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| 1.94 | The title is: Structural Diversity, Biosynthetic Pathways, Natural Sources, Molecular Targets, and Therapeutic Applications of Cardenolides: A Comprehensive Review |
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Title: Structural Diversity, Biosynthetic Pathways, Natural Sources, Molecular Targets, and Therapeutic Applications of Cardenolides: A Comprehensive Review
Biosynthetic Pathways of Cardenolides: PRISE Homolog Evolution and Progesterone 5β-Reductase Activity
The biosynthetic pathways of cardenolides are defined by key enzymatic steps and evolutionary adaptations, with a central focus on progesterone 5β-reductase activity and the evolution of PRISE (progesterone 5β-reductase/iridoid synthase) homologs. Lanosterol is proposed as the first committed intermediate, followed by cholesterol as a subsequent precursor, though experimental evidence for the lanosterol-to-cholesterol conversion in plants remains limited [1]. Progesterone, a critical pathway intermediate, can be detected via targeted metabolomics using ultra-performance liquid chromatography-mass spectrometry with selected ion monitoring for its m/z 315 [2].
Key Findings on Progesterone 5β-Reductase and PRISE Homologs
| Aspect | Details | Citation |
|---|---|---|
| Recombinant progesterone 5β-reductase activity | rAtStR2 from Arabidopsis thaliana enantioselectively converts progesterone to 5β-pregnane-3,20-dione | [3] |
| PRISE homolog substrate specificity | AtStR1 muteins with reduced methyl vinyl ketone (MVK) activity show enhanced preference for progesterone | [3] |
| PRISE phylogeny | Early duplications in land plants; clade V includes gymnosperm homologs, functionally characterized iridoid synthases (e.g., Catharanthus), and cardenolide-associated PRISEs (e.g., Digitalis) | [4] |
| PRISE progesterone 5β-reductase activity | All clades exhibit activity toward progesterone; homologs from cardenolide-free species (A. thaliana, Amborella trichopoda) also show this activity, suggesting an ancestral role | [4] |
| PRISE evolution | Gene duplication paralogues may have shifted to prefer larger substrates like progesterone, diverging from stress-related roles in detoxifying small reactive electrophile species (RES) | [3] |
| Digitalis purpurea progesterone 5β-reductase | Transcriptome analysis identified two contigs with >72% identity to characterized genes; their constitutive organ expression contrasts with tissue-specific cardenolide accumulation, implying other rate-limiting steps | [1] |
Phylogenetic and functional analyses highlight the evolutionary flexibility of PRISE homologs: their ancestral progesterone 5β-reductase activity may have coevolved toward specialized functions in cardenolide-producing plants, while paralogues from gene duplications adapted to larger substrates like progesterone. In Digitalis purpurea, the disconnect between constitutive progesterone 5β-reductase expression and tissue-specific cardenolide accumulation further underscores the complexity of pathway regulation beyond this key enzymatic step [1].
Structural Diversity and Natural Sources of Cardenolides: Isolation, Characterization, and Phytochemical Profiling
Cardenolides are a structurally diverse class of steroid derivatives defined by a five-membered unsaturated γ-lactone ring, distinguishing them from bufadienolides (six-membered lactone ring) [1]. They are predominantly isolated from select plant families, including Apocynaceae, Plantaginaceae (e.g., Digitalis species), Celastraceae, and Asclepiadaceae [1][5][6][7]. Historic sources like Digitalis purpurea L. and D. lanata Ehrh. have been used since 1785 for cardiac treatments [1], while other natural sources include Thevetia thevetioides (Apocynaceae) seeds [8], Salacia staudtiana (Celastraceae) seeds [5], Elaeodendron sp. (Celastraceae) wood [6], Calotropis species (C. gigantea and C. procera, Asclepiadaceae) [7][9][10], and restricted members of Coronilla and Securigera genera [11].
