Rosemary and Day Lily Blend Shows Antidepressant-Like Effects in Mice

A commercial herbal formulation combining rosemary leaves and day lily flowers reduced immobility time in the forced swim test among mice treated with the glucocorticoid dexamethasone, according to a preclinical study published in Clinical Phytoscience. The formulation, marketed as CLOCK and containing standardized extracts of Rosmarinus officinalis and Hemerocallis fulva, was also associated with the maintenance of selected hepatic redox-related parameters at levels comparable to those of untreated control animals. The researchers caution that the findings come from a short, exploratory animal experiment and warrant further investigation rather than any clinical application.

The study, conducted by a team led by Silvia Cristina Fagundes and Charise Dallazem Bertol, set out to evaluate both the behavioral and oxidative stress effects of the combined formulation in a dexamethasone-induced mouse model. Rosemary has a long history of use as a nervous tonic and cognitive stimulant, and previous animal studies have attributed antidepressant-like and neuroprotective effects to the plant, partly through compounds such as ursolic acid. Day lily flowers, traditionally known in some cultures as the plant of forgetfulness of sadness, have been employed to relieve anxiety, insomnia, and depressive symptoms, and have shown benefits in mood and sleep parameters in experimental and clinical studies. A formulation containing both species has previously been associated with increased levels of acetylcholine, choline, brain-derived neurotrophic factor, irisin, and melatonin, yet the combined antidepressant and antioxidant effects of the two plants remained insufficiently investigated.

For the experiment, forty isogenic black C57BL/6 mice, twenty males and twenty females aged 21 days and weighing between 16 and 35 grams, were provided by the University of Passo Fundo vivarium in Brazil. The study was approved by the institution’s Animal Use Ethics Committee under registration number 009/2023, and all procedures followed Brazilian CONCEA guidelines and the U.K. Animals (Scientific Procedures) Act of 1986. Because this was an exploratory preclinical study, no formal a priori sample size calculation was performed; the number of animals was instead defined based on previous studies using similar models and the ethical principles of the 3Rs. The mice were randomly allocated to four groups of ten animals each, five males and five females, housed in small cages segregated by group and sex under a 12-hour light/dark cycle at 18 to 22 degrees Celsius and 50 to 70 percent relative humidity, with food and water supplied ad libitum.

The first group served as an untreated control, receiving no dexamethasone and no treatment. The remaining three groups received a single subcutaneous dose of 64 micrograms per kilogram of dexamethasone 24 hours before treatments began, a protocol previously reported to induce depressive-like behavior in mice. Of these, group two received no treatment, group three received the herbal formulation at 30 milligrams per kilogram by gavage, and group four received the standard antidepressant fluoxetine at 15 milligrams per kilogram, also by gavage and diluted in saline. Saline was given by gavage to the control and depression-only groups to standardize administration routes. All treatments lasted seven consecutive days, and behavioral testing was conducted one day after completion.

Behavioral effects were assessed using two widely used rodent paradigms: the forced swim test and the tail suspension test, each lasting six minutes. In the forced swim test, each mouse was placed individually in a two-liter glass beaker filled with water at 23 to 28 degrees Celsius, and researchers recorded the latency to first immobility and the total immobility time. In the tail suspension test, mice were suspended by the tail tip on a fixed platform while the same measures were recorded. To minimize observer bias, one researcher selected and transferred the animals while a second, blinded to the treatment groups, conducted the tests. Immobility in these paradigms is generally interpreted as a behavioral correlate of despair or passive coping, although the authors note that it can also represent an adaptive energy-conserving strategy under acute stress.

The key behavioral result emerged in the forced swim test. Total immobility time during the six-minute test differed significantly between the herbal formulation group and the untreated depression group, with a P value of 0.0188, while no significant differences were observed among the remaining groups. Latency to immobility did not differ among any of the groups. In the tail suspension test, by contrast, no significant differences were observed between groups for either latency or total immobility time. The authors suggest that these divergent findings may reflect differences in the neurobiological mechanisms underlying each test, and note that environmental and procedural factors are known to influence tail suspension performance.

