possessing (4R,6R) absolute configuration. Binding of the conioidine diastereomers to calf thymus DNA (CT DNA) and human serum albumin (HSA) has been investigated by fluorescence spectroscopy and isothermal titration calorimetry (ITC). All stereoisomers display at least an order of magnitude weaker binding to DNA than the control compound netropsin; however, a strong association with HSA was observed for the (4R,6S) stereoisomer.

Mapping Solanum chacoense mediated Colorado potato beetle (Leptinotarsa decemlineata) resistance in a self-compatible F2 diploid population.

KEY MESSAGE: A major QTL on chromosome 2 associated with leptine biosynthesis and Colorado potato beetle resistance was identified in a diploid S. chacoense F2 population using linkage mapping and bulk-segregant analysis. We examined the genetic features underlying leptine glycoalkaloid mediated Colorado potato beetle (Leptinotarsa decemlineata) host plant resistance in a diploid F2 mapping population of 233 individuals derived from Solanum chacoense lines USDA8380-1 and M6. The presence of foliar leptine glycoalkaloids in this population segregated as a single dominant gene and displayed continuous distribution of accumulated quantity in those individuals producing the compound. Using biparental linkage mapping, a major overlapping QTL region with partial dominance effects was identified on chromosome 2 explaining 49.3% and 34.1% of the variance in Colorado potato beetle field resistance and leptine accumulation, respectively. Association of this putative resistance region on chromosome 2 was further studied in an expanded F2 population in a subsequent field season. Loci significantly associated with leptine synthesis colocalized to chromosome 2. Significant correlation between increased leptine content and decreased Colorado potato beetle defoliation suggests a single QTL on chromosome 2. Additionally, a minor QTL with overdominance effects explaining 6.2% associated with Colorado potato beetle resistance donated by susceptible parent M6 was identified on chromosome 7. Bulk segregant whole genome sequencing of the same F2 population detected QTL associated with Colorado potato beetle resistance on chromosomes 2, 4, 6, 7, and 12. Weighted gene co-expression network analysis of parental lines and resistant and susceptible F2 individuals identified a tetratricopeptide repeat containing protein with a putative regulatory function and a previously uncharacterized acetyltransferase within the QTL region on chromosome 2, possibly under the control of a regulatory Tap46 subunit within the minor QTL on chromosome 12.

Tropane alkaloids biosynthesis involves an unusual type III polyketide synthase and non-enzymatic condensation.

The skeleton of tropane alkaloids is derived from ornithine-derived N-methylpyrrolinium and two malonyl-CoA units. The enzymatic mechanism that connects N-methylpyrrolinium and malonyl-CoA units remains unknown. Here, we report the characterization of three pyrrolidine ketide synthases (PYKS), AaPYKS, DsPYKS, and AbPYKS, from three different hyoscyamine- and scopolamine-producing plants. By examining the crystal structure and biochemical activity of AaPYKS, we show that the reaction mechanism involves PYKS-mediated malonyl-CoA condensation to generate a 3-oxo-glutaric acid intermediate that can undergo non-enzymatic Mannich-like condensation with N-methylpyrrolinium to yield the racemic 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This study therefore provides a long sought-after biosynthetic mechanism to explain condensation between N-methylpyrrolinium and acetate units and, more importantly, identifies an unusual plant type III polyketide synthase that can only catalyze one round of malonyl-CoA condensation.

Enhancement of Solasodine Extracted from Fruits of Solanum nigrum L. by Microwave-Assisted Aqueous Two-Phase Extraction and Analysis by High-Performance Liquid Chromatography.

Background: Solasodine is a major bioactive ingredient in Solanum nigrum L. that has strong pharmacological characteristics. Therefore, the development of a simple and effective extraction method for obtaining solasodine is highly important. This study aims to provide a rapid and effective method for extracting solasodine from Solanum nigrum L. by microwave-assisted aqueous two-phase extraction (MAATPE). Methods: First, the high-performance liquid chromatography (HPLC) conditions were established for the detection of solasodine. Then, the aqueous two-phase system (ATPS) compositions were examined. On the basis of the results of single-factor experiments, for a better yield, response surface methodology (RSM) was used to optimize influential factors including the extraction temperature, extraction time and liquid-to-solid ratio. Results: The maximum extraction yield of 7.11 ± 0.08 mg/g was obtained at 44 °C, an extraction time of 15 min, and a liquid-to-solid ratio of 42:1 mL/g in the ATPS consisting of EtOH solvent, (NH4)2SO4, and water (28:16:56, w/w/w). The extraction yield of the alkaloid obtained using this method was markedly higher than those of microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE). Conclusions: In this work, solasodine was extracted by MAATPE for the first time and a high yield was obtained. MAATPE is a simple, rapid, and green technique for extraction from medical plants. Thus, the present study will enable the development of a feasible extraction method of active alkaloids from Solanum nigrum L.

