Calcific aortic valve disease (CAVD) is a highly prevalent valvular disorder and represents the third leading cause of cardiovascular morbidity worldwide [
1,
2]. It affects approximately 2% of individuals over the age of 70, contributing to an estimated 12.6 million cases annually on a global scale [
3,
4]. The pathogenesis of CAVD involves a multifactorial and progressive process, characterized by complex interactions among valvular endothelial cells, interstitial cells, and immune cells, ultimately driving structural remodeling of the aortic valve leaflets and disease progression [
5,
6]. Despite extensive clinical and basic research efforts, the underlying molecular mechanisms initiating and advancing CAVD remain incompletely understood. As a result, no pharmacological therapy has been approved to prevent or halt its progression, and surgical aortic valve replacement continues to be the only effective intervention currently available [
5,
7,
8]. These limitations underscore the urgent need for the development of novel therapeutic agents with the capacity to attenuate or reverse valvular calcification.
As part of our ongoing investigation into bioactive natural products from
Hypericum species [
9–
11], we employed a combined strategy integrating bioactivity-guided screening and molecular networking to explore
H. monogynum, which led to the isolation of hypermonane A (HMA,
1), a structurally unique bis-
seco polycyclic polyprenylated acylphloroglucinol (PPAP) bearing an unprecedented 3/6-5/6/5 pentacyclic scaffold, along with four known biosynthetic analogs (
2–
5). Herein, we reported the isolation, structural elucidation, and bioactivity evaluation of these compounds (
Fig. 1).
The residual 95% ethanol extract of
H. monogynum was suspended in water and subsequently partitioned with petroleum ether (PE). Silica gel column chromatography of the PE extract afforded six main fractions (Fr. A–Fr. F). To investigate potential anti-calcific activity, we first examined the expression of runt-related transcription factor 2 (RUNX2), a key osteogenic transcription factor, in human aortic valve tissues. Western blot (WB) analysis showed a marked upregulation of RUNX2 in CAVD samples compared to healthy controls (healthy:
n = 18; CAVD:
n = 24) (
Figs. 2A and B). Aortic valve samples in the control group were obtained from patients with dilated cardiomyopathy undergoing heart transplantation, with echocardiography confirming absence of calcification. In the calcification group, samples were collected from patients undergoing aortic valve replacement for calcific aortic valve disease (CAVD); exclusion criteria comprised rheumatic heart disease, infective endocarditis, or bicuspid aortic valve. Additionally, time-dependent elevation of RUNX2 expression was observed in human valvular interstitial cells (hVICs) treated with osteogenic medium (OM) (
Figs. 2C and D), indicating successful induction of the calcification phenotype. Using our in-house developed high-throughput screening platform, we assessed the anti-calcific potential of each fraction by monitoring RUNX2 protein levels in OM-treated hVICs [
11–
16]. Among all fractions, Fr. E exhibited the most significant inhibitory activity and was thus selected for further separation, yielding seven subfractions (Fr. E1–Fr. E7). Subsequent evaluation of these subfractions using the same screening strategy revealed that Fr. E6 showed the strongest inhibitory effect on OM-induced calcification (
Fig. 2E). Bioactivity-guided isolation from Fr. E6 ultimately led to the identification of five PPAPs, including one previously undescribed compound (
1) bearing a novel 3/6-5/6/5 pentacyclic scaffold, along with four known biosynthetic analogs (
2–
5). The molecular networking cluster containing the above compounds was expanded, with nodes labeled according to their parent
m/
z values (
Figs. 2F and G).
HMA (
1) possessed the molecular formula C
27H
38O
7, as determined by the
13C nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS) data (
m/
z 497.2508, [M + Na]
+, calcd. for C
27H
38O
7Na
+, 497.2515), indicating nine degrees of unsaturation. The
1H NMR spectrum (Table S1 in Supporting information) exhibited resonances for one
sec-butyl group (
δH 3.11, m; 1.08, d,
J = 7.2 Hz; 1.72, m; 1.34, m; 0.85, t,
J = 7.2 Hz) and five methyl singlets. The
13C and distortionless enhancement by polarization transfer (DEPT) NMR (Table S1) spectra showed 27 carbon resonances including seven methyls, six methylenes, four methines, and ten quaternary carbons. Comprehensive 2D NMR analysis revealed that HMA belonged to a PPAP derivative, comprising two characteristic structural units (units A and B,
Fig. 3A). The proton-proton correlation spectroscopy (
1H–
1H COSY) spectrum revealed three independent spin systems: H
3-25/H-24/H
2-26/H
3-27, H
2-18/H-19, and H
2-7/H-8/H-12/H
2-11/H
2-10 (
Fig. 3A). Structural elucidation of unit A was achieved through key heteronuclear multiple bond correlation (HMBC) correlations from H
3-17 to C-1/C-6/C-18, from H
3-21/H
3-22 to C-2/C-19, from H-19 to C-6, and from H
2-18 to C-1/C-2 (
Fig. 3A). Similarly, unit B was characterized by the HMBC correlations from H
3-15 to C-8/C-9/C-10 and from H
3-16 to C-12/C-13/C-14. Critical inter-unit connectivity was established
via the HMBC correlations from H
3-17 (unit A), H
2-7 (unit B), and H
2-14 (unit B) to the carbonyl carbon (C-5). The precise connectivity of the 2-methylbutyryl group with structural fragments A and B could not be unambiguously determined due to the absence of key correlation signals in NMR data. However, based on (1) C-2 being the sole remaining unsubstituted position in the molecular framework and (2) the well-documented prevalence of 2-methylbutyryl substitution at C-2 in PPAPs, we provisionally assigned this acyl group to C-2 of unit A. The planar structure of compound
1 was definitively confirmed by computational analysis using advanced chemistry development (ACD)/structure elucidator (
Fig. 3B) [
17–
19], and the proposed biosynthetic pathway further supported the rationality (
Fig. 3C).
