TCM-Driven Circadian Modulation in Cancer: Engineering Spatiotemporal Drug Delivery for Precision Oncology

Abstract

Circadian disruption constitutes a hallmark of cancer, driving tumorigenesis, progression, and therapy resistance via dysregulation of core clock genes. Emerging evidence indicates that traditional Chinese medicine (TCM), primarily through Chinese herbal medicine (CHM), exerts promising chronotherapeutic effects by modulating tumor-intrinsic circadian rhythms. Bioactive CHM constituents restore the oscillatory expression of clock genes (eg, BMAL1, PER2), thereby resynchronizing cell cycle control, DNA repair, metabolism, and immune surveillance to suppress malignancy. This review provides a comprehensive synthesis demonstrating that CHM not only corrects circadian dysfunction but also uncovers temporal vulnerabilities amenable to precision intervention. To address current translational gaps, we propose an integrated “multi-omics–intelligent delivery–preclinical validation–clinical translation” framework featuring: (1) AI-driven construction of individualized circadian maps to define optimal chronotherapeutic intervention windows; (2) development of tumor rhythm–responsive nanodelivery systems for spatiotemporally precise CHM release; (3) establishment of patient-derived circadian organoid-on-chip and chronotype-matched PDX platforms for multidimensional efficacy-toxicity profiling; and (4) staged clinical trials of chrono-guided CHM nanoformulations. By transforming oncology from static dosing to dynamic circadian modulation, this TCM-inspired paradigm pioneers a spatiotemporal precision medicine strategy that bridges holistic wisdom with cutting-edge chronobiology, thereby advancing the modernization of TCM.

Introduction

Malignant neoplasms represent a formidable global public health burden, necessitating the continuous innovation of therapeutic strategies as a central objective in biomedical research.^1–3 Although conventional chemotherapy and molecularly targeted therapies have markedly augmented patient prognosis, critical clinical barriers, including the development of drug resistance, systemic toxicities, and heterogeneous treatment responses, substantially limit therapeutic efficacy.^4–6 In recent years, the circadian rhythm, an evolutionarily conserved endogenous timekeeping system, has garnered increasing attention due to its profound implications in tumorigenesis and cancer progression.^7,8 This system orchestrates key biological processes, such as cell cycle regulation, DNA damage repair, and immune surveillance, through a core transcriptional-translational feedback loop mediated by the CLOCK/BMAL1 and PER/CRY axes. Notably, tumor-specific dysregulation of this circadian network has been robustly demonstrated to drive malignant transformation, disease advancement, and therapy resistance.^9–13 Consequently, targeting intrinsic circadian pathways in cancer cells to restore their temporal regulatory capacity has emerged as a pivotal strategy in anti-tumor therapy.^14–16

Chinese herbal medicine (CHM), serving as the fundamental material basis of traditional Chinese medicine (TCM), exhibits multidimensional regulatory advantages in cancer intervention through its distinctive network pharmacology characteristics. In contrast to conventional chemotherapeutic agents, CHM is characterized by multi-component synergy and relatively lower systemic toxicity. Its bioactive constituents often demonstrate superior safety profiles while maintaining robust therapeutic efficacy.^17–21 Contemporary research has revealed that specific CHM compounds can precisely modulate time-of-day-dependent cellular behaviors, such as proliferation, apoptosis, and metastasis, by restoring impaired circadian function in tumor cells (as detailed below). This chronomodulation strategy, grounded in tumor circadian modulation, not only effectively suppresses disease progression but also markedly reduces off-target damage to healthy tissues, thereby offering a novel paradigm for deciphering the temporal vulnerabilities of malignancy. Notably, the emergence of chronopharmacology has opened new avenues for synergizing natural products with modern anticancer therapies. By aligning drug administration with the intrinsic circadian phase of the tumor, the inherent advantages of CHM, characterized by a wide therapeutic window and a mild adverse effect profile, can be profoundly amplified to achieve concurrent optimization of efficacy and toxicity control.^22–25 Nevertheless, several critical scientific challenges remain unresolved. These include the insufficient characterization of individualized tumor circadian signatures, the mechanistic complexity of CHM’s multitarget action networks, and technological bottlenecks in delivery systems tailored for circadian-directed tumor intervention.

This article systematically elucidates the pharmacodynamic characteristics and molecular mechanisms through which bioactive constituents of CHM regulate impaired circadian functions and core gene networks within tumor cells, thereby influencing malignant progression. Furthermore, it explores the translational potential of CHM-based anticancer strategies grounded in chronopharmacology theory. To address current research bottlenecks, this review innovatively proposes an integrated framework encompassing multi-omics, intelligent delivery, preclinical validation, and clinical translation. Specifically, the proposed strategy involves: (1) the integration of multi-omics data from cancer patients with dynamic physiological parameters, such as activity rhythms and body temperature fluctuations recorded by wearable devices, combined with artificial intelligence (AI) computational modeling methods, including deep learning and temporal network analysis. This approach aims to construct personalized tumor circadian maps, enabling the precise definition of optimal chronotherapeutic intervention windows (OCIW) and supporting the development of AI-driven systems for circadian subtyping and dosing-time decision-making, thereby providing dynamic, deployable clinical decision support for time-programmed drug delivery; (2) the development of intelligent nanodelivery systems that are simultaneously adaptive to tumor-intrinsic circadian phases and responsive to the tumor microenvironment, facilitating the spatiotemporally precise controlled release of CHM bioactive constituents and enabling circadian-synchronized therapeutic intervention; (3) the establishment of parallel preclinical evaluation platforms based on patient-derived tumor-immune organoid-on-chip systems that simulate dynamic circadian rhythms, alongside rhythm-matched patient-derived xenograft (PDX) models, to conduct comprehensive multidimensional assessments of antitumor efficacy, immune modulation, circadian resynchronization effects, and toxicity profiles; and (4) the implementation of a staged clinical translation strategy through multicenter trials to validate the clinical benefit of chrono-tailored CHM nanoformulations. This final phase prioritizes conducting multicenter Phase I/II trials to evaluate the safety and preliminary efficacy of chrono-tailored CHM nanoformulations administered within individualized OCIW. Moreover, it emphasizes establishing clinical trial paradigms aligned with the CONSORT 2025 statement to assess how circadian-synchronized interventions contribute to tumor microenvironment remodeling and immune activation, while exploring specific biomarkers such as time-dependent intratumoral enrichment and circadian synchronization metrics of CD8⁺ T cell infiltration as predictors of treatment response. Ultimately, this strategy aims to advance an integrated, interdisciplinary oncology pathway that unifies tumor circadian diagnostics, intelligent nanodelivery, and chrono-precise therapy, thereby forging a novel paradigm at the intersection of TCM and modern biomedical science.

Core Role of Circadian Rhythm Regulation in Tumor Development and CHM Intervention

The Core Circadian Regulatory Network: From Physiological Homeostasis to Disease Pathogenesis

Circadian rhythms constitute an endogenous timing mechanism evolved to synchronize with the Earth’s rotational cycles. This system orchestrates physiological functions and metabolic homeostasis via periodic oscillations in gene expression. This intricate temporal regulatory network is indispensable for maintaining health, substantially governing critical processes such as sleep-wake cycles, aging, cognitive function, metabolic balance, motor coordination, and thermoregulation. Disruption of this network is notably implicated in the pathogenesis of diverse diseases, including metabolic disorders (eg, type 2 diabetes and obesity), cardiovascular diseases (eg, hypertension and atherosclerosis), neuropsychiatric conditions (eg, depression and Alzheimer’s disease), and immune dysfunctions.^26–32 Particularly noteworthy is the marked association between molecular clock perturbations and malignancies, which has emerged as a prominent frontier in contemporary medical research^33–38 (Figure 1).

Figure 1.

The circadian regulatory network in physiological homeostasis and disease. The central pacemaker in the suprachiasmatic nucleus (SCN) synchronizes peripheral oscillators via neural and hormonal signals to regulate key physiological processes, including metabolism, sleep-wake cycles, and cognition. Disruption of this rhythmic system contributes to the pathogenesis of multisystem disorders such as cancer, metabolic diseases, cardiovascular conditions, and neurodegeneration. (The figure is drawn by Figdraw).