Structure-based virtual screening leading to discovery of highly selective butyrylcholinesterase inhibitors with solanaceous alkaloid scaffolds.

According to recent research advance, it is interesting to identify new, potent and selective inhibitors of human butyrylcholinesterase (BChE) for therapeutic treatment of both the Alzheimer's disease (AD) and heroin abuse. In this study, we carried out a structure-based virtual screening followed by in vitro activity assays, with the goal to identify new inhibitors that are selective for BChE over acetylcholinesterase (AChE). As a result, a set of new, selective inhibitors of human BChE were identified from natural products with solanaceous alkaloid scaffolds. The most active one of the natural products (compound 1) identified has an IC50 of 16.8 nM against BChE. It has been demonstrated that the desirable selectivity of these inhibitors for BChE over AChE is mainly controlled by three key residues in the active site cavity, i.e. residues Q119, A277, and A328 in BChE versus the respective residues Y124, W286, and Y337 in AChE. Based on this structural insight, future rational design of new, potent and selective BChE inhibitors may focus on these key structural differences in the active site cavity.

Two-step Synthesis of Solasodine Pivalate from Diosgenin Pivalate.

A two-step synthesis of solasodine pivalate from diosgenin pivalate is described. The key transformation involves the reaction of diosgenin pivalate with benzyl carbamate (CbzNH2) promoted by TMSOTf. During the reaction the F-ring of the spiroketal moiety opens up with a simultaneous introduction of a Cbz-protected amino group in position 26. A one-pot deprotection of 26-amine with AcBr/BuOH followed by the N-cyclization affords solasodine pivalate in 45% overall yield.

Revision of the Structure of N, O-Diacetylsolasodine. Unusual Epimerization at the Spiro Carbon Atom during Acetylation of Solasodine.

The steroidal alkaloid solasodine (1) undergoes inversion of configuration at the C-22 spiro atom when treated with acetic anhydride-pyridine at ambient temperature. The basic solvolysis of the N, O-diacetyl derivative (2) reverses the reaction, yielding the starting solasodine (1). The mechanisms of both processes (acetylation and deacetylation) were studied in terms of possible reaction pathways.

Fe3O4-solamargine induces apoptosis and inhibits metastasis of pancreatic cancer cells.

Fe3O4-magnetic liposome (MLP) can deliver drugs to target tissues and can increase drug efficacy. The present study aimed to investigate the effects of solamargine (SM) and Fe3O4-SM in pancreatic cancer (PC). Cell viability was detected using a Cell Counting kit­8 assay. Apoptosis and cell cycle progression was tested using a flow cytometry assay. A scratch assay was used to examine cell metastasis. Quantitative polymerase chain reaction, western blot analysis or immunohistochemical analysis were performed to determine the expression of target factors. Magnetic resonance imagining (MRI) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling were conducted to detect tumor growth and apoptosis in vivo, respectively. It was demonstrated that Fe3O4-SM inhibited cancer cell growth via a slow release of SM over an extended period of time. SM was revealed to induce apoptosis and cell cycle arrest. Furthermore, SM decreased the expression of X-linked inhibitor of apoptosis, Survivin, Ki­67, proliferating cell nuclear antigen and cyclin D1, but increased the activity of caspase-3. It was also observed that SM inhibited tumor cell metastasis by modulating the expression of matrix metalloproteinase (MMP)-2 and TIMP metallopeptidase inhibitor-2. Furthermore, the phosphorylation of protein kinase B and mechanistic target of rapamycin was suppressed by SM. Notably, the effect of SM was enhanced by Fe3O4-SM. The malignant growth of PC was decreased by SM in vivo. Furthermore, the expression of Ki­67 was decreased by SM and Fe3O4-SM. Additionally, cell apoptosis was increased in the Fe3O4-SM group, compared with the SM group. The present study illustrated the antitumor effect and action mec-hanism produced by SM. Additionally, it was demonstrated that Fe3O4-SM was more effective than SM in protecting against PC.

Synthesis of nortropane alkaloid calystegine B2 from methyl α-d-xylopyranoside.

A new synthetic route for formation of a central cycloheptanone intermediate leading to the nortropane alkaloid calystegine B2 is described. The approach installs the desired ketone functionality directly in a ring-closing metathesis step. The target compound was prepared over 10 steps from commercially available methyl α-d-xylopyranoside.

