Cardiac glycosides: structural diversity, chemical ecology, bioactivity, and artificial synthesis

Contents

• 1. Introduction

• 2. Natural sources, ecological functions, and isolation
  • 2.1. Structure and distribution
  • 2.2. Ecological functions
  • 2.3. Extraction and isolation from natural sources

• 3. Biological activities
  • 3.1. Structure–activity relationship
  • 3.2. Therapeutic effect of CGs on cardiovascular diseases
  • 3.3. Anticancer effects of CGs
  • 3.4. Immunomodulatory and anti-inflammatory effects of CGs
  • 3.5. Antiviral effects of CGs
  • 3.6. Pharmacological activity of CGs in neuroprotection
  • 3.7. Toxicology and safety profile

• 4. Synthesis and biosynthesis
  • 4.1. (Semi)synthesis of CGs
  • 4.2. Biosynthesis of CGs

• 5. Conclusions and prospects

• 6. Author contributions

• 7. Conflicts of interest

• 8. Data availability

• Acknowledgements

• Notes and references

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Abstract

Covering: up to 2025

Cardiac glycosides (CGs), a class of metabolites found in nature, comprise sugar residues, unsaturated lactone rings, and steroidal cores. As renowned phytotoxins, they play vital roles in maintaining ecological balance. CGs have been widely used in the treatment of cardiovascular diseases such as heart failure and tachyarrhythmia for more than 200 years. Recent studies have revealed that CGs have numerous applications in various disease therapeutic areas, including anticancer, immunomodulatory, anti-inflammatory, antiviral, and neuroprotective effects. However, the medicinal resources of CGs are mainly reliant on natural plant and animal extracts, which not only limits their sustainable supply but also increases development costs. With the growing understanding of the pharmacological value of CGs and their increasing demand in the pharmaceutical industry, the sustainable supply of medicinal resources will become a bottleneck limiting their further development. Therefore, the artificial synthesis of target active ingredients, including chemical (semi)synthesis and biosynthesis, is becoming a hot topic among scholars worldwide. This paper presents the first systematic review of recent research advances in the structure, distribution, chemical ecology, biological activities, and artificial synthesis of CGs. Finally, we discuss the current challenges and urgent issues in this field, aiming to promote the widespread application of CGs in medicine through comprehensive pharmacological studies and exploration of synthesis techniques.

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Dian Jiao

Dian Jiao obtained her bachelor's degree in Traditional Chinese Pharmacy at Nanjing University of Chinese Medicine, China in 2024. Now she is pursuing her doctoral degree with Prof. Luqi Huang at China Academy of Chinese Medical Sciences. Her research focuses on elucidating biosynthetic pathways of cardiac glycosides and the heterologous production via synthetic biology.

Yibo Zhang

Yibo Zhang received his bachelor's degree in Biological Engineering at Jiaxing University, China in 2024. He is now pursuing his master's degree in Biological and Pharmaceutical Sciences at Hangzhou Normal University. His research subject is natural product biosynthesis.

Wending Guo

Wending Guo earned his bachelor's degree (2019) at Capital Medical University and master's degree (2022) at China Academy of Chinese Medical Sciences. Now he is completing his doctoral degree, and his research focuses on the elucidation of the biosynthetic pathways of plant-derived natural products, e.g. alkaloids.

Shuang Liu

Shuang Liu earned her bachelor's degree (2014) in Biological Science at Shanxi Agricultural University, and master's degree in Traditional Chinese Pharmacy (2017) at Beijing University of Chinese Medicine, China. She received her PhD degree in Pharmacognosy at Peking University, China in 2021. Then she joined Experimental Research Center of China Academy of Chinese Medical Sciences as an assistant professor. Her research interests are focused on biosynthesis of active ingredients in medicinal plants.

Ping Su

Ping Su earned his bachelor's degree (2012) and master's degree in Traditional Chinese Pharmacy (2015) at Capital Medical University, China. He received his PhD degree (2019) at China Academy of Chinese Medical Sciences with Prof. Luqi Huang, focusing on the elucidation of the biosynthetic pathways of plant-derived bioactive terpenoids. He then carried out his postdoctoral research on microbial natural product biosynthesis in Prof. Ben Shen's group at The Scripps Research Institute. In 2021, he joined China Academy of Chinese Medical Sciences as an associate professor. His research interests include natural product biosynthesis, chemoenzymatic synthesis, and synthetic biology.

Luqi Huang

Luqi Huang earned his bachelor's degree (1989) at Jiangxi University of Chinese Medicine and master's degree (1992) at China Academy of Chinese Medical Sciences. He received his PhD degree in Pharmacognosy at Peking University, China in 1995. He is the leader of the experts guiding group for the Fourth National Survey of Chinese Materia Medica (CMM) Resources, establishing a comprehensive database on CMM resources to improve their management, protection, development and utilization. Luqi Huang, the molecular pharmacognosist, is the president of China Academy of Chinese Medical Sciences, and his research interests include Chinese Materia Medica Resources and Molecular Pharmacognosy.

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1. Introduction

Cardiac glycosides (CGs) have been used in the clinical treatment of cardiovascular diseases such as heart failure and atrial fibrillation for more than 200 years. The use of foxglove extract for the treatment of patients with congestive heart failure was first described in 1785 by the English physician William Withering.¹ Over the following decade, its use in 163 patients with swelling led to the publication of the medical classic An Account of the Foxglove and Some of Its Medical Uses. Since then, foxglove has become one of the most important herbs in modern medicine. In recent times, this botanical has been replaced by its purified active ingredients—CGs, digitoxin,² and digoxin.³ With the development of chemical analytical techniques (including chromatography, mass spectrometry, and nuclear magnetic resonance), more CGs have been isolated, characterized, and synthesized with greater precision.^4,5 Some CGs are Food and Drug Administration (FDA)-approved Na⁺/K⁺-ATPase (NKA) inhibitors. NKA is a ubiquitous membrane protein found throughout the living world and abundantly present in cell membranes. It maintains the ionic concentration gradient across the cell membrane by actively transporting Na⁺ out of the cell and replacing it with K⁺. Since its discovery sixty years ago, NKA has been extensively studied, and its key physiological roles in regulating cardiac function and maintaining neural signaling have been established.^6,7 To date, hundreds of CGs have been identified from a wide range of angiosperms. Plants produce CGs that target the NKA of natural enemies, thereby serving as a self-defense mechanism.⁸ Notably, more than 100 insect species are known to have evolved mechanisms to tolerate CG toxicity.^9,10 This plant–insect interaction is an example of co-evolution, whereby plants produce CGs as a defense mechanism, while certain insects have adapted to use these plants as a food source. In addition, some animals consume CG-containing plants not to overcome plant defenses but to acquire toxins that protect them from predators.^11–13

Herbs containing CGs have been used in Traditional Chinese medicine (TCM) for thousands of years. For example, Periploca forrestii Schltr. is a commonly used folk medicine in TCM for the treatment of rheumatoid arthritis and wounds, suggesting that its main ingredient, CG, has a promoting effect on inflammation and wound healing. In recent times, numerous studies have found that, in addition to their well-known cardiac-strengthening effects, CGs possess considerable therapeutic potential as anticancer, anti-inflammatory, immunomodulatory, antiviral, and neuroprotective agents—providing a rich source of therapeutic leads for the pharmaceutical industry and ongoing inspiration for medicinal design. However, plants of the genus Digitalis are still the only viable source of CGs.¹⁴ The amount of CGs can vary among different organs of the same plant, and the yield is greatly affected by the harvesting time and environmental conditions.¹⁵ Some CGs are obtained through the biotransformation of precursor compounds extracted from toads. This method of extraction seriously disrupts the ecological balance, and the use of pesticides has also led to a decline in amphibian populations worldwide.^16,17 Therefore, relying solely on existing natural toad resources will inevitably lead to the decline or even extinction of wild species of toads. Breeding toads on an artificial scale not only requires specific growth cycles but also faces several challenges, including difficulties in product purification, species degradation, and heavy metal contamination.^18–20 Low natural availability of CGs in plants and animals, complicated extraction procedures, and environmental pollution by extraction solvents limit the broader application of CGs, and alternative production methods are needed. Currently, artificial synthesis using techniques such as biosynthesis and chemical synthesis is regarded as a promising alternative production method, attracting long-term interest and remaining one of the most exciting and dynamic topics. In particular, advancements in biosynthesis technology in the past two decades have provided theoretical and technological support for shifting the production of active ingredients from the traditional resource-based methods to a new industrial production mode that is highly efficient, green, stable, and controllable. Synthetic biology research on tanshinone,²¹ paclitaxel,²² artemisinin,²³ vinblastine,²⁴ and others has made great progress. The total synthesis of some CGs has been achieved.^25,26 In the case of CG biosynthesis, many key genes have also been identified, offering promising prospects for the heterologous synthesis of these natural compounds.

Research on the pharmacological effects, chemical composition, synthetic biology, and chemical synthesis of CGs has progressed rapidly in recent years, and these new findings offer fresh directions for their clinical application and drug development. Although there have been many reviews on CGs in the past, most of them have focused only on isolated aspect of pharmacological activity (e.g., anticancer effects). More than two hundred pharmacological studies on CGs are published annually. While previous reviews focused on pre-2022 findings, our analysis will integrate recent studies to emphasize the latest breakthroughs. Moreover, comprehensive reviews on the artificial synthesis of CGs remain scarce. Herein, we present the first systematic review of the recent research advancements on the structure, distribution, chemical ecology, biological activity, and artificial synthesis of CGs. We expect that this review will facilitate the translation of CGs from basic research to clinical applications and further support the exploration of their potential in treating a wide range of diseases.

2. Natural sources, ecological functions, and isolation

2.1. Structure and distribution

CGs comprise a steroid nucleus and sugars, with a basic skeleton containing 17 carbon atoms (Fig. 1). At the C-17 position, CGs have a five- or six-membered unsaturated lactone ring, a structural feature that confers unique pharmacological activity, based on which they can be categorized as cardenolides and bufadienolides. Steroid nuclei found in nature have four rings: A, B, C, and D. The A/B ring typically exhibits a cis-fused junction (e.g., digitoxigenin), and some CGs, like uzarigenin, also contain a trans-fused junction; the B/C ring displays a trans-fused junction, while the C/D ring exhibits a cis-fused junction. CGs have a β-hydroxyl group at the C-14 position, which is an important feature that distinguishes CGs from other steroidal compounds. Sugars are typically attached to the C-3 position of the steroid parent nucleus, predominantly in the β-configuration, and can be present either as monosaccharides or oligosaccharides. In terms of sugar composition, cardenolides exhibit considerable diversity in their sugar moieties, encompassing a wide range of monosaccharides and unique deoxy sugars, whereas bufadienolides are typically conjugated with more common sugars such as glucose or rhamnose. Currently, more than 20 types of sugar moieties in CGs have been reported. Based on the presence or absence of a hydroxyl group at the C-2 position, these sugars can be classified into α-hydroxy sugars and α-deoxy sugars.

Fig. 1 Main classification and structures of CG aglycons and sugar moieties.

2.1.1. CGs with butenolide ring (cardenolides). Cardenolides possess a five-membered unsaturated butyrolactone ring at the C-17 position. According to the Angiosperm Phylogeny Group system, CGs are mainly found in Scrophulariaceae (e.g., Digitalis²⁷), Apocynaceae (e.g., Nerium,²⁸Thevetia,²⁹Strophanthus,³⁰Calotropis,³¹Apocynum,³²Cryptolepis,³³Asclepias,³⁴Periploca,³⁵), Ranunculaceae (e.g., Adonis³⁶), Moraceae (e.g., Streblus³⁷), and other families (Fig. 2). Notably, the Apocynaceae represents the most significant source, with over 30 genera containing CGs, whereas the Scrophulariaceae, particularly the genus Digitalis (e.g., Digitalis purpurea L. and Digitalis lanata Ehrh.), comprises some of the most well-studied species.³⁸ Digitoxose, a α-deoxy sugar, serves as a hallmark constituent of CGs found in Digitalis species. It constitutes the canonical sugar moiety in well-known compounds such as digitoxin, digoxin, and lanatoside C. The presence of α-deoxy sugars (e.g., digitoxose and cymarose) represents a defining structural feature of CGs produced within the Scrophulariaceae and Apocynaceae families. Furthermore, the Asclepiadaceae family also serves as a major source of CGs, which are often characterized by the presence of aldehyde or hydroxymethyl groups at the C-10 position, thereby enhancing their biological activity.