In mammals, the core regulatory mechanism of the circadian clock is governed by an elaborate transcription-translation feedback loop involving both core and auxiliary regulatory genes. Environmental cues, such as light, diet, and temperature, modulate this system through neural signaling and hormonal secretion. Within the primary circadian loop, CLOCK and BMAL1 proteins form heterodimers that specifically bind to E-box elements in the promoters of PER and CRY genes, thereby activating their transcription.³⁹ Subsequently, the transcribed mRNAs are translated into PER and CRY proteins, which accumulate in the cytoplasm and assemble into complexes.⁴⁰ Upon reaching a critical threshold, these complexes translocate into the nucleus to inhibit the transcriptional activity of the BMAL1–CLOCK heterodimer, thus establishing the core negative feedback loop. Concurrently, auxiliary regulatory genes REV-ERB and ROR encode proteins that fine-tune BMAL1 expression by competitively binding to ROR response elements (ROREs) in its promoter. Specifically, REV-ERB represses transcription, whereas ROR enhances it. This secondary auxiliary feedback loop operates synergistically to maintain the precision and robustness of the circadian timing system^41,42 (Figure 2).

Figure 2.

The molecular mechanism and hierarchical outputs of the mammalian circadian clock. The core oscillator relies on a transcription-translation negative feedback loop driven by CLOCK/BMAL1 activators and PER/CRY repressors, with auxiliary REV-ERB/ROR loops fine-tuning rhythmic precision. This molecular machinery hierarchically regulates biological rhythms across cellular, tissue, and organismal levels, translating gene expression cycles into physiological phenotypes. (The figure is drawn by Figdraw).

Multidimensional Mechanisms Driving Tumor Progression Through Circadian Disruption

Circadian disruption operates through a central axis comprising the circadian gene network, metabolic reprogramming, and DNA repair mechanisms. This perturbation concurrently triggers multiple pathological pathways, including DNA repair deficiencies,⁴³ dysregulation of G1/S and G2/M checkpoints,^44,45 expansion of cancer stem cells,^46,47 metabolic reprogramming,^48,49 loosening of epigenetic controls,^50,51 intestinal microbiome imbalance,^52–56 attenuation of T-Cell Receptor (TCR)-Major Histocompatibility Complex Class I (MHC-I) immune surveillance,^57–59 and uncontrolled angiogenesis^60,61 (Figure 3). Studies have demonstrated that BMAL1 and CLOCK can directly bind to DNA double-strand break sites, recruiting repair factors to promote homologous recombination. Their downregulation not only leads to the accumulation of DNA damage in glioblastoma and liver cancer but also markedly impairs the therapeutic response to temozolomide and cisplatin.^62,63 In glioblastoma, CLOCK is preferentially overexpressed in glioma stem-like cells (GSCs), where it sustains self-renewal and blocks differentiation, both of which are essential for tumor maintenance and recurrence. Additionally, CLOCK drives aberrant angiogenesis by activating the HIF-1α/VEGF pathway, whereas targeted inhibition of CLOCK effectively disrupts both the vascular niche and GSCs propagation.^64,65 Furthermore, circadian disruption attenuates MHC-I antigen presentation and CD8⁺ T-cell infiltration, thereby weakening TCR-MHC-I immune surveillance and promoting the accumulation of myeloid-derived suppressor cells (MDSCs) at metastatic sites. Notably, this immunosuppressive microenvironment is closely associated with gut microbiota dysbiosis, which further exacerbates systemic inflammation and supports tumor progression.^66,67 In hepatocellular carcinoma, hypermethylation-mediated silencing of PER1 derepresses DNA polymerase β, thereby enhancing tumor stemness and invasiveness.⁶³ On the metabolic front, circadian perturbation induces substantial upregulation of lipogenic enzymes such as fatty acid synthase, rewiring tumor energy metabolism to support rapid proliferation.⁶⁸ Collectively, these findings reveal that the circadian–metabolic–DNA repair axis integrates genomic stability, stemness maintenance, immune evasion, and microenvironmental remodeling into a highly coordinated oncogenic hub.

Figure 3.

Multidimensional mechanisms by which circadian disruption drives malignant tumor progression. This figure centers on the core circadian clock and illustrates eight key pathological processes triggered by its dysregulation: DNA damage repair defects, cell cycle dysregulation, propagation of cancer stem cells, metabolic reprogramming, promotion of angiogenesis, decreased immune surveillance, aberrant epigenetic regulation, and gut dysbiosis. These pathways are functionally interconnected, forming a synergistic network that contributes to tumorigenesis. Each sector visually represents a specific biological process: DNA double-strand breaks indicate impaired repair; cell cycle diagrams show loss of G1/S and G2/M checkpoint control; uncontrolled proliferation of cancer stem cells indicates their propagation; mitochondrial metabolism and biosynthetic fluxes reflect metabolic rewiring; new blood vessel formation signifies angiogenic activation; disrupted TCR-MHC-I interaction highlights immune evasion; chromatin and epigenetic marks illustrate regulatory abnormalities; and altered gut microbiota composition symbolizes dysbiosis. The overall design underscores the central role of the circadian system as a master regulator in cancer development, providing an integrated framework for understanding the multifactorial nature of malignancy. (The figure is drawn by Figdraw).

Antitumor Potential of CHM Active Compounds as Circadian Modulators

TCM, with CHM as its foundational pillar, encompasses a rich repertoire of active constituents, including alkaloids, flavonoids, terpenoids, and polysaccharides. These bioactive components form a complex pharmacological network that exhibits unique advantages in holistic regulation during the treatment of malignant tumors.⁶⁹ Contemporary research indicates that these active compounds exert their therapeutic effects through synergistic mechanisms involving multiple targets, pathways, and hierarchical levels, thereby intervening in several critical stages of tumor development. On one hand, they directly suppress tumor growth by regulating the cell cycle, inducing apoptosis, and inhibiting angiogenesis. On the other hand, they indirectly augment therapeutic outcomes by modulating the tumor microenvironment and enhancing immune function.^70,71

Notably, accumulating evidence suggests that specific CHM-derived active compounds, such as flavonoids and terpenoids, can directly or indirectly regulate the expression and oscillation of core circadian genes (eg, CLOCK, BMAL1, PER, and CRY) within tumor cells. This modulation profoundly influences the temporal biological behaviors of tumor cells, as further elaborated below. This chronobiotic capacity not only endows CHM with its well-recognized multi-target pharmacological profile but also positions it as a potential circadian modulator capable of temporally synchronizing key antitumor processes, including DNA repair, metabolic rhythms, and immune surveillance. Such a network-based mode of action overcomes the limitations of single-target agents, which are highly susceptible to inducing drug resistance, and aligns remarkably well with the intricate pathophysiological landscape of malignancies.

Of particular interest is the complementary pharmacological spectrum established by diverse active components of CHM. Through target complementarity and pathway cross-regulation, these constituents generate integrated therapeutic effects. When this synergy extends to the regulation of tumor rhythms, it enables the precise targeting of temporal vulnerabilities within the tumor, thereby expanding the therapeutic window and substantially reducing systemic toxicity. Moreover, the multi-target nature of CHM helps mitigate adverse effects, offering novel avenues for developing highly effective and low-toxicity antitumor therapeutics. Ultimately, this dual capability of spatial multi-target regulation and temporal rhythmic modulation confers unique value and substantial potential upon CHM in comprehensive cancer therapy.

Advances and Translational Prospects of Chronopharmacological Anticancer Strategies Based on CHM

Molecular Mechanisms Underlying the Time-Dependent Antitumor Effects of CHM Bioactive Compounds via Circadian Pathway Modulation

Recent investigations have elucidated that bioactive constituents derived from CHM exert multilayered regulatory influences on the endogenous circadian machinery within tumor cells through both direct and indirect pathways. On one hand, specific components, particularly flavonoids and terpenoids, directly target core clock genes that are frequently dysregulated in malignancies, including CLOCK, BMAL1, PER, and CRY. By modulating transcriptional activity, protein stability, or nucleocytoplasmic shuttling dynamics, these compounds effectively re-establish tumor cell-specific circadian oscillations. On the other hand, certain agents, such as paclitaxel, do not directly perturb clock gene expression yet display markedly time-dependent efficacy. This phenomenon suggests that the local circadian apparatus in tumors governs the rhythmic expression of drug-metabolizing enzymes and molecular targets, thereby defining temporal windows of drug sensitivity.