Notably, the behavioral alterations induced by dexamethasone itself were modest, and no significant differences were detected between the dexamethasone-treated and control groups in the behavioral tests. The authors attribute this possibly to the short duration of dexamethasone exposure, although they acknowledge that other experimental factors cannot be excluded. This point matters for interpretation: the formulation reduced immobility relative to dexamethasone-treated animals, but the depression model itself did not produce a robust separation from controls, and the authors state that confirmation in additional experimental models of depression is warranted.

After the behavioral tests, the animals were euthanized and their livers collected and stored at minus 70 degrees Celsius for oxidative stress analysis. The researchers measured total protein by the Bradford method, lipid peroxidation via thiobarbituric acid reactive substances representing malondialdehyde levels, non-protein thiols using DTNB as an indicator of reduced glutathione, and nitric oxide via the Griess reaction as nitrite concentrations. Total hepatic protein levels were significantly higher in the untreated depression group (P = 0.014) and the fluoxetine group (P < 0.001) compared to controls, whereas the herbal formulation group did not differ significantly from the control group. No significant differences were observed in hepatic lipid peroxidation among groups. Nitric oxide levels differed significantly between the fluoxetine group and controls (P = 0.0002), with fluoxetine-treated animals showing lower levels, while the herbal formulation group remained comparable to controls. Non-protein thiol levels differed significantly between the fluoxetine group and both the control group (P = 0.0028) and the untreated depression group (P = 0.0357). No sex-based differences were observed, though the study was not designed or powered to evaluate sex-by-treatment interactions, and the limited number of animals per sex may have reduced the ability to detect such effects.

The authors interpret these biochemical patterns cautiously. The similarity of hepatic protein and non-protein thiol levels between the herbal formulation group and controls suggests preservation of hepatic redox homeostasis following treatment, and the two treatments appear to have differentially modulated nitric oxide metabolism. However, because conventional hepatic injury markers, histopathological evaluation, and direct measurements of antioxidant enzyme activity were not performed, the findings should be read as evidence of modulation of selected hepatic redox parameters rather than definitive evidence of hepatoprotection or global antioxidant activity. The proposed mechanisms behind the behavioral effects, including monoaminergic and serotonergic modulation, BDNF regulation, anti-inflammatory activity, and PI3K/AKT pathway involvement drawn from prior rosemary and Hemerocallis studies, were not directly investigated and should be considered possible explanations only.

Several further limitations frame the work. The seven-day treatment period is short, as conventional antidepressants typically require longer administration to achieve full therapeutic effects, although short-term designs may provide early evidence of pharmacological activity. The formulation consisted of commercially available whole-plant extracts standardized to 10 percent ursolic acid according to the manufacturer’s certificate of analysis, but information on the extraction method, drug-to-extract ratio, relative proportion of each plant extract, and complete phytochemical characterization was not provided. No overt signs of toxicity were detected, with stable body weight, unchanged feed intake, and no marked alterations in the evaluated oxidative stress parameters, yet the authors emphasize that this should not be interpreted as evidence of a definitive safety profile. Comprehensive toxicological evaluations, longer-term experimental designs, and clinical investigations are required to determine the formulation’s safety and therapeutic potential. Within those boundaries, the study stands as, to the authors’ knowledge, the first report of the behavioral and hepatic redox responses associated with combined administration of rosemary and day lily following a seven-day treatment protocol, and it supports continued investigation of whole-plant formulations as candidate complementary approaches in depression-related experimental models.

Subject of Research: Antidepressant-like and hepatic oxidative stress effects of a combined rosemary and day lily herbal formulation in a dexamethasone-induced mouse model

Article Title: Association of rosemary leaves and day lily flowers: evaluation of its antidepressant-like effects in mice

Article References: Fagundes, S. C., Alba, G. L., de Souza Rodrigues, N., Crestani, V. C., Tramontina, D., Soares, S. M., Ilha, V. Y. Z., Rossato-Grando, L., & Bertol, C. D. (2026). Association of rosemary leaves and day lily flowers: evaluation of its antidepressant-like effects in mice. Clinical Phytoscience, 12(1), Article 22. https://doi.org/10.1186/s40816-026-00435-3

Image Credits: AI Generated

DOI: 10.1186/s40816-026-00435-3

Keywords: rosemary, day lily, Hemerocallis fulva, Rosmarinus officinalis, antidepressant-like effect, forced swim test, tail suspension test, dexamethasone, oxidative stress, herbal medicine, preclinical study, mice