Solasodine reverses stemness and epithelial-mesenchymal transition in human colorectal cancer.

Adverse side effects of conventional chemotherapy, acquired resistance and fatal tumor metastasis of human colorectal cancer (CRC) are propelling the exploration for novel selective anticarcinogens. Solasodine is a main active component isolated from Solanum incanum L that exhibited a potent stemness and invasion inhibitory effect on human colorectal cancer HCT116 cells. Colony Spheroid formation assay showed that solasodine dose-dependently prohibited HCT116 cell stemness. CD133, CD44, Nanog, Oct-4 and Sox-2 were inhibited by solasodine to reverse stemness and similar mechanism was stimulated in vivo. Transwell and scratch wound assays revealed that solasodine impeded HCT116 cell invasion and migration potential strengthened by TGF-ß1. Moreover, solasodine attenuated TGF-ß1-induced EMT and decreased MMPs while in vivo study showed the same trend. The results of this study implied that solasodine may be a novel therapeutic drug for CRC treatment.

Analysis of calystegines in tomato-based products by liquid chromatography-Orbitrap mass spectrometry.

Taking into account the high consumption of tomato and tomato-based products, a study of the occurrence of 7 nortropane alkaloids (calystegines A3, A5, B1, B2, B3, B4 and C1) in this type of matrix has been performed. For that purpose, a simple and fast solid-liquid extraction with methanol/water (50/50, v/v) has been developed, and then liquid chromatography, using a HILIC-A as stationary phase, coupled to high resolution mass spectrometry (LC-HRMS-Orbitrap) has been used for their determination. The developed method was validated and recoveries ranged from 96 to 121%, and relative standard deviations lower than or equal to 16% were obtained. Limits of quantification (LOQ) were established at 0.1 (B4), 0.25 (B3) and 0.5 (A3, A5, B1, B2 and C1) mg/kg. Twelve different samples were analyzed, comprising 4 crushed tomatoes, 4 fried tomatoes and 4 jam tomatoes. Calystegines A3 and B2 were identified in all the analyzed samples, while calystegine B3 was determined in all the fried tomatoes as well as in two crushed tomatoes and one jam tomato at concentrations ranging from 0.4 mg/kg (calystegine B2) to 19.0 mg/kg (calystegine A3). Although calystegine A5 was also detected in all the samples, only 3 of them gave concentrations higher than LOQ. Additionally, calystegine B1 was found in one jam tomato at 1.9 mg/kg.

In silico and in vivo characterization of cabralealactone, solasodin and salvadorin in a rat model: potential anti-inflammatory agents.

BACKGROUND: The present study investigates the hepato- and DNA-protective effects of standardized extracts of Cleome brachycarpa (cabralealactone), Solanum incanum (solasodin), and Salvadora oleioides (salvadorin) in rats. MATERIALS AND METHODS: Hepatotoxicity was induced with intraperitoneal injection of carbon tetrachloride (CCl4) (1 mL/kg b.wt.) once a week for 12 weeks. The hepato- and DNA protective effects of the extracts in different combinations were compared with that of a standard drug Clavazin (200 mg/kg b.wt.). Tissue alanine aminotransferase, alpha-fetoprotein, tumor necrosis factor alpha (TNF-α), isoprostanes-2α, malondialdehyde, and 8-hydroxydeoxyguanosine, the significant hallmarks of oxidative stress, were studied. RESULTS: Histopathological findings of the liver sections from the rat group which received CCl4+cabralealactone, solasodin, and salvadorin demonstrated improved centrilobular hepatocyte regeneration with moderate areas of congestion and infiltration comparable with Clavazin. For in silico study, the identified compounds were subjected to molecular docking with cyclooxygenase-2 and TNF-α followed by a molecular dynamics study, which indicated their potential as anti-inflammatory agents. CONCLUSION: Cabralealactone, solasodin, and salvadorin confer some hepatoprotective and DNA-damage protective effects against CCl4-induced toxicity. They successfully restored the normal architecture of hepatocytes and have the potential to be used as inhibitor to main culprits, that is, cyclooxygenase-2 and TNF-α. They can combat oxidative stress and liver injuries both as mono and combinational therapies. However, combination therapy has more ameliorating effects.

Some observations on solasodine reactivity.