Notably, these bioactive compounds not only restore intrinsic circadian function within tumor cells but also substantially enhance chemosensitivity by synchronizing circadian phases across heterogeneous tumor cell populations. Furthermore, they possess the capacity to modulate the rhythmic activity of immune cells, thereby remodeling the immunological landscape of the tumor microenvironment. This multilevel and multiscale regulatory capability, synergized with the inherent polypharmacological nature of natural compounds, facilitates precise intervention in the disrupted circadian network at molecular, cellular, and tissue levels. Collectively, these attributes provide a robust pharmacological basis and novel conceptual paradigms for the development of chronotherapeutic strategies centered on circadian reprogramming in oncology. Representative TCM-derived bioactive compounds exhibiting circadian-modulating activities and their chemical structures are depicted in Figure 4, while their botanical origins, modes of circadian modulation, core molecular targets, and key mechanisms of action are summarized in Table 1.

Figure 4.

Chemical structures of representative bioactive compounds derived from traditional Chinese medicinal sources with potential circadian-modulating activities. The panel includes diverse natural molecules such as flavonoids (epigallocatechin-3-gallate, nobiletin), polyphenols (curcumin, resveratrol), amino acids (L-theanine), nucleosides (cordycepin), alkaloids (piperine, capsaicin), and the anticancer agent paclitaxel. All structures are rendered using standardized chemical notation to clearly depict their core molecular frameworks.

Table 1.

Chrono-Modulatory TCM-Derived Bioactive Compounds and Their Mechanisms in Cancer

TCM-Derived Bioactive Compounds	Botanical Source	Circadian Modulation Type	Circadian-Regulatory Molecular Targets	Key Mechanisms and References
Epigallocatechin‑3‑gallate	Camellia sinensis K.	Direct	BMAL1, CLOCK	Restores BMAL1 oscillation in HepG2 cells under metabolic stress;72 suppresses CLOCK in lung cancer stem cells to inhibit Wnt/β-catenin signaling73
L-Theanine	Camellia sinensis K.	Direct	BMAL1, p53	Upregulates BMAL1 to activate p53-dependent apoptosis and cell cycle arrest; suppresses melanoma progression and metastasis74
Curcumin	Curcuma longa L.	Direct	BMAL1, PER2, CLOCK, SIRT1	Enhances BMAL1 via PPARγ;75,76 induces PER2-dependent apoptosis in glioma with circadian timing;77 activates SIRT1 to modulate CLOCK-BMAL1 activity77
Melatonin	Ziziphus jujuba Mill. var. spinosa	Indirect	BMAL1, CLOCK, PER2, CRY, RORα, DBP	Upregulates PER2/CLOCK and suppresses BMAL1 in breast and prostate cancer; enhances PER/CRY expression to reinforce circadian repressor complex function78,79
Nobiletin	Citrus reticulata Blanco	Direct	BMAL1, RORα, HDAC3, SIRT1, FOXO3a	Suppresses PD-L1 via RORα–HDAC3 binding to the CD274 promoter, potentiating anti-CTLA-4 immunotherapy through GZMB⁺ CD8⁺ T cells;80 acts as an RORα agonist to enhance BMAL1 transcription;81,82
Mangiferin	Mangifera indica L., Anemarrhena asphodeloides Bunge	Direct	PER1	Reverses LPS-induced PER1 downregulation in NSCLC; inhibits migration via PER1 and blocks NLRP3 inflammasome/IL-1β axis83
Resveratrol	Vitis vinifera L., Arachis hypogaea L., Polygonum cuspidatum Siebold and Zucc.	Direct	BMAL1, CLOCK, SIRT1, REV-ERBα/RORγ, PER1/2	Activates SIRT1 to deacetylate BMAL1, enhancing CLOCK: BMAL1 activity;84 restores CD8+ T-cell infiltration and Th17/Treg balance85
Vanillic acid	Vanilla planifolia Jacks. ex Andrews, Olea europaea L.	Indirect	Antioxidant enzymes (SOD, CAT, GPx, GSH)	Restores circadian rhythmicity of redox markers (TBARS, SOD) in endometrial cancer; indirect clock output regulation via ROS scavenging86
Cordycepin	Ophiocordyceps sinensis (Berk.) G.H. Sung et al, Cordyceps militaris (L.) Fr.	Direct	BMAL1, RUVBL2	Targets RUVBL2 to reset BMAL1-driven circadian oscillator; enhances CD80 expression in dendritic cells for timed anti-tumor immunity87
Piperine	Piper nigrum L.	Direct	BMAL1, CLOCK, TRPV1	Upregulates BMAL1/CLOCK via TRPV1 activation in HepG2 cells; links circadian restoration to metabolic reprogramming in Hepatocellular Carcinoma88,89
Genistein	Glycine max (L.) Merr.	Indirect	PER1, SRC, GSK-3β	Inhibits noradrenaline-induced PER1 upregulation by blocking SRC–GSK-3β pathway; potential stabilization of glioma circadian rhythm90
Capsaicin	Capsicum annuum L.	Direct	BMAL1, CLOCK, PER1/2, CRY1/2, REV-ERBα	Reverses lipid-induced circadian gene dysregulation in HepG2; BMAL1 knockdown abolishes its anti-lipid effect, suggesting clock dependence91
Paclitaxel	Taxus brevifolia Nutt.	Indirect	BMAL1, EZH2, TERT	BMAL1 recruits EZH2 to suppress TERT, enhancing paclitaxel sensitivity; maximal efficacy at BMAL1 peak (ZT6–ZT10) in tongue cancer92

Epigallocatechin‑3‑gallate (EGCG)

EGCG, the predominant bioactive polyphenol in Camellia sinensis K., has attracted considerable scientific attention in recent years due to its extensive spectrum of biological activities. Accumulating evidence indicates that EGCG exerts dual therapeutic potential in ameliorating metabolic disorders and suppressing tumorigenesis by modulating the core circadian gene BMAL1. In hepatocellular carcinoma HepG2 cells, EGCG effectively reverses the suppression of BMAL1 expression induced by high-glucose or high-fat conditions, thereby restoring its circadian oscillation. This restoration subsequently activates the AMPK/PI3K-AKT signaling axis, promoting glucose uptake and glycogen synthesis, and ultimately alleviating insulin resistance.⁷² Notably, BMAL1, a pivotal node within the circadian regulatory network, is intimately associated with oncogenesis when dysregulated. In a complementary study, EGCG was shown to markedly downregulate the expression of another core clock gene, CLOCK, in lung cancer stem cells. This inhibition substantially reduced the proportion of CD133⁺ cells and disrupted the Wnt/β-catenin signaling pathway, thereby impairing the self-renewal capacity of tumor-initiating cells.⁷³ Collectively, these findings suggest that EGCG may function as a multitarget chronotherapeutic agent by coordinately modulating key clock genes such as BMAL1 and CLOCK. Its underlying mechanisms involve the restoration of mitochondrial function, suppression of oxidative stress, and regulation of cancer stem cell properties. Future investigations should aim to delineate the precise regulatory networks through which EGCG operates within the circadian–metabolism–tumor axis, thereby providing a robust theoretical foundation for its clinical translation.