Fig. 2 (A) Common plant distribution and representative compounds of cardenolides in nature. Species are alphabetically ordered for rapid cross-referencing. Representative species are enclosed within the red oval, with corresponding characteristic CGs labeled in the surrounding sectors. (B) The phylogenetic relationship of candidate plant species originated from TimeTree (https://www.timetree.org). Some taxa could not be resolved due to insufficient data in TimeTree. Glu: glucose; Gent: gentiobiose [β-D-Glu-(1→6)-D-Glu].

2.1.2. CGs with an α-pyrone ring (bufadienolides). Bufadienolides contain a six-membered unsaturated pyrone ring and are primarily found in Asparagaceae (e.g., Drimia³⁹), Ranunculaceae (e.g., Helleborus^40,41), Crassulaceae⁴² (e.g., Kalanchoe, Bryophyllum), Francoaceae⁴² (e.g., Melianthus, Bersama), and other families (Fig. 3).^39–50 In addition, these compounds have also been reported in species belonging to the Thesiaceae and Iridaceae families.⁵¹ This aglycon is present in animals such as toads and fireflies (e.g., Photinus ignitus). Bufadienolides, the main active ingredients in the rare TCM Chansu (Bufonis Venenum), are secondary metabolites found in Bufo gargarizans (Cantor, 1842) and Bufo melanostictus (Schneider, 1799).⁵² Lucibufagins, a class of phototoxic compounds found in fireflies, also belong to the bufadienolide family.⁵³ Unlike animal-derived bufadienolides that contain only cis-fused A/B ring junction and C-3 hydroxyl groups, plant-derived CGs have both cis- (compounds 1–54) and trans-fused A/B ring junctions (compounds 55–73) with a sugar moiety attached at the C-3 position.^50,54 CGs are also found in reptiles (e.g., Rhabdophis snakes), though these are diet-derived rather than endogenously synthesized.⁵⁵

Fig. 3 (A) Representative compounds of bufadienolides in nature. R₁–R₉: different substituent groups. If not specifically labeled, the sugar is linked by a 1,4 glycosidic bond. (B) Common plant distribution of bufadienolides in nature. Thev: thevetose; Glu: glucose; Rha: rhamnose; Gent: gentiobiose [β-D-Glu-(1→6)-D-Glu], 6dGlu: 6-deoxyglucose, historically termed glucomethylose.

2.1.3. Endogenous CGs. In 1991, an endogenous substance was first purified from human plasma that exhibited high-affinity binding to Na⁺/K⁺-ATPase (NKA) and was indistinguishable from ouabain, hence termed endogenous ouabain (EO).⁵⁶ Subsequent investigations led to the identification of other endogenous compounds which were structurally identical to digoxin, bufalin, marinobufagenin, telocinobufagin, marinobufotoxin, 19-norbufalin and proscillaridin A, collectively referred to as endogenous cardiotonic steroids.⁵⁷ Conventionally, quantification of EO in human extracellular fluid is primarily relied on immunoassays, and most of studies reported that immunoreactive EO plasma concentrations were below 1 nM in mammals.^58–60 However, measured levels vary considerably across laboratories due to differences in antibody specificity, extraction protocols, and elution conditions. This variability explains why some groups have even failed to detect EO in human plasma.^61,62 Notably, the threshold NKA inhibition concentration of humans is approximately 1 nM, which challenges the proposed role of these compounds as natriuretic steroid hormones in normal human physiology.⁵⁷ Thus, the existence, precise structure, and physiological concentration of putative endogenous CGs remain debated.^1,63 Nevertheless, significant progress has been made in understanding EO's interaction with its receptor (NKA) and its downstream effects in both brain and peripheral tissues. It is now widely accepted that endogenous NKA inhibitors represent physiologically important entities and contribute to the pathogenesis of several common diseases.^64,65

2.1.4. Special chemical structures of CGs. Besides the common CG structures mentioned above, some specific structures also exist (Fig. 4). From the stems of P. forrestii, periforgenin A-3-O-β-digitoxopyranoside (74)⁶⁶ and compounds 75–76,⁶⁷ which possess a rare modified C/D-ring steroid skeleton were discovered. Compound 76 exhibits potent cytotoxic activity against five human cancer cell lines. Moreover, compounds 77–81 were isolated from the aerial parts of Pergularia tomentosa L. and have been shown to be cytotoxic to human liver cancer cells.⁶⁸ With the exception of the C-3 position, specific structures at the C-5 position linking the sugar moiety include scilliglaucoside (82) and compounds 83–84.^43,44,49 Three bufadienolides were found to be present in Drimia maritima (L.) Stearn, whose main secondary metabolites are CGs. Ye et al. reported for the first time the biotransformation of cinobufagin using a Catharanthus roseus (L.) G. Don. cell culture system, which yielded three new compounds (85–87) with glycosylation occurring at the C-16 position.⁵² Additionally, a rare CG dimer (88) has been identified from Streblus asper Lour., formed through a C3–C4 linkage between the sugar units of two monomeric glycosides. This unique dimeric configuration significantly enhances its biological activity.⁶⁹

Fig. 4 Special chemical structures of CGs. (A) Special structures of cardenolides in plants; (B) special structures of bufadienolides in plants; (C) special structures of bufadienolides obtained by biotransformation of cinobufagin; (D) a rare CG dimer. R₁–R₃: different substituent groups. Dig: digitoxose; Glu: glucose; Cym: cymarose.

2.2. Ecological functions

The ecological roles of CGs primarily manifest in their function as plant-derived secondary metabolites, particularly in plant defense and interspecific interactions. These roles significantly shape the relationships between plants and other organisms (such as herbivores, insects, and microorganisms), as well as their dynamics within the broader ecosystem.

2.2.1. Chemical defense and pollinator mediation in plants. CGs from plants can inhibit the essential NKA in animal cells. The highly independent expression of CGs within and between plant tissues facilitates their heterogeneous evolution across populations and organs, enabling compartmentalized defense against specialized herbivores feeding on specific tissues.^8,10 Members of the genus Erysimum uniquely deploy dual defense systems consisting of glucosinolates and CGs to deter herbivory.⁷⁰ Large herbivorous mammals may be more sensitive to the bitterness of CGs than to their toxicity; thus mutant plant strains with more “cheaper” CG compounds exhibit enhanced protective effects.⁷¹ The wild species Solanum okadae Hawkes & Hjert. synthesizes CGs that effectively deter Colorado potato beetles (CPB), supporting hybridization breeding with wild relatives to enhance resistance in cultivated potato.⁷² This chemical defense diminishes damage inflicted by natural enemies, thereby enhancing plant fitness as an evolutionary adaptation. Moreover, plants use CGs to produce taste or long-term toxic effects to selectively exclude non-target pollinators. African milkweed (Gomphocarpus physocarpus E. Mey.) uses nectar cardenolides and floral volatiles to effectively attract Vespula germanica (Fabricius, 1793) wasps, thereby regulating pollination.⁷³

2.2.2. Toxin-driven adaptive strategies in other organisms. Phytotoxins such as CGs drive niche differentiation. These compounds restrict herbivory to species capable of detoxification while simultaneously providing an evolutionary refuge and exclusive resources for adapted specialists. This dynamic enhances ecosystem complexity. Target-site insensitivity to CGs evolved in some animals through amino acid substitutions within the NKA's conserved binding pocket.⁹ Interestingly, the behavior of monarch caterpillars (Danaus plexippus (Linnaeus, 1758)) shifts across developmental stages—from avoidance of toxic latex in younger to active consumption in later stages.¹¹ Both Asclepias plants, containing high concentrations of CGs, and monarch larvae that feed on them exhibit potent resistance against parasite infection.⁷⁴ Toxin tolerance, detoxification, and sequestration strategies exemplify the evolutionary arms race between plants and herbivores, with some specialists evolving to store CGs without physiological cost.^13,75 In contrast to other insects that sequester CGs internally, Lilioceris merdigera (Linnaeus, 1758) excretes ingested CGs in its feces, which form a deterrent fecal shield against predators such as Myrmica rubra (Linnaeus, 1758) for larval protection.⁷⁶ Moreover, some birds have also developed resistance to CGs through amino acid substitutions, allowing them to consume toxin-loaded insects without harm.¹²

2.3. Extraction and isolation from natural sources

The extraction and isolation of CGs remain challenging in natural product research, primarily due to their occurrence as complex mixtures of structurally analogous compounds with similar polarities, coupled with their susceptibility to degradation under high temperature, acidic or alkaline conditions, and enzymatic hydrolysis.⁷⁷ The solubility of CGs varies significantly with solvent choice; they are generally water-soluble but insoluble in non-polar solvents, except for chloroform and ethyl acetate. The degree of glycosylation and the number of hydroxyl groups both enhance hydrophilicity and influence pharmacokinetic properties. However, solubility cannot be accurately predicted based solely on molecular structure. For example, digitoxin—a compound containing five hydroxyl groups and three sugar units—is poorly soluble in water (1 : 100 000) yet highly soluble in chloroform (1 : 40). In contrast, ouabain, which possesses eight hydroxyl groups and one sugar unit, is readily soluble in water (1 : 75) but exhibits low solubility in chloroform.

Conventional extraction employs polar organic solvents such as ethanol,³⁰ methanol, or aqueous methanol/ethanol mixtures.⁶⁹ Preliminary enrichment is often achieved through liquid–liquid partitioning using petroleum ether–ethyl acetate–water or n-butanol systems.^27,78 Alternatively, dichloromethane extraction followed by washing with 1% NaCl solution can remove phenolic contaminants.³³ Bufadienolides are typically extracted with methanol.⁴⁰ Further purification is commonly performed using silica gel column chromatography (CC), preparative thin-layer chromatography (TLC), or high-performance liquid chromatography (HPLC). Normal-phase CC utilizing binary gradients of medium- to low-polarity solvents (e.g., chloroform, ethyl acetate, dichloromethane) mixed with methanol as a polar modifier effectively separates CGs of varying polarities.³³ HPLC, particularly reversed-phase C18 columns with acetonitrile–water⁷⁸ or methanol–water gradients,⁶⁸ is preferred over gas chromatography (GC) due to the non-volatility of CGs and no need for derivatization. Method selection depends heavily on the specific matrix and target compounds, often requiring optimization of solvent systems and gradient conditions to achieve high recovery and resolution. TLC remains useful for microgram-scale screening, with reagents like SbCl₃ employed for bufadienolides detection.⁴⁰ Although limited by low volatility, GC-MS has been applied in some cases, while high-performance capillary electrophoresis (HPCE) offers an alternative with high resolution and efficiency.⁷⁷

Structural elucidation of CGs is complicated by their chromatographic similarities and sensitivity, often requiring a combination of chromatographic and spectroscopic techniques to determine glycosylation patterns, stereochemistry, and substituent locations.^77,79 Acid hydrolysis followed by sugar derivatization further aids structural characterization.^27,34 Up to now, more than five hundred CG structures have been successfully elucidated through these integrated approaches.³¹ These elucidated structures provide a critical foundation for understanding structure–activity relationships, guiding the discovery of novel analogues with improved pharmacological profiles. Future efforts may focus on optimizing extraction and separation workflows to better preserve labile structures and enable the identification of even lower-abundance CGs. Moreover, integration with computational and bioactivity-guided approaches could accelerate the targeted discovery of compounds with desired therapeutic properties.