The relative configuration of
1 was elucidated through detailed analysis of its rotating-frame overhauser effect spectroscopy (ROESY) data (
Fig. 3A). A key correlation between H-19 and H
3-17 suggested their spatial proximity on the same molecular face, which was tentatively assigned as
α-oriented. This stereochemical assignment further indicated that the C-2 isovaleryl group and the cyclopropyl moiety were
α- and
β-oriented, respectively. Additional ROESY correlations of H-8/H-7
β, H-7
β/H
3-15, H-12/H-7
α, and H-12/H
3-16 established that H-8 and H
3-15 resided on the same molecular face, whereas H-12 and H
3-16 were located on the opposite face.
Due to restricted rotation around the bond adjacent to the C-6 carbonyl and the absence of detectable NMR signals for multiple conformers, compound
1 was deduced to exist as an atropisomer [
20,
21]. Structural modeling of two possible atropisomeric forms (
1 and
1a) revealed that only the 2
R*,4
S*,6
S*,8
S*,9
R*,12
R*,13
S*,19
R*,
M configuration of
1 was consistent with the observed ROESY cross-peaks, especially the spatial proximity between H-7
β and H-18
β (
Fig. 3D). To further validate the relative stereochemistry,
13C NMR chemical shifts for four candidate diastereomers (
1a–
1d) were computed using the Gauge-independent atomic orbital (GIAO) method. The calculated shifts were statistically analyzed and compared with experimental data
via the DP4+ method (
Fig. 3E and Fig. S1 in Supporting information), supporting the proposed configuration [
22]. The absolute configuration of
1 was subsequently confirmed by time-dependent density functional theory (TDDFT)-based electronic circular dichroism (ECD) calculations, which showed excellent agreement between the experimental and calculated curves (
Fig. 3F).
According to the previous reports, the known compounds were identified as chipericumin C (
2) [
23], chipericumin D (
3) [
23], monosescinol B (
4) [
24], and monosescinol C (
5) [
24].
Further investigations revealed that compound
1 (HMA) significantly downregulated the expression of osteogenic markers RUNX2 and alkaline phosphatase (ALP) and alleviated calcification in hVICs. Under OM conditions, escalating HMA concentrations (5–80 µmol/L) modulated RUNX2 and ALP expression (
Figs. 4A–C). The half maximal inhibitory concentration (IC
50) value of HMA against hVICs was determined to be 147.7 µmol/L
via cell counting kit-8 (CCK-8) viability assays (
Fig. 4D). During extended osteogenic culture, HMA also exerted anti-calcific effects. ALP activity assays revealed significantly reduced enzymatic activity in HMA-treated hVICs after 7-day OM culture (
Figs. 4E and F). Alizarin Red staining confirmed attenuated calcium deposition in HMA-treated cultures following 21-day osteogenic induction (
Figs. 4G and H).