L-Theanine

L-Theanine, a non-proteinogenic amino acid found in Camellia sinensis K., accounts for approximately 1–2% of the dry leaf weight and is characterized by notable biological activity coupled with an exceptional safety profile. Recent investigations have demonstrated that L-theanine exerts selective antitumor effects against melanoma by targeting the core circadian machinery.⁷⁴ Specifically, in both human A375 and murine B16-F10 melanoma cell lines, L-theanine substantially upregulates the expression of BMAL1, a pivotal circadian regulator, without inducing cytotoxicity in normal human melanocytes. Mechanistically, this compound enhances the transcriptional activity of the tumor suppressor p53 in a BMAL1-dependent manner, as validated by dual-luciferase reporter and Co-IP assays. Furthermore, L-theanine treatment disrupts cell cycle progression by downregulating Cyclin D1 and CDK2/4 (leading to G1/S arrest) and suppresses metastatic potential via inhibition of MMP2/9 and ICAM-1. These findings not only elucidate a novel chronobiological mechanism involving BMAL1-p53 activation but also provide a robust rationale for natural product-based chronotherapeutic strategies. Future inquiries should focus on validating these in vitro findings in systemic in vivo models to assess the translational potential of L-theanine in circadian-synchronized cancer therapy.

Curcumin

Curcumin, a polyphenolic bioactive compound derived from the rhizomes of Curcuma longa L., exhibits broad-spectrum antitumor activity. Recent investigations have demonstrated that curcumin exerts its anticancer effects, at least in part, through the modulation of circadian clock genes.⁷⁵ Specifically, curcumin markedly upregulates the expression of BMAL1, leading to activation of the PPARγ pathway and subsequent inhibition of Wnt/β-catenin signaling, thereby suppressing tumor cell proliferation.⁷⁶ In glioblastoma, curcumin modulates PER2 expression, inducing cell cycle arrest and apoptosis in a manner that displays pronounced circadian rhythmicity. Experimental evidence indicates that in C6 glioma cells treated with 5 μM curcumin, the peak of apoptosis occurs 6 to 11 hours before the maximal expression of PER2 protein, illustrating a robust temporal coupling between curcumin’s efficacy and the phase of the molecular clock. Furthermore, curcumin activates SIRT1, which in turn regulates the CLOCK–BMAL1 heterodimer and promotes deacetylation and degradation of PER2 protein, ultimately impairing the self-renewal capacity of cancer stem cells.⁷⁵ Notably, two curcumin analogs, demethoxycurcumin and bisdemethoxycurcumin, exhibit enhanced metabolic stability and may exert more sustained circadian-modulating effects within tumor tissues.⁷⁷ Collectively, these findings provide novel insights into chronotherapy-based oncology and suggest that synchronizing the administration of curcumin or its derivatives with endogenous circadian rhythms could substantially enhance their antitumor efficacy.^75,77

Melatonin

Melatonin, chemically designated as N-acetyl-5-methoxytryptamine, is an indoleamine hormone ubiquitous across diverse organisms. Although primarily recognized as an animal neurohormone synthesized within the pineal gland, its identification in the traditional medicinal plant Ziziphus jujuba Mill. var. spinosa establishes it as a biologically active component relevant to TCM-derived substances. Recent investigations have elucidated that melatonin exerts multifaceted antitumor effects by reprogramming the rhythmic expression of core circadian clock genes, such as PER2, CLOCK, and BMAL1. In both breast and prostate malignancies, melatonin markedly upregulates PER2 transcription while concurrently suppressing BMAL1 transcription, thereby rectifying a dysregulated circadian network.^78,79 Notably, in prostate cancer cells, melatonin further enhances CLOCK expression, cooperatively reinforcing the integrity of the molecular oscillator.⁷⁸ Mechanistically, this chronobiotic action involves the inhibition of positive regulators like RORα, which consequently attenuates BMAL1-driven pro-proliferative signaling.⁷⁹ Clinically, nocturnal melatonin secretion peaks are substantially diminished in breast cancer patients, accompanied by a marked reduction in circadian amplitude.⁹³ Importantly, exogenous melatonin administration has been shown in vitro to re-establish robust oscillatory patterns of circadian output genes, including DBP.⁷⁸ Collectively, these findings provide novel insights into circadian-based chronotherapeutic strategies for oncology.

Nobiletin

Nobiletin, a polymethoxylated flavonoid characterized by profound biological activity, is abundantly present in the peel of Citrus reticulata Blanco.⁹⁴ Recent investigations have elucidated that nobiletin exerts robust antitumor effects by modulating the core circadian gene network.^80,81,94 Functioning as a natural agonist of RORα, nobiletin markedly enhances the transcriptional activity of the core clock gene BMAL1, thereby stabilizing the circadian oscillator and ameliorating metabolic dysregulation.^81,82 Within the tumor microenvironment, nobiletin facilitates the assembly of an RORα-HDAC3 corepressor complex that specifically binds to the ROR response element (ATAAAGGTTA) in the CD274 promoter, leading to the transcriptional repression of PD-L1.⁸⁰ This regulatory mechanism is critically dependent on competitive interactions at the RORα ligand-binding domain: the overexpression of DDX3X displaces nobiletin from RORα, destabilizes the RORα–HDAC3 complex, and consequently relieves PD-L1 suppression, ultimately impairing CD8⁺ T cell-mediated antitumor immunity. In melanoma models, nobiletin substantially potentiates the efficacy of anti-CTLA-4 immunotherapy by increasing the proportion of tumor-infiltrating GZMB⁺ CD8⁺ T cells, an effect mediated through multilayered circadian regulation of the cancer-immunity cycle. Moreover, nobiletin activates the SIRT1/FOXO3a signaling axis to promote mitochondrial biogenesis and restore rhythmic metabolic function in skeletal muscle, providing a novel therapeutic avenue for malignancies associated with circadian and metabolic disruption.⁸¹

Mangiferin

Mangiferin, a C-glycosyl xanthone derivative (frequently classified as a C-glycosyl flavonoid) isolated from medicinal plants such as Mangifera indica L. and Anemarrhena asphodeloides Bunge, exhibits profound antitumor activity. Recent investigations have demonstrated that mangiferin suppresses the malignant progression of non-small cell lung cancer (NSCLC) by modulating the core circadian gene PER1.⁸³ Within a lipopolysaccharide (LPS)-induced tumor microenvironment, treatment with 25 μg/mL mangiferin substantially reversed the LPS-mediated downregulation of PER1 expression (P < 0.01). This functional significance was corroborated by PER1-siRNA knockdown experiments, which revealed that the loss of PER1 markedly enhanced tumor cell migration by 42%. Mechanistic studies elucidated that mangiferin operates via a dual regulatory axis: first, it upregulates PER1 expression, leading to the suppression of downstream cell cycle regulators; second, it inhibits the activation of the NLRP3 inflammasome, resulting in a 68% reduction in IL-1β secretion as quantified by ELISA. Notably, mangiferin also ameliorates LPS-induced mitochondrial membrane potential depolarization. JC-1 fluorescence assays indicated that the red-to-green fluorescence ratio was restored to 85% of that observed in untreated control cells, underscoring its protective role in preserving mitochondrial integrity. Collectively, these findings uncover a novel antimetastatic strategy wherein mangiferin concurrently targets circadian gene regulation and inflammatory microenvironment remodeling. This dual mechanism provides a compelling theoretical foundation for the development of natural product-based chrono-chemotherapeutic approaches in oncology.

Resveratrol

Resveratrol, chemically designated as 3,5,4’-trihydroxystilbene, is a polyphenolic compound widely distributed in botanical species such as Vitis vinifera L., Arachis hypogaea L., and Polygonum cuspidatum Siebold and Zucc. Recent investigations have demonstrated that resveratrol exerts robust antitumor effects by modulating the core circadian gene network.⁸⁴ Its primary molecular mechanism entails the activation of the SIRT1–BMAL1/CLOCK signaling axis. Specifically, resveratrol upregulates the expression of SIRT1, which is an NAD⁺-dependent deacetylase that directly deacetylates BMAL1. This process substantially enhances the transcriptional activity of the BMAL1/CLOCK heterodimer toward downstream clock genes (PER1/2) and cryptochrome genes (CRY1/2). Within the tumor microenvironment, this circadian reprogramming restores immune cell dysfunction induced by rhythm disruption. Notably, it achieves this by suppressing the accumulation of MDSCs and promoting CD8⁺ T cell infiltration, thereby reinforcing antitumor immunity.⁸⁵ Furthermore, resveratrol modulates the Th17/Treg balance via the REV-ERBα/RORγ nuclear receptor pathway and mitigates CXCL5–CXCR2 axis-mediated tumor metastasis. In breast cancer models, nocturnal administration of resveratrol markedly reduces tumor incidence. This chronopharmacological effect is intricately linked to the synchronization of endogenous PER2 protein oscillations and melatonin secretion rhythms.⁸⁴ Collectively, these findings characterize resveratrol as a potent chronobiotic agent that targets the circadian machinery to orchestrate antitumor immunity and suppress metastasis, thereby providing novel molecular targets for circadian rhythm-guided chronotherapy in oncology.