3. Biological activities

CGs exhibit a wide range of medicinal properties (Fig. 5 and Table 1). Digoxin is FDA-approved for intravenous use in acute heart failure or acute exacerbations of chronic heart failure. In ophthalmic diseases, compounded esculin and digitalis glycoside eye drops are used to treat visual fatigue and macular degeneration, and some experiments are underway to explore their use in the treatment of dry eye disease.⁸⁰ Clinical trials are underway globally to explore the therapeutic potential of CGs in anticancer, anti-inflammatory, antiviral activities, among others.

Fig. 5 Bioactivities, clinical and potential applications of CGs. The superscript * indicates findings based on clinical evidence, while others are based on in vivo and in vitro results.

Table 1 The structures, indications/bioactivity, and market share information of well-known CGs^a

CGs	Structure	Indications/bioactivity	Market information
a This table lists CGs, including both current and historical agents, with selected compounds under investigation for novel indications (Phase II and beyond). Abbreviations: HF, heart failure; NASH, nonalcoholic steatohepatitis. N/A, not applicable. US: United States; CN: China; EG: Egypt; GB: United Kingdom; NL: Netherlands.
Digoxin		Atrial fibrillation, HF, arrhythmias, tachycardia	In current use (Lanoxin tablet, digoxin injection)
		Anti-NASH activity	Phase II clinical trial (US: NCT06588699; CN: NCT04216693)
		Anti-rheumatoid arthritis activity	Phase II trial completion (EG: NCT04834557)
			Phase I trial completion (US: NCT03131973; GB: NCT01355354)
		Anti-cutaneous warts activity	Phase II trial completion (NL: NCT02333643)
Digitoxin		Arrhythmias, HF	Discontinued (FDA); available elsewhere (CRYSTODIGIN injection)
		Anti-tumor activity in pancreatic cancer	Phase II clinical trial (CN: CTIS2024-512128-12-01)
		Anti-cystic fibrosis activity	Phase II clinical trial (US: NCT00782288)
Deslanoside		Arrhythmias, HF, tachycardia	Discontinued (Cedilanid-D injection)
Ouabain (Strophanthin G)		HF	Discontinued (FDA); available elsewhere (Uabasin injection)
Strophanthin K		HF	Not approved (FDA); available elsewhere (Strophanthin K injection)
Convallatoxin		HF	Not approved (FDA); available elsewhere (Convallatoxin injection)
Lanatoside C		Arrhythmias, HF	Not approved (FDA); discontinued elsewhere (Lanatoside C injection)
Proscillaridin A		HF	Not approved (FDA); discontinued elsewhere (Prost tablet)
Digitalis glycosides (Digital purpurea total CGs)	N/A	Visual fatigue and macular degeneration	Not approved (FDA); available elsewhere (Esculin and Digitalis glycosides eye drops)
Rodealin (Rohdea japonica total CGs)	N/A	Atrial fibrillation, HF, tachycardia	Not approved (FDA); discontinued elsewhere (Rodealin injection)
Thevetosid (Thevetia peruviana total CGs)	N/A	Atrial fibrillation, HF	Not approved (FDA); discontinued elsewhere (Thevetosidum)

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In addition, CGs were found to promote wound healing. Digitoxigenin considerably improves skin condition by increasing the hydroxyproline content in the wound tissues.⁸¹ Periplocin notably promotes cell proliferation and migration and stimulates collagen production in fibroblast L929 cells through activation of the NKA-mediated Src/ERK and PI3K/AKT pathways.⁸² CG from Streptocaulon juventas (Lour.) Merr. induces collagen synthesis in predominantly human skin fibroblasts by protecting LLCPK1 cells from hypoxic injury.³²

CGs can markedly improve the level of homeostasis in the body. Digoxin can improve cerebral metabolic disorders caused by chronic cerebral hypoperfusion and attenuate cognitive deficits.⁸³ Ouabain reduces infrared-induced skeletal muscle dysfunction by preventing the reduction of α2 NKA function in rats.⁸⁴ Nanomolar concentrations of ouabain induce selective cytotoxicity in tissue-resident macrophages and enhance white adipose tissue homeostasis, highlighting its potential application in the treatment of metabolic syndrome, a condition characterized by pathogenic macrophage infiltration and activation.⁸⁵ In addition, ouabain is selectively cytotoxic to senescent cells, possibly inducing apoptosis by blocking ion pumps and depleting intracellular K⁺.⁸⁶

Recently, CGs have also been found to have therapeutic potential in reducing intraocular pressure⁸⁷ and improving reproduction.^88,89 Further pharmacological activities and mechanisms of action of CGs are being explored.

3.1. Structure–activity relationship

NKA represents the most well-established target of CGs and also functions as a signal transducer that regulates cell metabolism, survival, and death.⁹⁰ As the largest protein complex within the P-type cation pump family, it hydrolyzes ATP to drive the transport of K⁺ into and Na⁺ out of the cell at a 2 : 3 stoichiometric ratio, thereby establishing and maintaining the transmembrane electrochemical gradient.

The aglycone moiety of CG is considered the essential pharmacophore responsible for the pharmacological efficacy of CGs.⁹¹ X-ray crystallography has revealed that CGs embed into the CG-binding pocket of the NKA α-subunit. The conserved 14β-hydroxyl group mediates a common interaction mode with NKA across multiple CGs, primarily through hydrogen bonding with the carbonyl oxygen atom of Thr797.^92,93 Additional substituents interact with the protein via specific polar contacts.⁹³ The β-oriented surface of CGs facilitates extensive hydrogen-bonding networks with residues in the αM1, αM2, and αM6 helices of NKA.^30,33,94 A/B cis-junction is important for high activity.⁹⁵ Due to the hydrophobic nature of the B ring, the introduction of oxygen-containing groups at C-7 or C-8 can significantly enhance cytotoxic activity. Most substituents at C-10, C-13, and C-17 positions of naturally occurring CGs are in the β-configuration. The C-10 (e.g., 10-epi-uzarigenin) or C-13 (e.g., 13-epidigitoxigenin) α-configurations are typically achieved through chemical modification or semisynthesis, whereas the C-17 α-configuration (e.g., (+)-17β-hydroxystrebloside) is associated with a loss of cardiotonic activity. This is attributed to its ability to adopt at least three distinct conformations upon binding to the cation-binding site, leading to disrupted interaction with NKA.⁹² Substituents such as the C-10 angular methyl, aldehyde, or hydroxymethyl groups, the C-12 β-hydroxyl group, and the C-17β side chain are critical for mediating cardiotonic activity, whereas hydroxylation at C-16 is detrimental.²⁷ C1, C5, C11 and 19 hydroxyls may enhance the activity, but their presence is not essential.⁹⁵

A defining feature of CGs is the unsaturated lactone ring at the C-17β position, which distinguishes them from steroid hormones. This lactone contains two oxygen atoms that generate a highly negative electrostatic potential, enhancing both orientation and strength of hydrogen bonding. The interaction energy contributed by the lactone ring is comparable to that of the steroid core (−22.7 kJ mol⁻¹). Notably, α-pyrone-type lactones (as in bufadienolides, −27.6 kJ mol⁻¹) exhibit stronger interaction than butenolide-type rings (as in cardenolides, −20.5 kJ mol⁻¹), correlating with their higher cardiotonic potency.⁹⁶

The types and attachment sites of sugars considerably influence the solubility and biological activity of CGs. Aglycones are generally absorbed more rapidly and metabolized more easily than their glycosylated counterparts.⁹¹ Notably, glycosylation at the C-3 position often enhances cytotoxic activity several-fold compared to the aglycone form, indicating that the hydrophilic group at this position plays a critical role in potency.²⁷ Monoglycosides typically exhibit the highest cytotoxicity, while increased sugar chain length and molecular size tend to reduce toxic effects.⁹⁷

3.2. Therapeutic effect of CGs on cardiovascular diseases

CGs were initially employed in the treatment of cardiovascular diseases, particularly heart failure and arrhythmias. Their therapeutic effects are primarily mediated through specific binding to and inhibition of NKA, which subsequently alters Na⁺–Ca²⁺ exchanger (NCX) activity and promotes Ca²⁺ release from the sarcoplasmic reticulum. The resulting increase in intracellular Ca²⁺ concentration enhances myocardial contractility and improves cardiac output, thereby exerting positive inotropic effects and alleviating symptoms of heart failure. At nanomolar concentrations, CGs can activate protective signaling pathways (e.g., calcium oscillations, SRC kinase, MAPK) without significantly inhibiting ion pumping, suggesting potential for safer cardiovascular drugs.⁹¹

Pharmacological studies have shown that periplocin attenuates cardiac remodeling, improves cardiomyocyte contractility, and enhances cardiac diastolic function in heart failure rats, making it a potential treatment for heart failure with preserved ejection fraction.⁹⁸ C-reactive protein (CRP) is associated with inflammation in cardiovascular disease, and digoxin inhibits CRP synthesis. This is the first report on the inhibition of CRP by CGs.⁹⁹ Through live cell screening, Magadum et al. found that CGs can increase myocardial contractility and induce cardiomyocyte proliferation, thereby promoting cardiac repair and enhancing cardiomyocyte cycle activity.¹⁰⁰ Clinical studies have found that in an elderly population whose digoxin therapy was discontinued prior to hospital admission, prognosis was considerably affected even after receiving other medications, suggesting that digoxin continues to play an irreplaceable role in the treatment and prognosis of chronic heart failure.¹⁰¹ Digoxin may partially diminish the expected decrease in RV systolic function and increase in RV systolic size through its positive inotropic effect, suggesting that it may improve cardiac function and potentially reduce mortality.¹⁰²

3.3. Anticancer effects of CGs

Several studies have shown that CGs can be used as potential antitumor drugs and exhibit potent cytotoxicity against a wide range of cancer cells.^33,40,103 CGs exert anticancer activity across various body systems, mainly by targeting key signaling axes, affecting the transcription and expression of cancer cell metabolic pathway proteins, inhibiting the malignant behaviors of cancer cells (proliferation, migration, differentiation, and invasion),¹⁰⁴ inducing apoptosis and autophagy,¹⁰⁵ and ameliorating the clinical symptoms of cancer,¹⁰⁶ among other mechanisms.