To elucidate the underlying mechanisms by which compound HMA alleviates calcification in hVICs, we performed network pharmacology analysis (
Figs. 5A and B) and RNA sequencing (
Fig. 5C). Utilizing the Comparative Toxicogenomics Database (CTD) and GeneCards databases, 76 HMA-related target genes and 534 CAVD-related target genes were identified by identifying intersections (
Fig. 5A). Further cross-referencing of these two datasets revealed 65 overlapping target genes (
Fig. 5B). Transcriptomic profiling of hVICs under OM induction demonstrated profound dysregulation of calcification related signal pathways. Volcano plots identified key differentially expressed genes (DEGs), including actin, alpha 2, smooth muscle (ACTA2) (upregulated in OM
vs. CTR; downregulated in OM+HMA
vs. OM) (
Figs. 5D and E). Comparative analysis of upregulated genes highlighted HMA-specific suppression of extracellular matrix (ECM)-remodeling (collagen type Ⅰ alpha 1 chain, COL1A1; a disintegrin and metalloproteinase with thrombospondin motif 4, ADAMTS4) and osteogenic (alkaline phosphatase, ALP; twist-related protein 2, TWIST2) targets (
Figs. 5F and G). Functional enrichment analysis (
Figs. 5H and I) further confirmed HMA’s role in counteracting CAVD progression, with significant inhibition of biological processes critical to calcification: ECM organization, cellular response to stress, and sulfur compound metabolism. As validated by quantitative polymerase chain reaction (qPCR), HMA treatment significantly downregulated the mRNA expression levels of key calcification-related genes, including ADAMTS4, TWIST2, and COL1A1, in hVICs under osteogenic conditions (Figs. S2A–C in Supporting information). Furthermore, molecular docking analysis revealed strong binding affinities between HMA and the core transcription factors RUNX2 and TWIST2, with favorable docking scores of −6.6 and −6.9 kcal/mol, respectively (Figs. S2D and E in Supporting Information), suggesting a potential direct mechanism for its inhibitory action.
The wire injury model rapidly recapitulates the core pathological features of CAVD, characterized by sequential inflammation, fibrosis, and calcification, through controlled mechanical damage. This model provides a robust platform to investigate molecular mechanisms and therapeutic windows for phytomedicine components targeting aortic valve calcification [
13,
15]. To determine whether HMA administration ameliorates CAVD progression
in vivo, we established a wire injury-induced murine CAVD model and randomized animals into two cohorts: HMA-treated group: Intraperitoneal injections of HMA (20 mg/kg) every 72 h for 6 weeks (
n = 8); Vehicle control group: Equivalent volumes of saline administered on identical schedule (
n = 8). The Animal Care and Use Committee of the Wuhan Food and Drug Safety Evaluation Center sanctioned all animal studies (approval No. 202410342). Post-treatment assessments included echocardiography for cardiac function and histomorphometry for valvular calcification quantification (
Fig. 4I). HMA-treated mice exhibited significant functional improvements
vs. vehicle controls, with pronounced reductions in hemodynamic parameters (peak transvalvular pressure gradients; mean pressure gradients,
Figs. 4J–L). Pathological calcification was significantly attenuated in HMA-treated aortic valves, as evidenced by Alizarin Red staining (
Figs. 4M–O). Collectively, these data demonstrate that HMA attenuates CAVD progression by mitigating hemodynamic impairment and pathological calcification, highlighting its therapeutic potential.
In conclusion, by employing a bioassay- and molecular network-guided discovery strategy, HMA, a novel C-1/C-2, C-2/C-3 bis-seco PPAP derivative with a unique 3/6-5/6/5 pentacyclic scaffold, was identified from H. monogynum. Its absolute configuration was unequivocally established through an integrated approach combining computer-assisted structure elucidation (CASE) and GIAO-based NMR and ECD calculations. HMA exhibited potent anti-calcific activity in CAVD model, acting via dual inhibition of osteogenic differentiation (RUNX2/ALP) and extracellular matrix remodeling (COL1A1/ADAMTS4). In vivo studies using a wire injury-induced CAVD mouse model further validated its efficacy, demonstrating significant attenuation of valvular calcification. Structurally, the distinctive double-ring-opening of the phloroglucinol core appears pivotal to its bioactivity, as the precursor spirocyclic PPAP analogs showed negligible anti-calcific effects. This indicates that the formation of new core ring systems following ring-opening, particularly the generation of two lactone rings, plays a critical role in mediating anti-calcific activity. The lactone moieties in the structure are crucial for the drug molecule to exert its key biological activity and are recognized as essential pharmacophoric groups.
To position HMA within the current therapeutic landscape, we compared its mechanism with clinical-stage strategies. Unlike bisphosphonates, which primarily inhibit hydroxyapatite deposition, or NOTCH-pathway targeted agents focusing on a specific signaling axis, HMA offers a distinct dual-inhibitory advantage by concurrently suppressing both osteogenic differentiation and pathological ECM remodeling. This multifaceted action, targeting two key drivers of CAVD progression, may translate into a more comprehensive therapeutic effect for slowing disease advancement. Therefore, HMA presents a novel and promising lead compound for developing multi-target therapies against CAVD. These findings position HMA as a promising first-in-class small-molecule inhibitor for CAVD, addressing a critical unmet need for pharmacological intervention beyond surgical valve replacement. Future studies will focus on optimizing its pharmacokinetic properties and evaluating potential synergistic effects with anti-inflammatory agents.
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