Vanillic Acid

Vanillic acid, a phenolic derivative ubiquitously distributed across various edible plants and fruits, is predominantly derived from Vanilla planifolia Jacks. ex Andrews and Olea europaea L. In an MNNG-induced rat model of endometrial carcinoma, vanillic acid substantially ameliorates the circadian disruption of redox homeostasis.⁸⁶ Specifically, in both pretreatment and co-treatment groups, the median levels of lipid peroxidation products, namely thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides, declined from 7.3 and 6.0 to 2.0 and 3.7 nmol/dL, respectively. Concurrently, their peak phases shifted back from 10:20 and 16:07 to 03:51 and 14:22. Furthermore, vanillic acid markedly enhances both the median levels and the rhythmic amplitudes of the antioxidant defense system. This encompasses enzymatic antioxidants, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), alongside non-enzymatic antioxidants such as glutathione (GSH), vitamin C, and vitamin E. Notably, the acrophase of SOD activity is restored from 11:52 to 14:04. However, a key limitation of this study is the absence of direct evaluation of core clock gene expression. Consequently, the observed circadian modulation should be characterized as an indirect regulatory mechanism. The rhythmic normalization of redox parameters suggests that vanillic acid preserves redox balance, likely by modulating circadian output pathways or downstream clock-controlled genes, rather than targeting the central oscillator directly. This indirect chronomodulatory effect is mediated by the intrinsic antioxidant properties of vanillic acid, which scavenge MNNG-induced free radicals and mitigate lipid peroxidation damage. Collectively, these findings provide preliminary evidence that natural compounds like vanillic acid can exert antitumor effects through the modulation of circadian-regulated redox pathways.

Cordycepin

Cordycepin is predominantly isolated from the traditional medicinal fungi, including Ophiocordyceps sinensis (Berk.) G.H. Sung et al and Cordyceps militaris (L.) Fr., and is characterized by profound biological activity. Recent investigations have elucidated that cordycepin and its analogs, including N6-benzyladenosine and 3-deazaadenosine, exert robust antitumor effects by modulating core circadian clock genes.⁸⁷ Specifically, cordycepin targets the RUVBL2 protein, which directly interacts with BMAL1, a central component of the mammalian circadian machinery. This interaction promotes the disassembly of the circadian supercomplex, thereby initiating a new cycle of clock oscillation. This mechanism holds substantial implications for antitumor immunity: BMAL1 binds to the promoter region of the CD80 gene in dendritic cells, driving the rhythmic expression of CD80 and consequently shaping the temporal dynamics of antigen presentation and CD8⁺ T cell-mediated antitumor responses. In the B16-F10 melanoma mouse model, the loss of BMAL1 abolishes the time-dependent efficacy of immunotherapy, whereas cordycepin-mediated circadian resetting may optimize the therapeutic window for immune intervention. Notably, retrospective clinical analyses indicate that circadian-timed vaccination, particularly when administered in the morning, markedly improves treatment outcomes in patients with advanced melanoma. This underscores the potential of cordycepin as a chronobiotic agent to be combined with immunotherapy. Nevertheless, the precise molecular mechanisms of cordycepin in humans and its optimal dosing schedule remain to be fully elucidated.

Piperine

Piperine, the principal alkaloid constituent of the fruits of Piper nigrum L., has recently emerged as a potential modulator of circadian clock genes. In human hepatocellular carcinoma HepG2 cells, piperine markedly upregulates the mRNA and protein expression of the core circadian genes BMAL1 and CLOCK, thereby ameliorating metabolic disturbances induced by lipotoxicity or high-glucose conditions.^88,89 Mechanistic investigations indicate that the piperine-mediated regulation of BMAL1 is partially dependent on the activation of the transient receptor potential vanilloid 1 (TRPV1) channel, which subsequently modulates downstream calcium signaling and metabolic pathways.⁸⁹ Notably, BMAL1 is frequently downregulated in hepatocellular carcinoma, and its functional deficiency is intricately linked to dysregulated lipid and glucose metabolism, elevated oxidative stress, and uncontrolled cell proliferation. Restoring BMAL1 expression has been shown to suppress tumor growth and enhance chemosensitivity.^88,89 Thus, by targeting the TRPV1–BMAL1/CLOCK axis to reestablish circadian homeostasis, piperine not only improves metabolic phenotypes but may also indirectly exert antitumor effects through the correction of clock gene-driven metabolic reprogramming. Although this hypothesis has not yet been fully validated in direct tumor phenotype assays, it provides a compelling mechanistic link supporting the role of natural compounds in modulating the “circadian–metabolic–tumor” axis.

Genistein

Noradrenaline substantially induces the transient expression of the clock gene PER1 in C6 glioma cells via the activation of β2-adrenergic receptors. This process relies on two parallel signaling cascades: the PKA-CREB and SRC-GSK-3β pathways. Genistein, a natural isoflavone derived from Glycine max (L.) Merr., functions as a broad-spectrum tyrosine kinase inhibitor. It effectively blocks SRC phosphorylation and the subsequent activation of GSK-3β at Tyr216, thereby markedly suppressing noradrenaline-induced PER1 upregulation.⁹⁰ Although this study does not directly evaluate antitumor phenotypes, PER1 is widely recognized as a tumor suppressor in multiple malignancies, where it governs cell cycle progression and DNA repair. Aberrant perturbation of PER1 expression by neurotransmitters such as noradrenaline may therefore disrupt circadian homeostasis in glioma cells and influence their malignant behavior. Consequently, genistein may exert an indirect antitumor effect by stabilizing the circadian signaling network and antagonizing rhythm disruption induced by neurogenic stimuli within the tumor microenvironment. While this hypothesis is biologically plausible, its validation necessitates further clarification of the precise functional role of PER1 in glioma and definitive evidence of genistein’s chronomodulatory effects in vivo.

Capsaicin

Capsaicin, the principal pungent bioactive compound derived from the fruits of Capsicum annuum L., exhibits a broad spectrum of pharmacological activities. Recent investigations have revealed that capsaicin modulates core circadian clock genes to ameliorate metabolic dysregulation and potentially influence tumor-related signaling pathways.⁹¹ In human hepatocellular carcinoma HepG2 cells, treatment with 50 μM capsaicin substantially alleviates oleic acid-induced lipid accumulation and restores the disrupted rhythmic expression of key circadian genes, including BMAL1, CLOCK, PER1/2, CRY1/2, and REV-ERBα, at both the mRNA and protein levels. Notably, siRNA-mediated knockdown of BMAL1 markedly attenuates the inhibitory effect of capsaicin on lipid accumulation, indicating that its metabolic regulatory action is contingent upon the functional integrity of BMAL1. Although this study primarily focuses on lipid metabolism, core clock components such as BMAL1 are known to exert context-dependent tumor-suppressive or oncogenic functions across various malignancies. Furthermore, signaling pathways modulated by the circadian clock, such as Wnt/β-catenin and AKT/mTOR, are well-established drivers of tumorigenesis. Collectively, these findings indirectly suggest that capsaicin may influence cancer cell metabolism and proliferation by reprogramming the circadian network, thereby positioning it as a potential chronobiotic agent for circadian-targeted cancer intervention.