Breast cancer (BC) is the most common malignant tumor in women and the leading cause of female cancer incidence worldwide.¹⁰⁷ Lanatoside C, peruvoside, and strophanthidin ameliorate the dysregulation of EGR1 and downstream proteins of the MAPK/ERK signaling pathway and reduce the proliferation and invasion of MCF-7 BC cells.¹⁰⁸ Ouabain induces estrogen receptor α (ERα) degradation, thereby killing ERα-positive BC cells.¹⁰⁹ Oleandrin induces immunogenic cell death by stimulating the ER.¹¹⁰ In triple-negative BC cells, 3′-epi-12β-hydroxyfroside (hyfs) induces complete autophagic flux, and combination treatment with an autophagy inhibitor may enhance activity.¹¹¹ Preclinical 3D model screening has demonstrated that digoxin has therapeutic potential for treating dedifferentiated endometrial carcinoma.¹¹² Proscillaridin A and lanatoside C target UCP2 to increase ROS, inhibit uterine leiomyosarcoma cell growth, and induce cell death.¹¹³ Neriifolin exerts anticancer activity against prostate cancer cells through endoplasmic reticulum stress (ERS)–mediated DNA damage, G2/M blockade, and apoptosis induction.^114,115

CGs and their derivatives exhibit substantial anticancer activities in different cancer models and play a role in inhibiting tumor growth, overcoming drug resistance, and altering tumor microenvironmental aspects. Intrahepatic cholangiocarcinoma is a type of hepatocellular carcinoma with poor prognosis and high mortality. Digitoxin can inhibit the proliferation and migration of intrahepatic cholangiocarcinoma cells by targeting the NF-κB/ST6GAL1 signaling axis.¹¹⁶ CGs from Thevetia peruviana (Pers.) K. Schum. considerably induced apoptosis in human hepatocellular carcinoma HepG2 cells in a dose-dependent manner.¹¹⁷ Binding of ouabain, oleandrin, and digoxin to NKA downregulates the THADA–LAT1 pathway to inhibit the proliferation of HepG2 cells and human epidermoid carcinoma KB cells.¹¹⁸ CGs have agonistic and potentiating effects on RORγ/RORγT nuclear receptors, modulating Th17 or Tc17 lymphocyte differentiation.¹¹⁹ Periplocymarin synchronously activates apoptosis and initiates the AMPK/ULK1 and mTOR signaling pathways, leading to protective autophagy.¹²⁰ Lanatoside C downregulates the expression of signal transducer and activator of transcription 3 (STAT3), increases ROS levels, and decreases MMP, thereby slowing proliferation and inducing apoptosis in HuCCT-1 and TFK-1 cholangiocarcinoma cells.¹²¹ Ouabain at a nanomolar concentration interferes with spheroid growth and is cytotoxic to bile duct cancer cells.¹²² Digitoxin can promote apoptosis in colon cancer cells with drug-resistant KRAS mutations by decreasing HIF-1α and STAT3 levels, thereby inhibiting cellular proliferation and migration.¹²³ Proscillaridin A enhances the sensitivity of drug-resistant cells.¹²⁴ Moreover, digitoxin activates the ROS-induced RhoA/ROCK pathway, inhibits cancer cell proliferation and migration, and reverses the pro-angiogenic effects mediated by the tumor microenvironment.¹²⁵ Different phenotypes affect the rate of ROS production by digitoxin, which differentially induces apoptosis in four pancreatic cancer cell lines.¹²⁶ Oleandrin is a novel and potent inhibitor of ATM and ATR kinases that mediates DNA damage response to enhance the sensitivity of lung cancer to radiotherapy.¹²⁷ In human multiple myeloma AMO1 cells, periplocin considerably inhibited the oncogene c-MYC via α1-NKA.^128,129 Convallatoxin downregulates the PTHR1 and Wnt/β-catenin pathways, inhibiting the malignant development of osteosarcoma cells.¹³⁰ Ouabain induces apoptosis in leukemic stem cells by promoting the loss of Mcl-1 and c-Myc expression.¹³¹ 17β-neriifolin induces apoptosis by inhibiting the overexpression of HOXA9.¹³² A synthetic digitoxin derivative downregulates the phosphorylation of MEK1/2 and induces a cellular G2/M cell cycle arrest in leukemia cells, resulting in cell death.¹³³ Indoleamine-pyrrole 2′,3′-dioxygenase 1 (IDO1) plays an important role in cancer cell metabolism. Ouabain and digoxin downregulate IDO1 mRNA and protein levels at concentrations that do not affect cell viability.¹³⁴ CGs can downregulate the expression of the glucose transporter GLUT1 and affect glucose metabolism in various cancer cells by inhibiting NKA α3-isoform activity.¹³⁵

3.4. Immunomodulatory and anti-inflammatory effects of CGs

Neuroinflammation is present in virtually every neurological disorder and contributes to disease pathogenesis by promoting neuronal loss and blood–brain barrier dysfunction.^136,137 Ouabain promotes antioxidant effects in retinal cells and has been demonstrated for the first time to protect retinal ganglion cells through the activation of autophagic pathways.¹³⁸ Jansson et al. found that digoxin and lanatoside C attenuated meningeal and choroid plexus inflammatory responses and blocked inflammatory propagation, which was also confirmed in a 3D in vitro model.¹³⁹ Moreover, digoxin can reverse vascular dilation and pulp expansion, suggesting that it can attenuate pulp inflammation.¹⁴⁰

Respiratory diseases are common and prevalent conditions that constitute a major public health issue.¹⁴¹ COVID-19 is a pandemic of acute respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).¹⁴² Digitoxin and ouabain counteract the CFTR protein loss induced by SARS-CoV-2 infection, thereby reducing inflammation in the airway caused by COVID-19.¹⁴³ In addition to its role in acute models of inflammation, ouabain also acts on chronic inflammatory phenotypes. It inhibits the activation of the p38 MAPK signaling pathway, thus modulating allergic asthma.¹⁴⁴

Digoxin exhibits a protective effect against intervertebral disk degeneration by decreasing the transcriptional levels of proinflammatory cytokines (COX-2, iNOS, IL-1β, and IL-6). It inhibits destructive metalloproteinases and rescues extracellular matrix (ECM) loss, activating the AKT and ERK1/2 signaling pathways to promote ECM anabolism.¹⁴⁵ Osteoarthritis (OA), a common chronic joint disease with no known drugs to alter its progression, has become a leading cause of disability worldwide.¹⁴⁶ Jia et al. found that digoxin improves the OA inflammatory microenvironment and promotes chondrogenesis by downregulating the M1-like macrophage-derived exosome miR-146b-5p/Usp3&Sox5 axis.¹⁴⁷ Wang et al. demonstrated that ouabain and digoxin limited OA development in vitro. Low-density lipoprotein receptor-related protein 4 (LRP4), a novel target of digoxin, was targeted for regulating chondrocyte metabolism.¹⁴⁸

FDA-approved CGs have demonstrated potent anti-inflammatory activity in the development of a wide range of other inflammatory conditions. Ouabain downregulates the expression of the amino acid transporter SLC7A11 to inhibit glutathione synthesis. Excessive oxidative stress notably induces G2/M cycle arrest in human HaCaT keratinocytes, demonstrating anti-psoriatic activity.¹⁴⁹ Ouabain blocks electrophysiological disturbances and claudin-2 overexpression, offering potential to ameliorate ionizing radiation-induced intestinal dysfunction.¹⁵⁰ Minami et al. designed a specific perilipin 1-derived LD-targeting domain that binds to the optimized LIR-interacting domain, which recognizes digoxin as an activator of lipophagy to inhibit the transition to NASH in vivo.¹⁵¹ Multiple models of inflammation have been used to study CGs. Digoxin exhibits excellent anti-inflammatory activity by inhibiting soluble epoxide hydrolase activity and exerts antipyretic effects in rectal temperature measurements.¹⁵² Convallatoxin¹⁵³ and the synthesized CG BD-8 (ref. 154) also showed anti-inflammatory effects both in vivo and in vitro. The immunomodulatory and anti-inflammatory effects of CGs are shown in Table 2.

Table 2 Immunomodulatory and anti-inflammatory effects of CGs^a

CGs	Anti-inflammatory effects	Experimental subject	Administration mode and dosage
a Abbreviations: i.p., intraperitoneal; p.o., oral; i.a., intra-articular. N/A, not applicable (the administration mode and dosage were not addressed).
Ouabain^138	Reduce TNFR1/2, TLR4 and CD14 expression; increase TNFR2 levels; decrease the density of Iba-1 and the production of ROS	RGC	3 nM
Ouabain^149	Inhibit GSH synthesis, inducing oxidative stress in psoriatic keratinocytes	HaCaT	0–200 nM
Ouabain^144	Reduce migration of eosinophils, lymphocytes and macrophages; decrease IL-33, TSLP, IL-4, IL-1β, TNF-α and TGF-β; attenuate collagen deposition and mucus production	Female BALB/c mice	0.56 mg per kg per day, i.p.
Ouabain^150	Prevent occludin overexpression and pore-forming claudin-2 expression	Male Wistar rats	1 μg per kg per day, i.p.
Digoxin^140	Reverse ZOL-induced vasodilation/dilatation; reduce IL-17, TNF-α and TGF-β and increase of IL-6 expression	Male Wistar rats	1 μg per kg per day, i.p.
Digoxin^152	Elevate EETs levels, inhibiting inflammatory mediators; reduce lung edema, neutrophilic infiltration and alveolar septal thickening	Swiss albino mice, Wistar albino rats	0.1–0.2 mg kg^−1, p.o.
Digoxin^145	Suppress TNF-α-induced inflammation; attenuate ECM destruction; promote ECM anabolism	Male rats	50 nM, i.p.
		Human nucleus pulposus cells	10–100 nM
Digoxin^147	Reduce iNOS and IL-6 secretion; improve joint inflammatory microenvironment; promote chondrocyte production	RAW 264.7, OA patient synovial macrophages	20–80 μM
		Male C57BL/6 mice	0.02, 0.2 mg per kg per day, i.a.
Digoxin^151	Activate lipophagy; attenuate inflammatory and profibrotic gene expression	C57BL/6J mice	0.8 mg per kg per day, p.o.
		Human retrospective cohort study	N/A
Digitoxin, Ouabain^143	Prevent CFTR loss, reducing TNFα/NFκB- and ENaC-dependent inflammation in COVID-19 airways	Vero E6	50 nM
Digoxin, Ouabain^148	Stimulate chondrocyte differentiation and metabolism; relieve OA-associated pain; target LRP4 to promote chondrocyte anabolism	C3H10T1/2, C28I2, mouse primary chondrocytes	50–100 nM
		Male C57BL/6 mice	50–100 nM, i.a.
		Human retrospective cohort study	N/A
Digoxin, Lanatoside C^139	Reduce IL-1β-induced gCCL2, sICAM-1, sVCAM-1 and CX3CL1; block endothelial inflammatory secretion	Primary human brain mixed glial, pericyte, endothelial	0.1, 1 μM
BD-8 (ref. 154)	Inhibit NO production; decrease IL-1β; reduce phosphorylation of inflammation-associated signaling pathways	Primary mouse macrophages	0.01–100 μM
		Female Swiss albino mice	0.28, 0.56, 1.12 mg kg^−1, i.p.
Convallatoxin^154	Inhibit ZFP91-mediated pro-IL-1β ubiquitination and caspase-8 inflammasome activity, reducing IL-1β production	THP-1, BMM	3–100 nM
		C57BL/6 male mice	150 μg per kg per day, p.o.

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3.5. Antiviral effects of CGs

The binding of CGs to NKA is involved in antiviral activity against a wide range of viruses, either by activating signaling cascades or altering the concentration of intracellular ions.¹⁵⁵ CGs are involved in almost every step of the viral life cycle, including RNA synthesis, protein translation, and post-transcriptional processing, except viral attachment to the host cell.¹⁵⁶ CGs have been described as promising compounds in addition to ongoing clinical viral vaccines with strong immune responses.