Isolation, Characterization, and Key Compounds of Cardenolides from Natural Sources
| Source | Isolation Methods | Characterization Techniques | Key Compounds |
|---|---|---|---|
| T. thevetioides seeds | Degreasing with hexane, ethanol extraction | HPTLC (Kedde reagent), Q-TOF MS/MS, GC-MS (hydrolyzed glycosides) | Mature seeds: Triglycosides (thevetins A, B, C) and acetylated forms; aglycones cannogenin, digitoxigenin, yccotligenin Immature seeds: Monoglycosides (peruvosides A, B, C) and acetylated forms, diglycosides (thevebiosides A, B, C), thevetins [8] |
| S. staudtiana seeds | Column chromatography (Sephadex LH-20), preparative HPLC | ESI-HRMS, NMR, X-ray crystallography (compounds 1, 2, 4) | 14 compounds (1–14) [5] |
| Elaeodendron sp. wood | Not specified | HRFABMS, NMR (COSY, TOSCY, HMBC, ROESY) | Elaeodendroside T (with 1,4-dioxane ring between rings A and A′), elaeodendroside U [6] |
| Calotropis gigantea exudate | Solvent partitioning (hexane, ethyl acetate, water); silica gel, Sephadex LH20, ODS chromatography (guided by TCF/β-catenin assay) | Not specified | 6 cardenolides [10] |
| Unspecified source | Not specified | Not specified | 15 new and 18 known cardiac glycosides, including 8 19-nor-cardenolides (some rare 19-nor-10-hydroperoxycardenolides) [12] |
Structural diversity of cardenolides arises from variations in aglycones, sugar moieties, and substitutions. For example, Digitalis species produce mono-, di-, and tri-glycosides of aglycones such as digitoxigenin and digoxigenin [1]. Calotropis-derived cardenolides include 2-oxovoruscharin, whose semi-synthetic derivative UNBS1450 is in Phase I clinical trials [7]. Synthetic 5α-oleandrigenin analogues (A/B-trans steroid core) differ from natural A/B-cis oleandrin and oleandrigenin, with modifications to C3-O methylation, C14/C16 oxygen functions, and 17β-furyl groups [13]. Structure-activity relationship (SAR) analyses of Calotropis cardenolides indicate that six-membered sugar rings enhance cytotoxicity, C-10 formyl/methyl-hydroxyl groups increase activity, and 4'-OH/16-OH groups reduce it [7]. Additionally, the 1,4-dioxane ring in Elaeodendron cardenolides (e.g., elaeodendroside T) is critical for antiproliferative activity [6].
Cardenolide-Mediated Therapeutic Activities: Cytotoxicity, Antibacterial Effects, and Mechanisms of Action
Cardenolides exhibit potent, selective cytotoxic activity against various human cancer cell lines, with additional antibacterial properties reported for some compounds. Key findings on their biological activities, including cytotoxicity and associated mechanisms, are summarized below.
Cytotoxic Activity of Cardenolides and Cardiac Glycosides Across Cell Lines
| Compound(s) | Cell Lines Tested | IC₅₀ Range (μM) | Key Observations | Citation |
|---|---|---|---|---|
| Cardenolides (1—5, 8—18) | HT-1080, A549, LLC, B16-BL6 | HT-1080: 0.054—1.6; A549: 0.016—0.65; LLC/B16-BL6: >100 | Selective activity against human cell lines; non-cardenolides inactive | [14] |
| Compounds 13, 15 | HT-1080, A549 | 13: HT-1080 0.054, A549 0.020; 15: HT-1080 0.055, A549 0.016 | Activity comparable to doxorubicin (IC₅₀ HT-1080 0.059, A549 0.026) | [14] |
| Compounds 1, 11, 19, 22, 26, 28, 29 | P15, MGC-803, SW1990 | 0.01—0.62 | Isolated as 15 new (2−16) and 18 known (1, 17−33) cardiac glycosides; significant growth inhibition | [12] |
| 5α-oleandrigenin derivatives (1, 2, 5) | Not specified (general cytotoxicity) | 1 > 2 > 5 (order of magnitude difference) | Oleandrin (1) ~10x more active than oleandrigenin (2); 3-O-methyl oleandrigenin (5) less active than 2 | [13] |
| 16β-hydroxy derivative (20) | CEM | 14.1 ± 0.5 | Specific cytotoxicity towards CEM cells | [13] |
| 16β-acetoxy-14,15β-epoxide (24) | Multiple cancer cell lines, normal fibroblasts | Not specified (strong cytotoxicity) | Strong activity against cancer cells but affects normal fibroblasts (narrow therapeutic window) | [13] |
Morphological changes (stretching, shrinking, multi-blebbing) and ladder-like DNA fragmentation (indicating apoptosis) were observed in HT-1080 and A549 cells treated with cardenolides (e.g., echujin 15) [14]. Additionally, cardiac glycosides 1, 22, 26, and 28 triggered dose-dependent massive apoptosis in MGC-803 cells [12].
Structure-activity relationship (SAR) analyses have identified key features influencing cytotoxicity: six-membered ring sugar groups confer stronger inhibitory activity than five-membered rings; a formyl or methyl-hydroxyl group at C-10 enhances cytotoxicity, while 4'-OH or 16-OH groups decrease it [7]. Chemical modification of 2-oxovoruscharin (a Calotropis procera cardenolide) yielded UNBS1450, which exhibits more potent anti-proliferative activity, lower toxicity, and acts as a strong sodium pump inhibitor and non-apoptotic cell death inducer; UNBS1450 is currently in Phase I clinical trials [7].