Paclitaxel

While widely established as a cornerstone chemotherapeutic agent, Paclitaxel is fundamentally a diterpenoid compound originally isolated from the bark of Taxus brevifolia Nutt., representing a quintessential example of a plant-derived natural product with profound implications for chronotherapy. As a classical microtubule-stabilizing agent, its antitumor activity has recently garnered considerable attention due to its intricate connection with circadian clock gene regulation. Evidence demonstrates that the efficacy of paclitaxel exhibits pronounced chronodependence, a phenomenon closely linked to BMAL1.⁹² In tongue squamous cell carcinoma models, BMAL1 recruits EZH2 to the promoter region of telomerase reverse transcriptase (TERT) spanning positions −1857 to −1590 bp. This interaction forms a transcriptional repressor complex that substantially downregulates TERT expression (P < 0.01). Such repression induces S-phase cell cycle arrest (increased by 35%) and promotes apoptosis, as evidenced by a 2.1-fold elevation in the BAX/Bcl-2 ratio. Consequently, tumors with BMAL1 overexpression exhibit a 3.2-fold heightened sensitivity to paclitaxel. Preclinical investigations reveal that administering paclitaxel during the peak expression window of BMAL1 (ZT6 to ZT10) improves tumor growth inhibition by 58% compared with administration during the trough phase (ZT18 to ZT22) in xenograft models of tongue squamous cell carcinoma (P < 0.01). Notably, the reduction in tumor volume strongly correlates with fluctuations in BMAL1 mRNA levels (r = 0.82). Collectively, these findings uncover a novel mechanism by which paclitaxel enhances antitumor efficacy via the BMAL1–EZH2–TERT axis, providing a molecular rationale for chrono-optimized paclitaxel therapy guided by circadian rhythms.

Despite the promising evidence summarized above, it is crucial to acknowledge a significant caveat in the current body of research. A substantial proportion of studies rely on static measurements of clock gene expression (eg, mRNA or protein levels at a single time point) rather than rigorous validation of restored circadian oscillation (eg, period, phase, and amplitude analysis over a 24–48 hour cycle).

Furthermore, distinguishing the direct “circadian reprogramming effect” from the indirect “antitumor effect” remains a formidable challenge. Many CHM compounds possess potent intrinsic activities, such as anti-inflammatory, antioxidant, or metabolic regulatory properties. These activities may secondarily influence the expression or stability of clock proteins rather than directly targeting the core oscillator. Therefore, future investigations must employ dynamic circadian reporter systems to determine whether these compounds act as true zeitgebers (entraining agents) or merely modulate clock-controlled outputs as a consequence of their general pharmacological actions.

Chrono-Synchronized Nanodelivery System for CHM Based on Personalized Tumor Circadian Profiling

To achieve precise chronotherapeutic intervention with active constituents of CHM within the optimal time window, the primary prerequisite is accurate identification of the intrinsic circadian phase of an individual’s tumor. Conventional dosing strategies based on behavioral rhythms, such as sleep and wake cycles, or peripheral blood biomarkers, often fail to reflect the true state of the local circadian clock within the tumor. To address this limitation, there is an urgent need to establish a patient-centered, personalized tumor circadian profile. This profile integrates multi-omics data, including transcriptomic, genomic, proteomic, epigenomic, and metabolomic profiles from tumor tissues, with dynamic physiological monitoring (for example, wearable devices that record locomotor activity rhythms) and clinical pathological features. Advanced AI approaches, such as deep learning and time series network analysis, are then applied to decode the oscillatory patterns of core clock genes (for instance, CLOCK, BMAL1, PER2) within the tumor microenvironment, thereby enabling precise delineation of each patient’s OCIW. Beyond serving as a decision support tool for chronotherapy, this circadian profile functions as the core input signal for intelligent drug delivery systems. Guided by the individualized OCIW, time-programmed drug release strategies can be engineered to synchronize therapeutic delivery with the tumor’s intrinsic rhythm. Specifically, administration timing is anchored to the most therapeutically sensitive phase within the standardized circadian time coordinate system (CT0 to CT24), ensuring dynamic alignment between intervention timing and the tumor’s vulnerability window (Figure 5).

Figure 5.

Schematic illustration of a chrono-synchronized nanodelivery system for bioactive compounds derived from TCM based on Personalized tumor circadian profiling. This approach integrates multi-omics data from tumor tissues, including transcriptomic, genomic, proteomic, epigenomic, and metabolomic profiles, with dynamic physiological monitoring such as locomotor activity rhythms captured via wearable devices to construct a patient-specific circadian clock landscape. Subsequently, AI algorithms, notably deep learning and time-series network analysis, are employed to decode the oscillatory patterns of core clock genes within the tumor microenvironment, enabling robust identification of the OCIW for each individual. Guided by this Personalized OCIW, a time-programmed drug release strategy is implemented, anchoring therapeutic administration to the most sensitive phase within the standardized circadian time coordinate system (CT0–CT24). This ensures dynamic alignment between treatment timing and the intrinsic vulnerability window of the tumor, thereby providing a technological framework for precision chronotherapy. (The figure is drawn by Figdraw).

However, even with the identification of optimal intervention timing, active components of CHM still face common challenges in practical applications, including poor solubility, low bioavailability, inadequate in vivo stability, and limited targeting, which severely constrain the full realization of their chronopharmacological effects. In recent years, advances in multifunctional nanocarrier technologies have offered promising solutions to these limitations.^95–98 Notably, the rhythmic expression of core clock genes (eg, CLOCK, BMAL1, PER2, CRY1) provides a novel design dimension for developing “circadian sensing” intelligent delivery systems. Smart drug delivery platforms constructed from diverse advanced nanocarriers, such as liposomes, polymeric micelles, mesoporous silica, metal–organic frameworks, exosome-mimetic vesicles, and near-infrared-responsive gold nanocarriers, not only enable high-efficiency loading of multiple classes of CHM-derived bioactive compounds, such as flavonoids, terpenoids, alkaloids, and polyphenols,^99–102 but also achieve dynamic synergy with the circadian network through the following mechanisms: (1) significantly enhancing aqueous solubility and plasma stability, thereby prolonging systemic circulation half-life to maintain therapeutic drug concentrations throughout the OCIW; (2) promoting tumor accumulation via passive targeting (eg, the enhanced permeability and retention effect, EPR) and improving specific recognition and cellular uptake by clock protein overexpressing tumor subpopulations (such as BMAL1-positive or PER2-positive cells) through active targeting strategies like ligand conjugation or tumor cell membrane biomimetic coating; (3) responding to tumor microenvironment specific stimuli (eg, acidic pH, elevated ROS, or matrix metalloproteinase activity) and further integrating circadian gated release logic, for instance, triggering payload release during BMAL1-driven metabolic peaks or synchronizing the delivery of DNA damaging agents with PER2-mediated troughs in DNA repair capacity; (4) co-delivering adjuvants such as photosensitizers, immunomodulators, or nucleic acid based clock modulators (eg, siRNA against REV-ERBα or RORα agonists) to actively reprogram the tumor circadian machinery and enable synergistic chronotherapeutic intervention (Figure 6).

Figure 6.

Design framework of a multifunctional intelligent nanodelivery system tailored for the tumor circadian network. The schematic is divided into four functional modules: (1) Circadian cue-guided tumor targeting (Top Left): The membrane-cloaked nanoplatform utilizes the EPR effect for passive accumulation and achieves active targeting via receptor-mediated internalization through a circadian-responsive surface interface. (2) TME-triggered phase-gated drug release (Top Right): The system responds to specific tumor microenvironment features (hypoxia, acidity, high GSH, immunosuppression, and aberrant vasculature) and circadian-regulated effector molecules to trigger precise drug liberation. (3) Smart multicomponent co-assembly (Bottom Left): The nanocarrier encapsulates diverse bioactive constituents, including CHM-derived circadian modulators, photosensitizers, sonosensitizers, chemotherapeutic agents, immunomodulatory antibodies, gene-regulatory payloads, and theranostic moieties. (4) Spatiotemporal theranostics for cancer (Bottom Right): By integrating external stimuli responsiveness (thermal, pH, ultrasound, laser, magnetic/electric fields) with clock gene oscillation monitoring, the platform enables precise chronopharmacological intervention and real-time theranostics. (The figure is drawn by Figdraw).