This article presents new transformations of solasodine - a representative steroid alkaloid sapogenin from the Solanum family. Oxidation of N,O-diacetylated solasodine with either NaNO2/BF3·Et2O or t-BuONO/BF3·Et2O resulted in partial degradation of the side chain to (20S)-3ß-acetoxypregn-5-ene-20,16ß-carbolactone (vespertilin acetate). The same starting compound when treated with TMSOTf afforded the corresponding pseudosapogenin after aqueous work-up. However, when the crude reaction mixture was directly subjected to purification on a silica gel column, efficient autoxidation to pregna-5,16-dien-3ß-ol-20-one acetate was observed. One-step synthesis of this important drug intermediate from spirosolan alkaloids may be potentially exploited for large-scale production of steroid hormones.

Aglycone solanidine and solasodine derivatives: A natural approach towards cancer.

Over the past few years, it was suggested that a rational approach to treat cancer in clinical settings requires a multipronged approach that augments improvement in systemic efficiency along with modification in cellular phenotype leads to more efficient cell death response. Recently, the combinatory delivery of traditional chemotherapeutic drugs with natural compounds proved to be astonishing to deal with a variety of cancers, especially that are resistant to chemotherapeutic drugs. The natural compounds not only synergize the effects of chemotherapeutics but also minimize drug associated systemic toxicity. In this review, our primary focus was on antitumor effects of natural compounds. Previously, the drugs from natural sources are highly precise and safer than drugs of synthetic origins. Many natural compounds exhibit anti-cancer potentials by inducing apoptosis in different tumor models, in-vitro and in-vivo. Furthermore, natural compounds are also found equally useful in chemotherapeutic drug resistant tumors. Moreover, these Phyto-compounds also possess numerous other pharmacological properties such as antifungal, antimicrobial, antiprotozoal, and hepatoprotection. Aglycone solasodine and solanidine derivatives are the utmost important steroidal glycoalkaloids that are present in various Solanum species, are discussed here. These natural compounds are highly cytotoxic against different tumor cell lines. As the molecular weight is concerned; these are smaller molecular weight chemotherapeutic agents that induce cell death response by initiating apoptosis through both extrinsic and intrinsic pathways.

Solasodine-3-O-ß-d-glucopyranoside kills Candida albicans by disrupting the intracellular vacuole.

The increasing incidence of fungal infections and emergence of drug resistance underlie the constant search for new antifungal agents and exploration of their modes of action. The present study aimed to investigate the antifungal mechanisms of solasodine-3-O-ß-d-glucopyranoside (SG) isolated from the medicinal plant Solanum nigrum L. In vitro, SG displayed potent fungicidal activity against both azole-sensitive and azole-resistant Candida albicans strains in Spider medium with its MICs of 32 µg/ml. Analysis of structure and bioactivity revealed that both the glucosyl residue and NH group were required for SG activity. Quantum dot (QD) assays demonstrated that the glucosyl moiety was critical for SG uptake into Candida cells, as further confirmed by glucose rescue experiments. Measurement of the fluorescence intensity of 2',7'-dichlorofluorescin diacetate (DCFHDA) by flow cytometry indicated that SG even at 64 µg/ml just caused a moderate increase of reactive oxygen species (ROS) generation by 58% in C. albicans cells. Observation of vacuole staining by confocal microscopy demonstrated that SG alkalized the intracellular vacuole of C. albicans and caused hyper-permeability of the vacuole membrane, resulting in cell death. These results support the potential application of SG in fighting fungal infections and reveal a novel fungicidal mechanism.

The Impact of Steroidal Glycoalkaloids on the Physiology of Phytophthora infestans, the Causative Agent of Potato Late Blight.

Steroidal glycoalkaloids (SGAs) are plant secondary metabolites known to be toxic to animals and humans and that have putative roles in defense against pests. The proposed mechanisms of SGA toxicity are sterol-mediated disruption of membranes and inhibition of cholinesterase activity in neurons. It has been suggested that phytopathogenic microorganisms can overcome SGA toxicity by enzymatic deglycosylation of SGAs. Here, we have explored SGA-mediated toxicity toward the invasive oomycete Phytophthora infestans, the causative agent of the late blight disease in potato and tomato, as well as the potential for SGA deglycosylation by this species. Our growth studies indicate that solanidine, the nonglycosylated precursor of the potato SGAs α-chaconine and α-solanine, has a greater physiological impact than its glycosylated forms. All of these compounds were incorporated into the mycelium, but only solanidine could strongly inhibit the growth of P. infestans in liquid culture. Genes encoding several glycoside hydrolases with potential activity on SGAs were identified in the genome of P. infestans and were shown to be expressed. However, we found no indication that deglycosylation of SGAs takes place. We present additional evidence for apparent host-specific adaptation to potato SGAs and assess all results in terms of future pathogen management strategies.