Mechanisms of cytotoxicity include apoptosis induction, Na⁺/K⁺-ATPase inhibition, Wnt signaling modulation, and microtubule stabilization. Cardenolides induce apoptosis via caspase activation and PARP cleavage—for example, 5α-oleandrigenins 5, 23, and 24 induce strong caspase-3/7 activity and PARP-1 cleavage in CEM cells [13]. Na⁺/K⁺-ATPase inhibition is linked to cytotoxicity, with glycosides generally more inhibitory than aglycones (oleandrin (1) is ~25% more active than oleandrigenin (2) in this context) [13]. However, discrepancies exist: compound 15 induces apoptosis but shows no ATPase inhibition [13]. Cardenolides also modulate the Wnt pathway: calotropin (4) from Calotropis gigantea inhibits TCF/β-catenin transcriptional activity, decreases β-catenin and c-myc levels, and induces β-catenin proteasomal degradation via CK1α-dependent phosphorylation [10]. Breastin (a cardenolide-containing preparation with oleandrin and odoroside H) enhances tubulin polymerization similarly to paclitaxel, as confirmed by confocal microscopy and in vitro assays; molecular docking revealed binding to tubulin’s drug-binding sites, supporting microtubule stabilization [15].
Beyond cytotoxicity, some cardenolides exhibit antibacterial activity: compounds 1 and 3 from Salacia staudtiana seeds, along with the crude extract, showed distinct activity against B. subtilis and S. aureus [5].
Molecular Targets of Cardenolides: Na+/K+-ATPase Inhibition, Wnt Signaling Suppression, and Tubulin Interaction
Cardenolides are highly specific inhibitors of the sodium pump (Na⁺/K⁺-ATPase)—an essential ion carrier critical for maintaining plasma membrane potential and ionic balance across cell membranes [1][16][13]. These compounds bind to Na⁺/K⁺-ATPases at nanomolar concentrations [1], with glycosides typically exhibiting higher inhibitory activity than their corresponding aglycones [13]. Molecular modeling confirms that cardenolides bind to the experimentally determined ouabain-binding site, mediated by hydrogen bonds and hydrophobic interactions with residues including I315, F316, F783, F786, L793, and T797 [13]. Beyond direct inhibition, Na⁺/K⁺-ATPase acts as a signal transducer: some cardenolides (e.g., juventasoside B from Streptocaulon juventas) can stimulate the Na⁺/K⁺-ATPase/Src receptor complex at concentrations inducing <10% enzyme inhibition, selectively activating downstream pathways to alter prostate cancer cell growth [17]. Notably, the regulation of cell viability by such cardenolides correlates with their ligand-binding affinity to the Na⁺/K⁺-ATPase receptor, not their inhibitory potency [17].
Cardenolides also suppress Wnt signaling, as demonstrated by six compounds isolated from Calotropis gigantea exudate, which strongly inhibit TCF/β-catenin transcriptional activity (TOP activity) at nanomolar concentrations without significant non-selective inhibition (FOP activity) [10]. These cardenolides are cytotoxic to Wnt-dependent colon cancer cell lines (SW480, DLD1, HCT116) but not Wnt-independent RKO cells [10]. Calotropin, a representative compound from this set, induces dose-dependent decreases in nuclear and cytosolic β-catenin levels via proteasomal degradation [10]. This effect is mediated by upregulated CK1α mRNA expression (increasing CK1α protein levels), which triggers β-catenin phosphorylation at the CK1α site (S45), followed by GSK3β-mediated phosphorylation (S33, S37, T41) and subsequent ubiquitination [10]. Calotropin-induced apoptosis in SW480 cells is CK1α-dependent, as siRNA knockdown of CK1α abrogates both β-catenin degradation and apoptosis [10].
Additionally, cardenolides interact with tubulin, as shown by Breastin—a preparation of monoglycosidic cardenolides (oleandrin, oleandrigenin sarmentoside, neritaloside, odoroside H, odoroside A) from Nerium oleander leaves. Breastin enhances tubulin polymerization in U2OS osteosarcoma cells expressing α-tubulin-GFP (comparable to paclitaxel) [15], and in vitro assays confirm that high Breastin concentrations increase light scattering at 350 nm, mirroring paclitaxel’s activity [15]. Molecular docking studies reveal that Breastin’s cardenolides (including odoroside H and neritaloside) bind to tubulin’s main drug-binding sites (Vinca alkaloid-, taxane-, and colchicine-binding sites), supporting tubulin interaction as a mechanism for their anticancer effects [15]. In a MAXF 401 tumor xenograft model, Breastin (80 mg/kg/day) reduces tumor growth (to 770% of initial size over 39 days, vs. 2836% in controls) and enhances paclitaxel efficacy when co-administered, preventing paclitaxel resistance [15].