Particularly crucial is that when nanodelivery strategies are aligned with an individual’s OCIW, spatiotemporal co-release of therapeutics can be achieved precisely during the tumor’s most vulnerable circadian phases, such as troughs in DNA repair capacity, peaks in metabolic activity, or periods of heightened immune surveillance, thereby maximizing antitumor efficacy while minimizing off-target toxicity to healthy tissues. For example, administration of a curcumin nanoformulation during the BMAL1 expression peak, typically corresponding to circadian time CT6 to CT10, synergistically enhances its inhibition of the Wnt/β-catenin pathway and concurrently activates the SIRT1–BMAL1 positive feedback loop; similarly, delivery of EGCG during the PER2 high expression window, for instance, CT18 to CT22, more effectively induces G1/S phase arrest and mitochondrial-dependent apoptosis. This integrated paradigm, in which circadian profiling guides time-programmed dosing and nanocarriers execute spatial targeting, not only overcomes the pharmaceutical limitations of CHM but also elevates chronopharmacology from empirical timed administration to mechanism-driven, circadian-synchronized precision intervention, thereby establishing a critical technological foundation for developing robust preclinical evaluation frameworks and advancing stepwise clinical translation.

Clinical Translation Pathway: Establishing a Chronotherapy Evaluation Framework for CHM Nanomedicines Guided by Circadian Rhythms

Breakthroughs in basic research urgently require matched preclinical and clinical validation paradigms. The current translational bottleneck for chronotherapy based on bioactive constituents of CHM stems not only from the lack of standardized tools for assessing tumor circadian rhythms but also from the absence of multidimensional efficacy-toxicity evaluation systems specifically designed for CHM nanocarriers. To address this, we propose a three-tier validation framework centered on organoids–PDX–clinical trials, focusing on systematic evaluation of circadian-synchronized CHM nanomedicines to bridge the gap between mechanistic discovery and clinical application.

Before preclinical model validation, it is essential to establish a dedicated workflow for the discovery and confirmation of chronobioactive compounds from CHM tailored for chronopharmacological applications (Figure 7A). This pipeline begins with traditional medicinal herbs, such as Scutellaria baicalensis Georgi, Panax ginseng C. A. Mey., and Curcuma longa L., from which bioactive constituents are systematically extracted and enriched to generate a comprehensive compound library. Subsequently, a high-throughput circadian reporter cell system is employed under standardized light-dark entrainment conditions to rapidly evaluate the modulatory effects of individual fractions on the period, phase, and amplitude of core clock gene oscillations. This enables the preliminary identification of candidate molecules exhibiting notable potential for circadian rhythm intervention. These candidates are then subjected to in vivo functional validation in tumor-bearing mouse models to assess their actual regulatory impact on circadian phenotypes within tumor tissues. Furthermore, multi-omics profiling, including transcriptomics and proteomics, is integrated to systematically elucidate the molecular mechanisms by which these compounds modulate the circadian network and associated signaling pathways in the tumor microenvironment. Collectively, this workflow aims to efficiently pinpoint lead chronobioactive compounds that are both mechanistically well-defined and pharmacologically robust from the complex matrix of CHM, thereby providing a high-quality, evidence-based foundation for subsequent nanomedicine development and chronotherapy translation.

Figure 7.

A comprehensive clinical translation framework for chronotherapeutic nanomedicines derived from CHM. (A) Integrative screening and system design: This phase combines high-throughput screening of CHM-derived bioactives using circadian reporter systems with the rational design of intelligent chrono-adaptive nanodelivery carriers. (B) Preclinical evaluation via chrono-nano synergy: Efficacy and toxicity are assessed using a dual-platform approach comprising dynamic patient-derived organoids cultured on microfluidic chips and immune-humanized PDX models guided by the OCIW. (C) Clinical roadmap for translation: Guided by OCIW and tumor circadian oscillations, the framework outlines a stepwise progression from Phase I to IV clinical trials, integrating formulation optimization to ensure safety, patient stratification, and standardization. (The figure is drawn by Figdraw).

At the preclinical evaluation level, advanced models capable of dynamically simulating patient-specific tumor rhythms and compatible with nanodrug assessment should be established (Figure 7B). On one hand, patient-derived tumor immune organoid on-chip platforms can be developed by integrating microfluidic perfusion, controllable light-dark cycles, and real-time imaging to enable dynamic monitoring of tumor penetration, drug release kinetics, oscillatory responses of core clock genes, and immune microenvironment remodeling induced by CHM nanoformulations across different circadian phases. On the other hand, circadian-matched PDX models should be implemented, where dosing time in recipient mice is aligned with the donor patient’s tumor circadian profile, to simultaneously evaluate in vivo biodistribution, tumor accumulation efficiency, and circadian-dependent antitumor activity of nanocarriers, thereby highlighting the synergistic potential between nanodelivery and optimal timing.

In the early phase of clinical translation, a systematic, stepwise research pathway should be designed around the distinctive characteristics of CHM-based nanomedicines (Figure 7C). This pathway takes individualized OCIW-guided nano-CHM therapy as its central starting point, thereby facilitating an orderly and mechanism-informed transition from foundational discovery to clinical application.

Firstly, during Phase I clinical trials, the emphasis should be on assessing the safety profile, maximum tolerated dose, and pharmacokinetic characteristics of the nanoformulation within each patient’s personalized OCIW. Particular attention must be given to nanomaterial-associated immunogenicity, hepatic and renal accumulation, and potential time-dependent toxicities. These assessments are essential for providing a solid foundation for safe medication use in subsequent studies.

Subsequently, moving into Phase II clinical trials, multi-omics rhythmic profiling, including tumor tissue transcriptomics, plasma metabolomics, and physiological rhythm data collected via wearable devices, should be introduced to achieve precise patient stratification. This phase aims to compare the efficacy of “rhythm-matched nano-dosing regimens” against conventional dosing strategies. Beyond traditional endpoints such as ORR and PFS, it is crucial to explore novel surrogate biomarkers that reflect the synergistic effects of rhythm and nanomedicine. Examples include: (1) evaluating the rhythmic enrichment of nanodrugs in tumors using imaging tracers; (2) PER2 amplitude recovery rates; and (3) the synchronization index of CD8⁺ T-cell infiltration rhythms, thereby enhancing the sensitivity and specificity of efficacy evaluations.

Building on this foundation, large-scale, multi-stage clinical trials (Phase III and Phase IV) should be conducted to further validate the confirmatory efficacy and long-term safety, incorporating real-world data for post-marketing surveillance. Throughout the clinical translation process, formulation development and optimization remain pivotal. Not only must continuous improvements be made based on clinical feedback to enhance the physicochemical stability, targeting ability, and batch consistency of nanocarriers, but standardized formulation processes suitable for various indications must also be established to ensure the reproducibility of clinical evidence and regulatory feasibility.

To promote standardized development and facilitate regulatory approval of CHM-based chrono-nanotherapeutics, we propose a conceptual extension to the existing CONSORT 2025 statement, specifically tailored for rhythm-guided nanomedicines. This proposed framework aims to incorporate four key reporting domains: (1) physicochemical characterization and batch-to-batch consistency of nanocarriers; (2) methodologies for patient-specific circadian phenotyping and criteria for defining the OCIW; (3) correlation between dosing time and in vivo pharmacokinetics or pharmacodynamics of nanoformulations; and (4) monitoring protocols for time-dependent nanotoxicity, such as complement activation-related pseudoallergy or immune responses within specific circadian phases. Critically, regulatory agencies, including the FDA, EMA, and NMPA, currently lack dedicated evaluation pathways for therapies that integrate multicomponent herbal medicines, nanoscale delivery systems, and individualized chronotherapeutic scheduling. Early dialogue with regulators, coupled with the development of reference standards for CHM nanoformulations and validated biomarkers of circadian response, will be essential to ensure reproducibility, comparability, and global regulatory feasibility. Only through such coordinated efforts can the promise of circadian-reprogramming nanomedicine transition from scientific innovation to clinical reality. In summary, chrono-pharmacological research on CHM for anticancer therapy is entering a new phase characterized by deep integration of mechanisms, technologies, and clinical applications. By integrating personalized tumor rhythmic profiles, smart responsive nanodelivery systems, multidimensional evaluation platforms compatible with nanomedicine, and standardized clinical pathways, we aim to establish an integrated approach combining Western and Chinese medicine for cancer treatment. This includes “tumor rhythm diagnosis–smart nano-delivery–chrono-precision therapy,” offering a novel strategy for malignant tumors that combines spatiotemporal precision with the distinctive features of TCM.