As FDA-approved inhibitors targeting NKA, ouabain and digoxin inhibit Japanese encephalitis virus (JEV) infection during the viral replication phase.¹⁵⁷ Amarelle et al. found that ouabain inhibits influenza A virus (IAV) replication in alveolar epithelial cells by decreasing intracellular potassium levels.¹⁵⁸ Zika virus (ZIKV) is a novel mosquito-borne flavivirus associated with neurological disorders.¹⁵⁹ Ouabain and digoxin have been shown to reduce the viral burden of ZIKV in adult mice and prevent ZIKV infection-induced microcephaly, which is fatal in pregnant mice.¹⁶⁰ Ouabain reduces ZIKV replication by decreasing RNA copy number, while NS5-RdRp and NS3-helicase proteins are suggested to be the targets of its direct action on ZIKV proteins.¹⁶¹ Moreover, Du et al. demonstrated that ouabain, digoxin, lanatoside C, and digitoxin have anti-Ebola virus (EBOV) replicative and transcriptional activities, which may specifically inhibit the entry of EBOV into host HeLa cells by mediating intracellular calcium disorder.¹⁶² Compared to drugs such as cyclosporin A, sunitinib, and chloroquine, digoxin has the lowest inhibitory concentration (IC₅₀) to block Bunyamwera virus (BUNV) infection by inhibiting NKA-induced high electron-density and swollen cristae in mitochondria.¹⁶³ Ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) specifically bind to the α1 subunit of the NKA (ATP1A1) and blocks EGFR Tyr845 phosphorylation, thereby inhibiting infection caused by efficient uptake of human respiratory syncytial virus (RSV).¹⁶⁴ Prion diseases are rapidly progressing and ultimately fatal neurodegenerative disorders in mammals.¹⁶⁵ CGs are targeted NKA inhibitors that reduce cellular prion protein (PrP^C) levels.¹⁶⁶ C4′-Dehydro-oleandrin reaches higher concentrations in the brain and exhibits lower toxicity than other CGs.¹⁶⁷ In 2024, Wu et al. found that lanatoside C inhibited Herpes simplex virus type 1 (HSV-1) replication both in vitro and in vivo by inducing the localization of intracellular nuclear factor erythroid 2-related factor 2 (NRF2) at the periphery of the cell nucleus.¹⁶⁸ In addition, ten unreported CGs from S. asper exhibited potent antiviral effects against HSV-1 in vitro.⁷⁸ Lytic reactivation of Epstein–Barr virus (EBV) plays an important role in virus-driven malignancies. Cai et al. reported for the first time that two CGs from Sonchus asper (L.) Hill could effectively inhibit early antigen expression in vitro to limit EBV lytic replication.³⁷ Notably, Yang et al. revealed that the notable antiviral effects of CGs on porcine transmissible gastroenteritis coronavirus (TGEV) and human HCoV-OC43 were dependent on an NKA-independent signaling axis. Ouabain inhibits the viral activities of coronaviruses by activating Ndfip1/2 and NEDD4, leading to Janus kinase 1 (JAK1) proteolysis and downregulation.¹⁶⁹ Cutaneous warts are caused by the human papillomavirus (HPV).¹⁷⁰ Topical ionic contra-viral therapy with digoxin considerably reduced the diameter of cutaneous warts and HPV load compared with placebo.¹⁷¹ In addition, CGs inhibited the expression of both early and late vaccinia virus (VACV) proteins at different concentrations.¹⁷²

The SARS-CoV-2 epidemic has led to growing interest in the potential therapeutic applications of CGs for COVID-19. Souza et al. found that CGs reduced viral replication and inhibited NF-κB from directly interfering with SARS-CoV-2 yield and inflammatory cytokine production.¹⁷³ Digoxin and ouabain treatments considerably inhibited SARS-CoV-2 replication by more than 99% in vitro, demonstrating greater efficacy than approved drugs such as chloroquine and remdesivir.¹⁷⁴ Pollard et al. found that digitoxin notably reduced the levels of cytokines TNFα, GRO/KC, MIP2, MCP1, and IFNγ in cotton rats infected with influenza viruses.¹⁷⁵ As the initiation of SARS-CoV-2 infection requires the receptor binding domain (RBD) on the viral spike protein to bind to the host receptor ACE2 protein, CGs (ouabain, digitoxin, and digoxin) may prevent viral penetration and reduce infectivity by first inhibiting ACE2:RBD binding.¹⁷⁶ Oleandrin also demonstrates potent prophylactic and therapeutic activity in inhibiting SARS-CoV-2. A defined extract of Nerium oleander L. applied to the relevant hamster model of COVID-19 showed a substantial reduction of nasal viral load in the nasal turbinates.¹⁷⁷ In addition, oleandrin inhibits the expression of 78 kilodalton glucose-regulated protein (GRP78), which can reduce SARS-CoV-2, Beta, and Delta, as well as highly infectious Omicron variants. When combined with oleandrin at low nanomolar dosages, the treatment of COVID-19 is enhanced by remdesivir or nirmatrelvir.¹⁷⁸ The antiviral effects of CGs are shown in Table 3.

Table 3 Antiviral effects of CGs^a

CGs	Virus	Mechanism	Experimental subject	Dosage
a Abbreviations: i.p.,intraperitoneal; p.o., oral; t.c., topical cutaneous; s.l., sublingual; s.c., subcutaneous; N/A, not applicable (the mechanism was not addressed).
Ouabain	JEV^157	Attenuate virus replication via NKA blockade	Vero, Huh-7, U251	49–784 nM
			BALB/c mice	3 mg per kg per day i.p.
	IAV^158	Attenuate virus replication via NKA blockade	Alveolar epithelial cells	1–20 nM
	RSV^164	Inhibit viral uptake by targeting ATP1A1	A549, HSAEC	25 nM
Ouabain	EBOV^162	Inhibit virus entry and replication	Hela, HEK293T, THP-1	50–500 nM
	ZIKV (ref. 160 and 161)	Attenuate virus replication via NKA blockade	Vero	12.25–784 nM
			Adult mice, pregnant mice	2 mg per kg per day, i.p.; 3 mg per kg per day, p.o.
		Attenuate virus replication via NS5-RdRp/NS3-helica	Vero	2.5–20 nM
	TGEV/HCoV-OC43 (ref. 169)	Attenuate virus replication through JAK1_STAT1/3	ST, HCT-8	30–300 nM
	SARS-CoV-2 (ref. 174 and 176)	Attenuate virus replication	Vero	100 nM
		Reduce the ACE2/RBD binding	A549	80–120 nM
Digoxin	JEV^157	Inhibit viral RNA synthesis via NKA blockade	Huh-7	49–784 nM
	ZIKV^160	Attenuate virus replication via NKA blockade	Vero	12.25–784 nM
	EBOV^162	Inhibit virus entry and replication	Hela, HEK293T, THP-1	50–500 nM
	BUNV^163	Attenuate virus replication via NKA blockade	Vero, HEK293T, BHK-21	0–500 nM
	HPV^171	Attenuate virus replication	Human study	5–30 mg per day, t.c.
	SARS-CoV-2 (ref. 174 and 176)	Attenuate virus replication	Vero	150 nM
		Reduce the ACE2/RBD binding	A549	80–120 nM
Lanatoside C	HSV-1 (ref. 168)	Attenuate virus replication via NRF2	ARPE-19, Vero	50–800 nM
	EBOV^162	Inhibit virus entry and replication	Hela, HEK293T, THP-1	50–500 nM
Digitoxin	EBOV^162	Inhibit virus entry and replication	Hela, HEK293T, THP-1	50–500 nM
	SARS-CoV-2 (ref. 175 and 176)	Decrease THFα, GRO/KC, MIP2, MCPl, and IFNy	Cotton rat	30–300 μg per kg per day, i.p.
		Reduce the ACE2/RBD binding	A549	80–120 nM
Oleandrin	SARS-CoV-2 (ref. 177 and 178)	Inhibit NKA	Vero	0.005–1.0 μg mL^−1
			Golden Syrian hamster	0.0325, 0.325, 3.25 mg per day, s.l.
		Induce viral infection through GRP78 blockade	Vero E6-ACE2, Caco-2-ACE2, A549-ACE2	4.38–100 nM
Rostafuroxin (PST2238)	RSV^164	Inhibit viral uptake by targeting ATP1A1	A549, HSAEC	20 nM
C4′-Dehydro-oleandrin (KDC203)	PrPC^167	Inhibit NKA	T98G, LN-229, 104C1, GPC-16, Guinea pig neurons/astrocytes/cardiomyocytes	12–800 nM
			Guinea pig	0.15, 0.225, 0.3 mg per kg per day, s.c.
CGs from Streblus asper	HSV-1 (ref. 78)	N/A	Vero	0.19–1.03 μM
CGs from Sonchus asper	EBV^37	Inhibit NKA	Raji, A549, THP-1, C666-1	0.35–5.8 nM
CGs	VACV^172	Attenuate virus replication	Hela	5–100 nM

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3.6. Pharmacological activity of CGs in neuroprotection

As the α3 subunit of NKA serves as a neuronal receptor for agrin, its activation is implicated in various physiological activities in the nervous system.^179,180 As FDA-approved NKA inhibitors, CGs trigger signaling pathways or regulate Ca²⁺ levels in a concentration-dependent manner to maintain neuronal homeostasis,¹⁸¹ and are therefore frequently used in studies of neurotransmitter-related diseases. Multiple sclerosis, a central nervous system autoimmune disease, causes loss of nerve myelin, and digoxin stimulates myelin regeneration.¹⁸² By combining both anti-inflammatory and neuroprotective activities, digoxin exerts a healing effect on peripheral nerve injuries.¹⁸³ Neuroinflammation drives the development of epilepsy, and digoxin exhibits a clear anticonvulsant potential.¹⁸⁴ Low doses of digoxin stimulate dendritic spine formation or recirculation and promote motor learning ability.¹⁸⁵ Moreover, digoxin reduces hippocampal neuronal death, neuroinflammation, and cholinergic deficits, which can help in the treatment of cognitive impairment and Alzheimer's disease.¹⁸⁶ At subnanomolar concentrations, ouabain counteracts calcium overload and neuronal apoptosis during excitotoxic stress.¹⁸⁷ Dysregulation of microRNAs (miRNAs) is involved in the development of a wide range of diseases; however, no clinical approvals have been granted for the treatment of neurological disorders by modulating miRNAs. Nguyen et al. screened CGs as novel miR-132 inducers capable of protecting rodent and human neurons, suggesting a promising therapeutic approach for neurodegenerative diseases.¹⁸⁸ In addition, CGs have been shown to restore WDR45 autophagy abnormalities and exhibit neuroprotective effects during hypoxia and glucose starvation, with therapeutic potential for beta-propeller protein-associated neurodegeneration.¹⁸⁹

3.7. Toxicology and safety profile

Despite their broad therapeutic potential, CGs face a major challenge in clinical translation due to their narrow therapeutic index—the fine margin between effective and toxic doses. Their mechanism, mediated through NKA inhibition, induces intracellular calcium overload and predisposes to arrhythmias. This cardiotoxicity severely limits systemic use and necessitates meticulous dosing and therapeutic drug monitoring.

Digoxin is currently the only FDA-approved CG still in clinical use for humans, indicated for conditions such as atrial fibrillation, heart failure, arrhythmias, and tachycardia,¹⁹⁰ as well as in veterinary medicine.¹⁹¹ Other CGs like digitoxin and deslanoside have been discontinued by the FDA due to high toxicity and have been replaced by alternative agents. Digoxin is commercially available in capsules, tablets, and ampules for oral and intravenous administration.¹⁹² Thus, using digoxin as a representative example, the therapeutic window in humans is well established at 0.5–2.0 ng mL⁻¹ (equivalent to 0.6–2.6 nM).¹⁹³ Published data on acute toxicity report toxic doses ranging from 50 to 200 μg kg⁻¹ in humans, varying by sex, age, and route of administration. The frequency and severity of side effects—50% cardiac and 25% gastrointestinal—depend on dosage, patient condition, and concomitant medications.^194,195 In recent human trials, therapeutic drug monitoring has detected digoxin levels below the quantitation limit (300 pg mL⁻¹).¹⁷¹ Given that clinical trials for their emerging pharmacological effects (e.g., anticancer, antiviral) remain scarce and largely in early stages, the majority of reported in vivo data are still derived from preclinical animal models. In mice, acute toxic doses vary widely: 8.15 μg kg⁻¹ (subcutaneous), 124–7670 μg kg⁻¹ (other injections), and up to 17 780 μg kg⁻¹ (oral). Rats generally exhibit higher toxicity thresholds than mice.¹⁹⁴ Many in vivo studies demonstrate efficacy at non-toxic doses, though high or prolonged exposure may worsen health outcomes. These findings support the therapeutic potential of CGs at low doses, over short durations, or with altered administration routes—strategies that may reduce toxicity and side effects.¹⁵⁸ Combination therapy is also under consideration.¹⁹³ However, much of the evidence remains limited to cellular models, underscoring the need for further validation. Additionally, toxicological data for other CGs remain limited. Subtle structural variations can significantly alter their toxicological profiles, clinical manifestations, and histopathological outcomes,¹⁹² underscoring the need for more comprehensive studies to support clinical translation.