Environmental and Cultivation Influences on Cardenolide Production: Rhizobacterial, Light Quality, and CO2 Effects
Environmental and cultivation conditions exert distinct effects on cardenolide production across plant species. For milkweeds—known for synthesizing cardenolides that disrupt Na+/K+‐ATPase in animal cells [18]—elevated CO₂ modulates both constitutive and induced cardenolide profiles, while light quality and rhizobacterial inoculation influence accumulation in other species. Broader environmental factors (e.g., temperature, rainfall, altitude, soil conditions) and post-harvest practices (collection, drying, storage) also affect cardenolide levels in seeds [19].
Cardenolide Responses to Elevated CO₂ in Milkweed Species and Light Quality in Digitalis purpurea
| Species | Condition | Parameter | Effect/Value | Citation |
|---|---|---|---|---|
| Asclepias incarnata | Elevated CO₂ | Constitutive cardenolide concentration | Reduced by 37% | [18] |
| A. syriaca | Elevated CO₂ | Constitutive cardenolide concentration | Reduced by 10% | [18] |
| A. curassavica | Elevated CO₂ | Constitutive cardenolide concentration | Reduced by 5% | [18] |
| A. speciosa | Elevated CO₂ | Constitutive cardenolide concentration | Increased by 22% | [18] |
| Four milkweed species | Elevated CO₂ | Mechanically induced cardenolide concentration | Increased by 28% across species | [18] |
| Multiple milkweed species | Elevated CO₂ | Constitutive cardenolide diversity | Increased by 24% across species | [18] |
| A. incarnata | Elevated CO₂ | Induced cardenolide diversity | Declined by 70% | [18] |
| A. speciosa | Elevated CO₂ | Induced cardenolide diversity | Declined by 11% | [18] |
| Digitalis purpurea | Red:blue (R:B) 2:8 LEDs | Digitoxin content | 0.427 mg g⁻¹ DW (highest level) | [20] |
| Digitalis purpurea | Red:blue (R:B) 2:8 LEDs | Digoxin content | 0.16 mg g⁻¹ DW (highest level) | [20] |
| Digitalis purpurea | Blue LEDs | Cardenolide levels | Lower than R:B 2:8 LED treatment | [20] |
In Digitalis purpurea—a rich source of cardenolides [20]—the effect of light quality is linked to blue light photoreceptors and phytochrome, which regulate cardenolide biosynthesis [20]. Rhizobacterial inoculation further enhances production: in D. lanata, strains T1F11, P1F2, and P1V21 increased lanatoside C content by almost 8-fold compared to non-inoculated controls [21].
References
[1] Wurtele, Eve Syrkin; Chappell, Joe; Jones, A Daniel; Celiz, Mary Dawn; Ransom, Nick; Hur, Manhoi; Rizshsky, Ludmila; Crispin, Matthew; Dixon, Philip; Liu, Jia; P Widrlechner, Mark; Nikolau, Basil J, Medicinal Plants: A Public Resource for Metabolomics and Hypothesis Development, 2012, Metabolites
[2] Cuello C, Jansen HJ, Abdallah C, Zamar Mbadinga DL, Birer Williams C, Durand M, Oudin A, Papon N, Giglioli-Guivarc'h N, Dirks RP, Jensen MK, O'Connor SE, Besseau S, Courdavault V., The Madagascar palm genome provides new insights on the evolution of Apocynaceae specialized metabolism, 2024, Heliyon
[3] Klein J, Ernst M, Christmann A, Tropper M, Leykauf T, Kreis W, Munkert J., Knockout of Arabidopsis thaliana VEP1, Encoding a PRISE (Progesterone 5β-Reductase/Iridoid Synthase-Like Enzyme), Leads to Metabolic Changes in Response to Exogenous Methyl Vinyl Ketone (MVK), 2021, Metabolites
[4] Trinh-Don Nguyen; O'Connor, Sarah E., The Progesterone 5β-Reductase/Iridoid Synthase Family: A Catalytic Reservoir for Specialized Metabolism across Land Plants, 2020, ACS CHEMICAL BIOLOGY
