The aging population in high-income countries presents a significant societal and economic burden due to increased age-related diseases. Improving healthspan, the years lived in good health, is a key research focus. Cellular senescence, a stable arrest of cell division, is a major contributor to aging. Senescent cells accumulate in tissues and release inflammatory factors (SASP), accelerating aging. Selective removal of senescent cells improves healthspan in animal models. Botanical extracts, rich in phytochemicals with potential anti-aging properties, offer a valuable resource for discovering new therapies. Previous high-throughput screening identified a *Salvia haenkei* extract (SH) that delays senescence in vitro. This study investigates the in vivo anti-aging potential of a standardized SH extract called Haenkenium (HK).

Extensive research has explored the role of senescent cells in aging and age-related diseases. Studies show that the accumulation of senescent cells contributes to inflammation, tissue damage, and reduced healthspan. Senolytics, which selectively eliminate senescent cells, and senomorphics, which modulate the function of senescent cells, have shown promise in extending healthspan in animal models. Botanical extracts have a long history of use in traditional medicine for age-related conditions, and many phytochemicals have been identified with potential anti-aging properties, acting through various mechanisms such as antioxidant defense, mitophagy, sirtuin induction, or mTOR inhibition. Previous research has shown the *Salvia haenkei* extract to delay senescence in vitro. This work aims to build on this finding and test the in vivo effects of the extract.

Aged mice (starting at 20 months) received daily oral administration of HK (0.5 mg/kg body weight) in their drinking water until the end of their lifespan. Control groups received regular water. Lifespan, healthspan (assessed via a physical appearance score), and various tissue-specific aging phenotypes were evaluated. The chemical composition of HK was analyzed using mass spectrometry, identifying luteolin as a key component. In vitro studies using different cell types (fibroblasts, cardiomyocytes, renal cells) investigated the effects of HK and luteolin on senescence induced by various stressors (UV-B radiation, doxorubicin). Molecular mechanisms were explored using RNA sequencing, RT-qPCR, immunohistochemistry, surface plasmon resonance (SPR), and proximity ligation assay (PLA) to investigate the p16-CDK6 interaction.

HK treatment significantly extended the median lifespan of mice (32.25 months vs. 28 months in controls). Treatment also improved healthspan, reducing age-related phenotypes such as fur thinning, kyphosis, and tumor development. HK improved several tissue-specific aging markers: hair follicle size in skin, bone mineral density in femurs, reduced cartilage degeneration in knee joints, improved grip strength, reduced glomeruli size and fibrosis in kidneys. RNA sequencing revealed that HK treatment downregulated a gene expression signature associated with inflammation and senescence in muscle tissue. HK also significantly reduced senescence induced by doxorubicin, a chemotherapeutic agent, both systemically (in p16Luc reporter mice) and in human cardiomyocytes, mitigating doxorubicin-induced cardiotoxicity. Luteolin, the most abundant flavonoid in HK, effectively reduced senescence in vitro across various cell lines and stressors. Mechanistically, luteolin disrupted the p16-CDK6 interaction, preventing cell cycle arrest associated with senescence, as confirmed by SPR and PLA assays. Pharmacokinetic analysis showed that luteolin was detectable in the plasma of mice after oral HK administration.

This study demonstrates that HK treatment effectively extends both lifespan and healthspan in mice, suggesting a significant gerosuppressive effect. The observed improvements across various tissues and age-related phenotypes indicate a systemic impact of HK, primarily through senescence modulation. The identification of luteolin as an active component disrupting the p16-CDK6 interaction provides a compelling mechanistic explanation. This interaction is critical in regulating cellular senescence, and its disruption could alleviate several age-related pathologies. The relatively low dose of HK required to achieve these effects is promising for future translational research and development of HK or luteolin as therapeutic agents for age-related diseases. The findings support the potential of botanical extracts as a source of effective interventions for healthy aging.

This study provides strong evidence for the anti-aging potential of HK, a standardized *Salvia haenkei* extract. HK significantly extends lifespan and healthspan in mice by reducing age-associated senescence. Luteolin, a key component of HK, disrupts the p16-CDK6 interaction, suggesting a novel mechanism for senescence inhibition. These findings highlight the therapeutic potential of HK and luteolin for treating age-related diseases. Future studies should focus on clinical trials to evaluate the efficacy and safety of HK or luteolin in humans, as well as further investigation into the precise molecular mechanisms and the potential synergistic effects of other HK components.

The study used a single inbred strain of mice (C57BL/6), which might limit the generalizability of the findings. While luteolin was identified as an active component, the complex nature of HK means that synergistic effects of other compounds might contribute to the observed benefits. Further research is needed to determine the long-term effects of HK treatment and fully elucidate the specific molecular mechanisms underlying its geroprotective effects. Finally, the in vivo experiments primarily focused on acute doxorubicin-induced senescence, warranting further exploration of chronic exposure scenarios.