4. Synthesis and biosynthesis

4.1. (Semi)synthesis of CGs

The structural diversity of CGs arises from variations in C-3, C-5, and C-17 stereoconfigurations, as well as the presence or absence of oxidizing groups and double bonds, ultimately influencing their biological efficacy.⁷⁷ Extracts from natural plants have struggled to meet the market demand, and owing to the incomplete and immature understanding of their biosynthetic pathways, structural modification of these compounds remains limited and challenging. Therefore, chemical synthesis remains the predominant synthetic approach for CGs at present.¹⁹⁶ Baran and co-workers accomplished the seminal total synthesis of ouabagenin from adrenosterone. This work pioneered a solution to the long-standing challenge of selective C–H hydroxylation at saturated carbon centers, achieving controllable oxidation processes and enabling access to diverse ouabagenin analogues.^197,198 By 2019, the total chemical synthesis of the cardiac aglycons (+)-digitoxigenin¹⁹⁹ and ouabagenin,^25,26,200 alongside the CGs ouabain^25,26 and rhodexin A,^201,202 had been accomplished. In the past five years, total syntheses of other cardenolide aglycones, including 19-hydroxysarmentogenin,²⁰³ ouabagenin,²⁰³ and cinobufagin,²⁰⁴ have also been reported. Detailed chemical (semi)synthetic strategies for accessing cardiac aglycones and glycosides can be found in Table S1.

The optimization of steps and synthetic conditions in recent years has led to the total chemical synthesis of the CGs cannogenol 3-O-α-L-rhamnoside (89),⁹⁵ acovenoside A (90) and its congeners,⁹⁷ rhodexin B (91),²⁰⁵ oleandrin (92), and beaumontoside²⁰⁶ (Fig. 6). The raw materials for chemical synthesis are primarily commercially available steroids or those obtained through simple, rapid syntheses of common steroids. Then, the process focuses on introducing a C-14 β-hydroxyl group and an unsaturated endolactone ring at the C-17 position, followed by a glycosylation reaction. The steps in the reaction flow to modify the steroidal parent nucleus are selected in the appropriate order to introduce specific functional groups—primarily hydroxyl or acetyl groups—onto the target CGs. The current reactive inertia of the C(sp³)–H bond at the C-14 position of CGs as well as spatial site-blocking effects makes direct C–H hydroxylation to install a hydroxyl group at C-14 extremely challenging. Most programs choose to introduce hydroxyl groups by first installing a double bond in ring D, followed by a hydration reaction. In 2019, Khatri et al. employed a Cu(II)-catalyzed diastereoselective Michael/aldol cascade approach to achieve rapid assembly of functionalized C-14 hydroxyl group-containing steroidal skeletons.⁹⁵ The modification of C-17 is primarily achieved by subjecting the precursor to Barton vinyl iodide synthesis, followed by Stille or Suzuki coupling with a commercially available tin reagent. The modification of the steroidal scaffold primarily involves stereoselective protection and deprotection, epoxidation, and ring-opening reactions. The glycosylation step emphasizes the selection and optimization of catalysts and reaction conditions, considering the inherent differences in the reactivity of alcohol hydroxyl groups at various positions on the steroid scaffold. Most synthetic schemes select phosphine salts as catalysts, and different phosphine salts of various strong acids lead to different levels of α:β selectivity. Liu et al. used gold(I)-catalyzed glycosylation with superarmed glycosyl ortho-alkynylbenzoates as donors.⁹⁷ The final step of the chemical synthesis is to perform global deprotection to obtain the target CG.

Fig. 6 Total chemical synthesis of CGs in recent years. TBS: tert-butyldimethylsilyl; TIPS: triisopropylsilyl; TMS: trimethylsilyl.

In addition to conventional purely chemical synthesis, a chemoenzymatic approach has emerged. By combining the advantages of biocatalysis and chemical synthesis, this strategy demonstrates high regioselectivity and stereoselectivity, providing low-cost and eco-friendly synthesis routes to natural products that were previously difficult or inaccessible to produce using only contemporary chemical methods. This efficient approach to synthesizing natural products and their derivatives has become a growing trend.^207–209 In recent years, researchers have begun exploring chemoenzymatic approaches to tackle the key steps in the synthesis of complex CGs, achieving notable progress (Fig. 7). The underlying synthetic principles of chemoenzymatic and chemical methods are similar. Currently, the main biocatalytic step is the formation of C-14 hydroxyl group, which offers considerable advantages over the cumbersome steps and low yields associated with chemical catalysis. Two P450 enzymes, Calotropis gigantea (L.) W. T. Aiton CYP11411 and B. gargarizans CYP44476, directly convert androstenedione (AD) to 14α-OH-AD. Subsequent chemical reactions produce key 14α-hydroxyl group steroid intermediates, ultimately enabling CG synthesis through the modular installation of a five- or six-membered lactone ring at the C-17 position. When the desired 17β-substituted orientation cannot be achieved through the above coupling reaction, an S_N2-type-free radical reaction can be employed to control the configuration at C-17, guided by NOE correlations between the C-18 methyl group and the C-20 hydrogen atom.²¹⁰ Moreover, a novel steroidal C14α-hydroxylase (CYP14A) with high catalytic activity and broad substrate specificity was mined and identified from Cochliobolus lunatus R.R. Nelson & F.A. Haasis (strain CGMCC 3.3589). Based on RoseTTaFold de novo prediction and molecular docking, a binding model for the CYP14A steroidal substrate was developed. By applying fixed-point saturating mutagenesis and a combined mutation strategy, mutants with notably improved catalytic efficiency and regioselectivity were obtained. The synthesis of cardenolide periplogenin, (+)-digitoxigenin, and its three diastereomers was further achieved through chemical methods.²¹¹

Fig. 7 The chemoenzymatic synthesis pathways of CGs. TBS: tert-butyldimethylsilyl; TMS: trimethylsilyl.

4.2. Biosynthesis of CGs

The elucidation of the biosynthetic pathways of CGs is one of the most promising research areas for the future. It not only helps to reveal the molecular mechanisms underlying their natural synthesis but also provides a theoretical foundation and technical support for their efficient production. Biotransformation employs biological systems—such as bacteria, fungi, plant tissues, or isolated enzymes—to structurally modify exogenous compounds under mild conditions. This approach enables diverse chemical transformations that are often challenging in conventional organic synthesis, facilitating access to novel structural analogues.²¹² Although historically applied in the modification of CGs,²¹³ biotransformation has been increasingly supplanted by advances in biosynthetic methods. Currently, based on advancements in genomics, transcriptomics, metabolomics, and enzyme functional studies, the hypothesized and partially elucidated biosynthetic pathways of CGs involve complex metabolic processes. These primarily include the synthesis of the steroidal backbone, formation of sugar moieties, and glycosylation of the aglycone with sugar units. The NCBI accession numbers of the relevant enzymes and their species information are provided in Table S2.

4.2.1. Biosynthesis of the steroid nucleus. The biosynthetic pathway of CGs has not been fully elucidated, and the currently hypothesized pathway involves multiple enzymatic gene regulations (Fig. 8). The biosynthetic pathway proceeds from carbon sources via the cytoplasmic mevalonate (MVA) pathway,²¹⁴ leading to cycloartenol through a series of enzymatic reactions.²¹⁵

Fig. 8 Putative biosynthetic pathways of steroid nucleus.

β-Sitosterol, campesterol, and cholesterol biosynthesis. Cycloartenol serves as the common precursor for both phytosterols (e.g., campesterol and β-sitosterol) and cholesterol.²¹⁶ The pathways diverge through early enzymatic steps: the phytosterol pathway is initiated by sterol C-24 methyltransferase 1 (SMT1)^217,218 and phytosterol-specific C4 sterol methyl oxidase 1 (SMO1),²¹⁹ while the cholesterol pathway begins with C24 sterol side-chain reductase 2 (SSR2)^220,221 and pathway-specific C4 sterol methyl oxidase 3 (SMO3).²²² A set of intermediate enzymes—cyclopropylsterol isomerase (CPI),²²³ sterol C-14 demethylase (CYP51),²²⁴ sterol C-14 reductase (C14-R),²²⁵ and sterol 8,7 isomerase (8,7-SI)²²⁶—are common to both pathways. Following these shared steps, the pathways diverge again. In the phytosterol branch, sterol C-24 methyltransferase 2 (SMT2) catalyzes a second methylation to form the C24-ethyl group characteristic of β-sitosterol.²²⁷ This pathway subsequently utilizes sterol methyl oxidase 2 (SMO2),²²⁸ sterol C-5(6) desaturase 1 (C5-SD1),²²⁹ 7-dehydrocholesterol reductase 1 (7-DR1),²³⁰ and 24-sterol reductase (SSR1)²³¹ to complete demethylation, dehydrogenation, hydrogenation, and side-chain reduction, yielding either β-sitosterol or campesterol. Conversely, cholesterol biosynthesis is completed through the actions of sterol methyl oxidase 4 (SMO4), sterol C-5(6) desaturase 2 (C5-SD2), and 7-dehydrocholesterol reductase 2 (7-DR2).²¹⁶
Pregnenolone biosynthesis. The conversion of three sterols to pregnenolone involves the mitochondrial cytochrome P450 side-chain cleavage enzyme (P450_scc) catalyzing the hydroxylation of C-22 and C-20 on the side chain and the break between the two carbons,²³² which is the first and rate-limiting step in the control of steroid biosynthesis.²³³ P450_scc has been characterized in both animals and plants. In animals, the Homo sapiens (Linnaeus, 1758) CYP11A1 gene²³⁴ and the B. gargarizans CYP11A1 gene²³⁵ encode cholesterol P450_scc, which catalyzes the conversion of cholesterol to pregnenolone. Although the activity of plant P450_scc on different sterol substrates was demonstrated decades ago,²³² it was not definitively identified and characterized until 2023. As CGs are present only in leaves and not in roots, CYP87A4 from D. lanata was identified through differential gene expression analysis and confirmed to act on cholesterol or campesterol via yeast heterologous expression experiments.²³⁶ In the same year, on the basis of isotope labeling that confirmed pregnenolone as a precursor in CG biosynthesis, two P450_scc enzymes—CYP87A106 from D. purpurea and CYP87A103 from Calotropis procera (Aiton) W. T. Aiton—were identified through differential expression analysis between leaf and root transcriptomes, along with BLAST searches. This finding was demonstrated in further experiments showing that both enzymes can produce pregnenolone using cholesterol, campesterol, and β-sitosterol as substrates. Additional CYP87A homologs identified across plant species via BLAST similarly exhibit sterol side-chain cleavage activity.^237,238 The evolutionary mechanism by which CYP87A family proteins in plants acquire the catalytic activity required for pregnenolone biosynthesis remains unclear. These proteins differ in sequence from their mammalian counterpart, CYP11A1. Notably, amino acid substitutions such as A355V or L357A, based on CYP11A1, result in the loss of side-chain cleavage activity in DlCYP87A4 and EcCYP87A126. This suggests that mammalian P450_scc is specialized for steroid hormone biosynthesis, whereas plant P450scc has evolved to synthesize specific metabolites.^236,239,240
Progesterone biosynthesis. The current study suggests that the conversion of pregnenolone to progesterone occurs in two steps: the first step involves the oxidation of the C3-hydroxyl group to produce isoprogesterone, catalyzed by NAD⁺-dependent 3β-hydroxysteroid dehydrogenase (3βHSD), and the second step involves the migration of the double bond from position 5 to position 4. The currently characterized plant 3βHSDs^241,242 belong to the short-chain dehydrogenase/reductase gene family,²⁴³ and serve to oxidize and isomerize the precursor compound sterol. The second step of isomerization to progesterone is irreversible.
Pregnanolone biosynthesis. The types of CGs reported in plants are mostly in the C-3 β-configuration and have a cis-fused A/B ring junction. In these plants, progesterone is converted by NADPH-dependent co-reactive progesterone-5β-reductases (St5βR)^244,245 and 3βHSD to pregnanolone (3β-hydroxy-5β-pregane-20-one). Enzymes exhibiting St5βR activity are present in numerous plants that both produce and do not produce CGs. Recent studies have revealed that these enzymes also function as iridoid synthases, and are now collectively referred to as PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes).^246,247 In plants producing CGs, the presence of progesterone-5α-reductases (St5αR),²⁴⁸ 3βHSD, and 3α-hydroxysteroid 5β-oxidoreductases (3αHSD)²⁴⁹ enables the generation of the corresponding products with a C-3 α-configuration or trans-fused A/B ring junction. Notably, enzymes with confirmed 3αHSD activity remain limited and generally demonstrate low catalytic efficiency—an issue that still requires resolution.
Downstream biosynthesis processes for the steroid nucleus. In addition to the two CYP450 s used in the chemoenzymatic synthesis described above, which catalyze the 14α-hydroxylation of precursors, enzymes involved in the 14β-hydroxylation of pregnanolone in plants have not been identified. In 2022, Zhao et al. identified the key P450 enzyme (CYP11411) from C. gigantea that catalyzed the direct C–H activation at the C-14 position of AD, resulting in the retention of the 14α-OH-AD product configuration via the canonical “oxygen rebound” mechanism.¹⁵⁶ To date, all reported enzymatic hydroxylation reactions of tertiary carbon centers in natural products have maintained their stereoconfigurations, as shown in the previous example.²⁵⁰ Therefore, 14β-hydroxylation of tertiary carbon centers in progesterone may occur through hydrogen isomerization (α-H to β-H) followed by hydroxylation, or via direct hydroxylation through a stereoinversion, which involves hydrogen atom abstraction, electron transfer, the formation of a stable cation intermediate via isomerization, and the subsequent attack of the cation by water. The 21-hydroxylase CYP21A1 has been reported only in mammals (e.g., Mus musculus (Linnaeus, 1758)).²⁵¹ 21-Hydroxypregnane 21-O-malonyltransferases (21MAT) transfer the malonyl group from malonyl-CoA to the C-21 hydroxyl group of the pregnane precursor 5β-pregnane-3β,14β,21-triol-20-one generated in the above two steps. Among the tested substrates, the free ketol exhibited the lowest acceptance in this acyltransferase reaction, whereas the 3-O-glycosylated ketol demonstrated significantly higher enzyme affinity, supporting the hypothesis that glycosylation may occur at an early stage in CG biosynthesis.²⁵² The enzymes involved in the subsequent cyclization to generate unsaturated five-membered or six-membered lactone rings at C-17 remain to be elucidated. Notably, under physiological conditions, analogues of 21-O-malonyl-5-pregnane-3β,14β-diol-20-one undergo only very slow lactonization, whereas those lacking the 14β-hydroxyl group show negligible reactivity, indicating that 14β-hydroxylation precedes lactone formation.²⁵³