[5] Kamtcha, Duplex Wetadieu; Tene, Mathieu; Bedane, Kibrom Gebreheiwot; Knauer, Lena; Brieger, Lukas; Strohmann, Carsten; Tane, Pierre; Kusari, Souvik; Spiteller, Michael, Cardenolides and dihydro-β-agarofuran sesquiterpenes from the seeds of Salacia staudtiana, 2019, FITOTERAPIA
[6] Cao, S.; Brodie, P.J.; Miller, J.S.; Ratovoson, F.; Callmander, M.W.; Randrianasolo, S.; Rakotobe, E.; Rasamison, V.E.; Suh, E.M.; TenDyke, K.; Kingston, D.G.I., Antiproliferative Cardenolides of an Elaeodendron sp. from the Madagascar Rain Forest, 2007, Journal of Natural Products
[7] Chan, Eric Wei Chiang; Sweidan, Nuha I.; Wong, Siu Kuin; Chan, Hung Tuck, Cytotoxic Cardenolides from Calotropis Species: A Short Review, 2017, RECORDS OF NATURAL PRODUCTS
[8] Vázquez-Martínez J, Bravo-Villa P, Molina-Torres J., Thevetia thevetioides Cardenolide and Related Cardiac Glycoside Profile in Mature and Immature Seeds by High-Resolution Thin-Layer Chromatography (HPTLC) and Quadrupole Time of Flight–Tandem Mass Spectrometry (Q-TOF MS/MS) Reveals Insights of the Cardenolide Biosynthetic Pathway, 2024, Molecules
[9] Kinda, P.T.; Nacoulma, A.P.; Samson, S.; Compaore, M.; Djandé, A.; Lagnika, L.; Kiendrebeogo, M., The Metabolomic study of Calotropis procera Ait. from Burkina Faso, based on chemical functional groups profiling using FTIR, 2020, Journal of Complementary and Integrative Medicine
[10] Ishibashi, Masami, Screening for natural products that affect Wnt signaling activity, 2019, JOURNAL OF NATURAL MEDICINES
[11] Wink, M., Evolution of secondary metabolites in legumes (Fabaceae), 2013, SOUTH AFRICAN JOURNAL OF BOTANY
[12] Tian, D.-M.; Cheng, H.-Y.; Jiang, M.-M.; Shen, W.-Z.; Tang, J.-S.; Yao, X.-S., Cardiac Glycosides from the Seeds of Thevetia peruviana, 2016, Journal of Natural Products
[13] Michalak, K.; Raŕová, L.; Kubala, M.; Čechová, P.; Strnad, M.; Wicha, J., Synthesis and evaluation of cytotoxic and Naþ/Kþ-ATP-ase inhibitory activity of selected 5a-oleandrigenin derivatives, 2019, European Journal of Medicinal Chemistry
[14] Ueda, JY; Tezuka, Y; Banskota, AH; Le Tran, Q; Tran, QK; Sam, I; Kadota, S, Antiproliferative Activity of Cardenolides Isolated from Streptocaulon juventas, 2003, BIOLOGICAL & PHARMACEUTICAL BULLETIN
[15] Rashan LJ, Özenver N, Boulos JC, Dawood M, Roos WP, Franke K, Papasotiriou I, Wessjohann LA, Fiebig HH, Efferth T., Molecular Modes of Action of an Aqueous Nerium oleander Extract in Cancer Cells In Vitro and In Vivo, 2023, Molecules
[16] Dobler, S.; Petschenka, G.; Pankoke, H., Coping with toxic plant compounds – The insect’s perspective on iridoid glycosides and cardenolides, 2011, Phytochemistry
[17] Xu, Y.; Xu, J.; Zhu, W.; Yan, Y.; Jiang, X.; Xie, Z.; Feng, F.; Zhang, J., Bioassay-Guided Fractionation and Biological Activity of Cardenolides from Streptocaulon juventas, 2023, Planta Medica
[18] Decker LE, Hunter MD., Interspecific variation and elevated CO2 influence the relationship between plant chemical resistance and regrowth tolerance, 2020, Ecol Evol
[19] Bhattacharya, S., Seeds as Herbal Drugs, 2020, Nuts and Seeds in Health and Disease Prevention
[20] Verma SK, Gantait S, Jeong BR, Hwang SJ., Enhanced growth and cardenolides production in Digitalis purpurea under the influence of different LED exposures in the plant factory, 2018, Sci Rep
[21] Mañero, FJG; Ramos, B; García, JAL; Probanza, A; Casero, MLB, Systemic induction of the biosynthesis of terpenic compounds in Digitalis lanata, 2003, JOURNAL OF PLANT PHYSIOLOGY