Conclusions

This study systematically demonstrates that circadian disruption serves as an intrinsic temporal driver of tumor progression, and that bioactive constituents of CHM, by targeting the core circadian clock network, not only suppress tumor growth but also possess a unique capacity to actively reprogram the temporal order of tumors. This “circadian modulation” mechanism marks a paradigm shift in CHM-based anticancer action from conventional “spatial multitarget modulation” to “precision intervention along the temporal dimension”, elevating CHM beyond typical natural products by endowing it with intrinsic chronobiological regulatory functions and spatiotemporal synergistic therapeutic potential. Thus, the TCM principle of “treatment in accordance with circadian timing” receives a modern scientific interpretation through chronobiology: the therapeutic goal shifts from “tumor eradication” to “restoration of temporal homeostasis”, reflecting a paradigm shift from antagonism to harmonization and from static suppression to dynamic remodeling. This integration not only offers novel insights for addressing key challenges such as tumor heterogeneity, drug resistance, and immune evasion but also establishes an epistemological bridge between the TCM holistic perspective and modern precision medicine.

Furthermore, addressing the clinical landscape of CHM, it is important to distinguish between current market availability and future therapeutic potential. While numerous CHM-derived bioactive compounds, such as curcumin, resveratrol, and EGCG, are widely available as dietary supplements, and traditional formulations like Kanglaite or Aidi injections are clinically utilized as adjuvant therapies, their application as precision oncology agents remains limited by suboptimal pharmacokinetic profiles and a lack of temporal control. The emergence of nano-chronotherapy, as proposed in this review, aims to bridge this gap. By encapsulating these market-available natural compounds into intelligent nanocarriers and synchronizing their release with the tumor’s intrinsic circadian rhythm, we can transform these traditional remedies into next-generation precision medicines. This strategy not only leverages the safety and multi-target advantages of CHM but also overcomes the translational barriers that currently restrict the clinical efficacy of CHM.

While the potential of this integrative strategy is significant, realizing its full clinical impact requires overcoming current translational barriers. Crucially, the ultimate “spatiotemporal” precision hinges on the seamless integration of temporal control with robust spatial delivery. Addressing these multifaceted challenges is essential to translate the elegant theory of circadian reprogramming into clinical reality.

Limitations and Future Perspectives

Despite these promising insights, this emerging paradigm faces significant scientific and translational constraints. The majority of current evidence derives from in vitro cell models or murine studies, with a lack of direct validation of the relationship between circadian dynamics and drug responses within the authentic human tumor microenvironment. Specifically, the field currently grapples with the lack of standardized circadian experimental designs, making cross-study comparisons difficult. Furthermore, the profound tumor-specific circadian heterogeneity across different cancer types and individuals remains a major obstacle to the precise determination of OCIW.

To achieve the aforementioned spatiotemporal precision, a critical bottleneck is the still-weak integration between nanomedicine and circadian biology. There is an urgent need to develop intelligent nanocarriers that are not just passive carriers but active sensors of circadian cues, ensuring precise tumor accumulation to fulfill the spatial prerequisite for temporal control. Additionally, while organoid models are powerful, their current limitations in fully mimicking the systemic circadian network and the dynamic tumor-immune interactions restrict their predictive power. Moreover, clinical data on the long-term safety, immunogenicity risks, and time-dependent toxicities of CHM nano-chronotherapy are extremely scarce, and no standardized monitoring metrics or regulatory frameworks currently exist. Finally, although AI-driven circadian prediction models show promise, algorithms for multi-omics data integration and computational modeling linking wearable device signals to intrinsic tumor rhythms remain underdeveloped.

Looking ahead, the advancement of TCM chronopharmacology will critically depend on the synergistic integration of three pillars: the depth of mechanistic understanding, the breadth of multidisciplinary technological convergence, and the feasibility of clinical translation pathways. Only through the deep integration of chronobiology, nanomedicine, AI, and clinical oncology can a patient-centered, rhythm-guided “diagnosis–delivery–intervention” closed-loop system be established. This will advance TCM chronotherapy from empirical “timed drug administration” to mechanism-driven “circadian-synchronized precision intervention.” Specifically, this technological leap provides the long-sought “bridge” for the TCM principle of “treatment in accordance with circadian timing” (Yin Shi Zhi Bing). By transforming the subjective concept of “timing” into the objective metrics of OCIW and circadian-synchronized release, this framework effectively showcases the unique value of Chinese wisdom through the rigorous methodology of modern precision medicine. Ultimately, this integrative paradigm holds promise for delivering an innovative therapeutic strategy against malignant tumors that combines spatiotemporal precision, enhanced efficacy with reduced toxicity, and the distinctive features of TCM within the global landscape of precision medicine.

Funding Statement

This research was funded by the National Natural Science Foundation of China (Grant number 82004118), Natural Science Youth Foundation of Nanjing University of Chinese Medicine (Grant number NZY82004118), Medical Scientific Research Project of Jiangsu Provincial Health Commission (Grant number Z2020015), Nanjing Medical Science and Technique Development Foundation (Grant number ZDXX25166).

Abbreviations

AI, Artificial Intelligence; AMPK, AMP-Activated Protein Kinase; APC, Antigen-Presenting Cell; AKT, Protein Kinase B; BMAL1, Brain and Muscle ARNT-Like 1; CDK, Cyclin-Dependent Kinase; CHM, Chinese Herbal Medicine; CLOCK, Circadian Locomotor Output Cycles Kaput; CONSORT, Consolidated Standards of Reporting Trials; CRY, Cryptochrome; CXCL5, C-X-C Motif Chemokine Ligand 5; CXCR2, C-X-C Motif Chemokine Receptor 2; DBP, D-Box Binding Protein; DNA, Deoxyribonucleic Acid; EPR, Enhanced Permeability and Retention; EGCG, Epigallocatechin-3-Gallate; EZH2, Enhancer of Zeste Homolog 2; GSCs, Glioma Stem-Like Cells; GSH, Glutathione; GSK-3β, Glycogen Synthase Kinase-3 Beta; HDAC3, Histone Deacetylase 3; HIF-1α, Hypoxia-Inducible Factor 1-Alpha; IL-1β, Interleukin-1 Beta; LPS, Lipopolysaccharide; MDSCs, Myeloid-Derived Suppressor Cells; MHC-I, Major Histocompatibility Complex Class I; NAD⁺, Nicotinamide Adenine Dinucleotide; NLRP3, NOD-, LRR- and Pyrin Domain-Containing Protein 3; NSCLC, Non-Small Cell Lung Cancer; OCIW, Optimal Chronotherapeutic Intervention Window; ORR, Objective Response Rate; PDX, Patient-Derived Xenograft; PER, Period; PFS, Progression-Free Survival; PI3K, Phosphoinositide 3-Kinase; PPARγ, Peroxisome Proliferator-Activated Receptor Gamma; ROR, Retinoic Acid Receptor-Related Orphan Receptor; REV-ERB, Nuclear Receptor Subfamily 1 Group D; ROREs, ROR Response Elements; SIRT1, Sirtuin 1; SCN, Suprachiasmatic Nucleus; TCM, Traditional Chinese Medicine; TCR, T-Cell Receptor; TBARS, Thiobarbituric Acid Reactive Substances; TERT, Telomerase Reverse Transcriptase; Th17, T Helper 17 Cell; TRPV1, Transient Receptor Potential Vanilloid 1; VEGF, Vascular Endothelial Growth Factor; Wnt/β-catenin, Wingless-Type MMTV Integration Site Family / β-Catenin; ZT, Zeitgeber Time.

Data Sharing Statement

The data that support the findings of this study are available from the corresponding author, Zhengguang Zhang, upon reasonable request.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.