Studies on the biosynthetic reaction mechanisms, reaction sequences, and related enzyme genes of bufadienolides have been rarely reported. The putative biosynthetic pathway of bufadienolides in plants initiates from the precursor 5β-pregnane-3β,14β,21β-triol-20-one. This intermediate undergoes acylation with oxaloacetyl-CoA, followed by aldol condensation, decarboxylation, reduction, and elimination reactions, ultimately producing a six-membered unsaturated lactone ring.⁵¹ Isotope tracing revealed that cholesterol is the biosynthetic precursor of marinobufotoxin and marinobufagenin.²⁵⁴ Unlike in plants, the biosynthesis of bufadienolides in toads does not involve the cleavage of cholesterol side chains.²⁵⁵ Thus, pregnenolone does not act as a precursor.^256,257 These findings align with the aforementioned evolutionary implications regarding P450_scc and further suggests the existence of an alternative biosynthetic pathway in animals. Based on transcriptome analysis, the cholesterol-bile acid–bufadienolide pathway is currently proposed. In toads, the process begins with the MVA pathway, then, lanosterol undergoes a series of reactions to produce the key intermediate, cholesterol. CYP27A1 initiates the conversion of cholesterol to lithocholic acid, a bile acid salt intermediate,²⁵⁸ which is subsequently converted into bufadienolides through a series of unknown steps.²⁵⁹ The enzymes 3βHSD²⁶⁰ and steroid 5β-reductase (SRD5β),²⁶¹ which modify the steroid skeleton, have also been reported in toads. In addition, Lei et al. identified five CYP450 enzymes from toads,²⁶² which are similar to the previously reported fungal CYP enzyme Sth10 from Thanatephorus cucumeris (A.B. Frank) Donk (starin NBRC 6298).²⁶³ This enzyme catalyzes the hydroxylation of bufalin and resibufogenin at different sites to yield compounds 93–105.

4.2.2. Glycosylation. Unlike the poor regioselectivity and stereoselectivity, as well as the challenges related to the protection and deprotection of functional groups in the chemical synthesis of CGs,²⁶⁴ glycosyltransferases (GTs) serve as biocatalysts with substrate and sugar donor promiscuity.²⁶⁵ GTs stabilize the product, modulate its physiological activity, and control its intracellular distribution,²⁶⁶ offering great potential in the field of drug discovery. Some reported GTs can glycosylate CG precursors in vitro,²⁶⁷ suggesting that the substrate promiscuity of steroid 3-O-glycosyltransferases (S3GTs) may explain why glycosylation occurs at different stages. Thus, glycosylation should no longer be considered a terminal step in biosynthesis. S3GTs that catalyze the transfer of glucosyl fragments to the C-3 hydroxyl group of three sterols to produce sterol glycosides have been reported.^268–270

S3GTs capable of glycosylation modification on intact CGs have been identified, including microbial-derived OleD from Streptomyces antibioticus (Waksman & Woodruff, 1941) Waksman & Henrici, 1948 (ref. 271) and YjiC1 from Bacillus subtilis (Ehrenberg, 1835) Cohn, 1872,²⁷² but suffer from low catalytic efficiency and poor regioselectivity. Three plant GTs catalyze the glycosylation of substrates 106–117 to form their corresponding CGs (Fig. 9). In 2017, Wen et al. reported for the first time that UDP-glycosyltransferase (UGT) 74AN1 from Asclepias curassavica Lour., a plant UDP-GT with substrate promiscuity, catalyzes the formation of 3-O-β-D-glucosides from various intermediates in the CG biosynthesis pathway and is the first to catalyze cardiac steroid 3α-hydroxyglycosylation of GT. UGT74AN1 accepts UDP-Glc as the primary sugar donor and only minimally utilizes UDP-GlcNAc.²⁶⁷ In addition, based on the use of suspension cultures of plant cells from several non-cardiac steroid-producing species for CG biotransformation,⁵² the team identified the first UGT74AN3 from the non-CG-producing plant C. roseus that accepts only UDP-Glc as its major sugar donor and is catalytically active toward eight structurally distinct CTSs and phenolic compounds.²⁷³ In 2022, Huang et al. identified and characterized the plant S3GT UGT74AN2 from C. gigantea, capable of generating various cardiotonic steroid 3-O-glycosides using UDP-Glc, UDP-GlcNAc, and UDP-Gal as sugar donors, and through structure- and sequence-based engineering, yielded the triple mutant UGT74AN2 I284R/W390H/V391G, which exhibits broader sugar donor specificity and enhanced catalytic activity.²⁷⁴

Fig. 9 Glycosylation process of CGs. R₁–R₄: different substituent groups. (A) Glycosylation of UDP-Glc as sugar donor; (B) glycosylation of other sugar donors.

4.2.3. Synthetic biology of CGs in microbial cell factories. With advances in synthetic biology and metabolic engineering, biosynthesis utilizes the powerful and diverse biochemical reaction networks of microorganisms to convert low-value, renewable resources into high value-added natural compounds.²⁷⁵ Various precursors of cardiac steroidal compounds have been synthesized (Table 4), laying the foundation for subsequent CG biosynthesis.

Table 4 Biosynthesis of CGs precursors in microbial cell factory

Products	Microbial strains	Fermentation vessels	Carbon source	Yield	References
Campesterol	Y. lipolytica	5 L bioreactor	Sunflower seed oil	942 mg L^−1	277
	S. cerevisiae	5 L fermenter	Glucose	916.9 mg L^−1	281
	Y. lipolytica	5 L fermenter	Glucose	837 mg L^−1	280
	C. jadinii	Shake flask	Glucose	92.1 mg L^−1	282
		5 L bioreactor		807 mg L^−1
	S. cerevisiae	96-Well plates	Glucose	18.4 mg L^−1	279
β-Sitosterol	S. cerevisiae	96-Well plates	Glucose	2 mg L^−1	278
Cholesterol	S. cerevisiae	5 L fermenter	Glucose	5.5 g L^−1	283
	C. jadinii	Shake flask	Glucose	81.8 mg L^−1	282
		5 L bioreactor		1.52 g L^−1
	S. cerevisiae	5 L bioreactor	Glucose	1.78 g L^−1	288
Pregnenolone	Y. lipolytica	5 L bioreactor	Glucose or sunflower seed oil	78 mg L^−1	287
	S. cerevisiae	5 L bioreactor	Glucose	0.83 g L^−1	288
Progesterone	M. neoaurum	Shake flask	Glucose	235 mg L^−1	290
	S. cerevisiae	5 L fermenter	Glucose and ethanol	1.06 g L^−1	238

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In 2016, Du et al. introduced an exogenous 7-dehydrocholesterol reductase (DHCR7) into Yarrowia lipolytica (Wick., Kurtzman & Herman) Van der Walt & Arx, using sunflower seed oil as a carbon source. This enabled campesterol production with a yield of 453 ± 24.7 mg L⁻¹via high-cell-density fermentation in a 5 L bioreactor.²⁷⁶ In the following year, they screened DHCR7 from Danio rerio (Hamilton, 1822) and enhanced the expression of the key gene POX2, which increased the yield of campesterol to 942 ± 50.1 mg L⁻¹.²⁷⁷ By 2020, Xu et al. reconstituted the multi-enzyme pathway for synthesizing various phytosterols in Saccharomyces cerevisiae (Desm.) Meyen through metabolic engineering, strain evolution, and fermentation engineering, achieving a campesterol yield of 7 mg L⁻¹ and β-sitosterol yield of 2 mg L⁻¹ through fermentation in 96-well plates,²⁷⁸ and the campesterol yield was increased to 18.4 mg L⁻¹ in 2023 by partially restoring the activity of the sterol acyltransferase ARE2 in yeast and enhancing the upstream FPP supply.²⁷⁹ In 2020, Qian et al. similarly achieved the de novo synthesis of campesterol in Y. lipolytica by knocking down multifunctional β-oxidation protein (Mfe1) and peroxisomal biogenesis factor 10 (PEX10) and heterologously introducing DHCR7, to obtain a yield of 837 mg L⁻¹ through incubation in a 5 L fermenter for 144 h.²⁸⁰ In 2021, Zhou et al. achieved a campesterol yield of 916.9 mg L⁻¹ through fermentation by overexpressing a screened heterologous DHCR7 gene combined with an endogenous promoter engineering strategy, using a 5 L fermenter for fed-batch fermentation process.²⁸¹ By 2023, Gu et al. developed a highly efficient CRISPR/Cas9 technique for metabolic pathway modification in the polyploid industrial yeast Cyberlindnera jadinii (Sartory, R. Sartory, J. Weill & J. Mey.) Minter 2009, achieving yields of 92.1 and 81.8 mg L⁻¹ of campesterol and cholesterol, respectively, via shake flask fermentation. High-density replenishment of batch fermentation further increased the yields to 807 mg L⁻¹ and 1.52 g L⁻¹, marking the first gram-scale production of a steroidal compound in C. jadinii.²⁸² In addition, Xu et al. achieved a cholesterol yield of 5.5 g L⁻¹ during high-density replenishment batch fermentation, enabling the production of 2.03 g L⁻¹ of diosgenin through synthetic-pathway optimization, fine-tuning of gene expression, and elimination of competing pathways.²⁸³

Except for sterols, the de novo synthesis of pregnenolone in S. cerevisiae was achieved as early as 1988, yielding 60 mg L⁻¹ using glucose as the carbon source;²⁸⁴ however, no substantial breakthrough has been reported to date. The P450_scc system comprises the P450 monooxygenase CYP11A1 and its natural redox partners adrenodoxin (Adx) and adrenodoxin reductase.²⁸⁵ CYP11A1 catalyzes the conversion of sterols to pregnenolones, which is the first and limiting step in steroidogenesis. By 2013, Makeeva et al. constructed a stable transgenic Escherichia coli (Migula, 1895) Castellani & Chalmers, 1919 strain with a functionally reconstructed bovine cholesterol pathway, yielding 0.42 ± 0.015 mg L⁻¹ of pregnenolone from 500 μM (193 mg L⁻¹) of cholesterol in 24 h.²⁸⁶ In 2019, Zhang et al. introduced the mammalian P450_scc CYP11A1 and its electron transport chain into campesterol-producing engineered Y. lipolytica strains developed in their earlier study,²⁷⁷ achieving a pregnenolone yield of 78.0 mg L⁻¹.²⁸⁷ In 2025, Chen et al. achieved cholesterol and pregnenolone titers of 1.78 g L⁻¹ and 0.83 g L⁻¹, respectively, in engineered yeast via rational engineering of DHCR7 and RgCYP87A3 guided by computational simulations and QM/MM analysis, coupled with mitochondrial co-localization of electron transfer components and pathway optimization.²⁸⁸

In 2014, Strizhov et al. achieved a progesterone yield of 25 mg L⁻¹ from 7.5 mM cholesterol using Mycolicibacterium smegmatis (Trevisan 1889) Gupta et al. 2018 (strain mc²155).²⁸⁹ By 2022, Liu et al. reported that CYP11A1 can also catalyze the side-chain cleavage of 4-HBC to produce progesterone. They introduced CYP11A1 into Mycobacterium neoaurum Tsukamura, 1972, a strain with high 4-HBC yield, and further accelerated the electron transfer to achieve the first green and sustainable fermentation production of phytosterols to progesterone, achieving a yield of 45 mg L⁻¹ under light-emitting diode light-driven conditions. By combining engineered M. neoaurum and InP nanoparticles to form a novel inorganic–biological hybrid system, a yield up to 235 ± 50 mg L⁻¹ was achieved.²⁹⁰ Moreover, an engineered M. smegmatis mc²155 strain produced 85.2 ± 4.7 mol% 3-methoxymethyl-pregnenolone within 48 hours. Subsequent acid hydrolysis removed the protecting group, yielding high-purity pregnenolone via crystallization.²⁹¹ Over the past two decades, yeast-engineered strains have remained suboptimal for progesterone production compared to microbial conversion. In 2025, Li et al. screened P450_scc, cytochrome P450 reductase (CPR), and HSD from Marsdenia tenacissima (Roxb.) Wight & Arn. By minimizing intracellular shuttling of substrates and enzymes in engineered S. cerevisiae yeast, they achieved progesterone production of 1.06 g L⁻¹via fermentation using simple carbon sources.²³⁸

Optimization of secondary metabolite production of CGs in plant factories is also a key research direction. In D. purpurea stem cultures, the accumulation of digitoxin and digoxin was increased up to 9.1-fold and 11.9-fold, respectively, through induction and feeding with the precursor progesterone (200–300 mg L⁻¹).²⁹² By expressing P5βR from Arabidopsis thaliana (L.) Heynh. in D. purpurea cultured in vitro, digitoxin and digoxin contents were increased up to 3.8-fold and 2.2-fold, respectively.¹⁴

5. Conclusions and prospects

CGs have been a much publicized and controversial compound since their discovery. Achieving a balance between the therapeutic efficacy and side effects of CGs remains a critical concern. The pharmacological activity of CGs in the treatment of cardiovascular diseases was among the earliest areas to be investigated and applied in the clinic. Digoxin is not per se cheap, while digoxin-based medications are often administered at very small doses, making it accordingly low-cost medications available for the treatment of heart failure; however, its frequency of use is currently declining dramatically.²⁹³ Contrastingly, for patients with atrial fibrillation who cannot control their heart rate with other medications, CGs remain one of the most commonly used medications worldwide.²⁹⁴ With the deepening research on the pharmacological activities of CGs in multidisciplinary medical fields, their wide range of pharmacological effects has been gradually discovered, which is expected to lead to the development of new therapeutic drugs for clinical diseases across various fields, including oncology, neurology, respiratory diseases, and immune system disorders. Besides the widely-known NKA, several studies have found that CGs act on various new drug targets and are expected to be the most promising broad-spectrum drugs for application. Notably, given the pleiotropic effects of CGs, their optimal concentration as drugs warrants further investigation to ensure the stability and survival of all animal cells for maximum cost-effectiveness; it is essential to avoid cytotoxicity to functionally important differentiated cells while targeting harmful tumor or senescent cells.

Although hundreds of CGs are found in plants, only 20 to 30 of them are clinically applicable, with merely six or seven being commonly used. An examination of pharmacologic studies of CGs revealed that there is adequate data only for digoxin and digitoxin. The complex chemical structure and diversity of sugar residues of CGs enhance their variety and biological activity as drugs. Low solubility and severe cardiotoxicity pose serious limitations to the therapeutic use of CGs.²⁹⁵ Nonetheless, C-3 glycosylation modification enhances the solubility and reduces the toxicity of this class of drugs, which is considered a practical approach to improve their pharmacodynamic and pharmacokinetic properties and enhance their biological activity.^296–298 Studies on different structural CGs for the treatment of clinical diseases can help us better understand the intrinsic link between structure and pharmacodynamics. Large-scale, high-quality prospective efficacy studies or clinical trials with low bias and minimal randomization error are needed in the future. These studies should utilize evidence from ongoing pharmacokinetic and pharmacodynamic studies to comprehensively evaluate the safety, efficacy, and therapeutic effects of CGs on pharmacological actions, as well as assess the efficacy of combination therapeutic strategies.

Furthermore, since the 1980s, discoveries and pharmacological studies on endogenous CGs have enhanced the understanding of human physiological and pathophysiological processes, opening new avenues for therapeutic targeting. Numerous studies have confirmed that endogenous ouabain and marinobufagenin, two of the most prominent steroid hormones, play an important role in the development of cardiovascular diseases such as hypertension and heart failure.⁶⁴ However, their existence, structure, biosynthetic pathways, and mechanisms of action remain unclear or controversial.^1,63 Urgent research priorities for endogenous CGs include elucidating their biosynthetic pathways, defining their physiological and pathophysiological roles in cardiovascular and metabolic regulation, deciphering NKA-mediated signaling mechanisms, standardizing detection methods, and exploring their therapeutic targeting potential. The resolution of these questions will not only advance drug discovery but also improve our understanding of evolutionary biology and adaptive physiology.

The traditional methods of obtaining active ingredients from TCM, such as raw plant extraction, do not fully meet the clinical needs and have a negative impact on the natural environment. The scarcity of medicinal plant resources and the incomplete analysis of biosynthetic pathways continue to constrain the study of CGs. Although the total chemical synthesis of some CGs has been achieved, challenges remain, such as cumbersome steps, low yields, and harsh reaction conditions. Especially, the synthesis of the C-14 β-hydroxyl group and C-17 unsaturated lactone rings—the key structures of CG compounds—remains challenging due to spatial site resistance. Most chemically synthesized raw materials, including unsaturated lactone rings and different types of sugar donors, are still obtained commercially and cannot be produced cost-effectively. Currently, biocatalysts are applied in chemoenzymatic synthesis approach for the synthesis of the C-14 hydroxyl group, which considerably simplifies the synthesis steps and demonstrates great potential for future applications. In nature, many biologically active CGs are decorated with specific functional groups such as hydroxyl, carbonyl, and glycosyl moieties at various positions. Representative examples including digoxin, ouabain, and convallatoxin possess characteristic hydroxylations at C-12 or C-19, which significantly contribute to their potent bioactivities. Chemically introducing these functional groups—especially the C-19 hydroxyl—remains challenging due to issues such as poor regio- and stereoselectivity, lengthy synthetic routes, and low yields. As a promising alternative, chemoenzymatic synthesis offers a more efficient and selective approach. For instance, a CYP450 capable of catalyzing C-19 hydroxylation of steroidal substrates has been reported, providing a potential biocatalytic tool for modifying CGs at this position.²⁶³ This enzyme and similar biocatalysts could be harnessed in the future for the synthesis of complex natural CGs and their analogues. Furthermore, there is a continuing need to discover and characterize novel enzymes that can functionalize distinct sites (e.g., C-5, C-14, and C-19) of the cardenolide scaffold. Expanding this biocatalytic toolbox will facilitate precise structural diversification and efficient synthesis of CGs.

Access to rare sugar donors through biosynthesis has also been explored to some extent,²⁹⁹ suggesting that biosynthesis could be the most promising and widely applied alternative method to produce CGs. In recent years, new technologies and methods, such as high-throughput screening, molecular probes, and genome-wide association studies, have emerged, accelerating the elucidation of the complete biosynthetic pathway of CGs. Low-cost genome sequencing, advances in computational tools, and the application of artificial intelligence and machine learning technologies are revolutionizing the ability to identify, understand, and manipulate biosynthetic pathways. Currently reported P450 enzymes have low activity, and heterologous biosynthesis is still limited; therefore, the modification of enzyme function must be addressed. Methods and applications based on structure and machine learning-guided enzyme design make enzymes programmable and predictable, enabling them to execute chemical reactions that do not occur naturally. This advancement will greatly contribute to the popularization of biocatalysis.³⁰⁰ Protein engineering utilizing tools such as AlphaFold for protein structure prediction³⁰¹ and protein crystal structure resolution technology,³⁰² enables the precise modification of the catalytic properties of enzymes with low catalytic activity. This approach will lay the foundation for the heterologous production of CGs in microorganisms by using synthetic biology methods. In summary, synergistic breakthroughs in underlying technology clusters and intelligent design of enzyme functions will lead to a digital revolution in biosynthesis, facilitating the discovery of new CG drugs, more sustainable production methods, and a deeper understanding of complex biological processes.

6. Author contributions

Conceptualization, L. H., P. S., and S. L.; formal analysis and investigation, D. J.; writing – original draft preparation, D. J., Y. Z., and W. G.; writing – review and editing, D. J., Y. Z., P. S., and S. L.; visualization, D. J., and W. G.; project administration, P. S., and S.L. All authors have read and agreed to the published version of the manuscript.

7. Conflicts of interest

There are no conflicts to declare.

8. Data availability

This review synthesizes existing data from publicly available sources. No primary research results, software or code have been included and no new data were generated or analysed as part of this review. Drug approval and clinical trial data were extracted from: WHO International Clinical Trials Registry Platform (https://www.who.int/ictrp), U.S. FDA Drugs Database (https://www.accessdata.fda.gov/scripts/cder/daf), China Center for Drug Evaluation (https://www.cde.org.cn), U.S. National Library of Medicine ClinicalTrials.gov (https://clinicaltrials.gov). All data supporting the conclusions are derived from the cited references and repositories, with access details provided in the reference list.

Supplementary information: detailed chemical synthetic approaches to cardiac aglycons and glycosides and the NCBI accession numbers for relevant enzymes. See DOI: https://doi.org/10.1039/d5np00050e.

9. Acknowledgements

This work was supported by National Natural Science Foundation of China (82204568, 82304672), the Fundamental Research Funds for the Central Public Welfare Research Institutes (ZZ16-YQ-037, ZZ16-YQ-042, ZZXT202201), key project at central government level: the ability establishment of sustainable use for valuable Chinese medicine resources (2060302), and Scientific and Technological Innovation Project of the China Academy of Chinese Medical Sciences (CI2023E002, CI2024C004YN).

10. Notes and references

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