Valerian root extracts are widely used as mild sedatives to promote sleep, with clinical studies confirming their efficacy. Their sleep-promoting effects are associated with the adenosine A1 receptor (A1AR), a key regulator of sleep through neural activity inhibition. Adenosine, a neuromodulator that accumulates during wakefulness, activates A1ARs to facilitate sleep transitions. Using advanced analytics, we detected adenosine at 0.05% in the valerian extract Ze 911, supporting direct A1AR activation in vitro. Additionally, we explored A1ARs’ allosteric sites for modulatory activity. Valerenic acid and pinoresinol, key constituents of Ze 911, were identified as positive allosteric modulators (PAMs) of A1ARs. Valerenic acid exhibited strong PAM activity, with high cooperativity (αβ = 4.79 for adenosine and αβ = 23.38 for CPA) and intrinsic efficacy (τB = 5.98 for adenosine and τB = 3.14 for CPA). Pinoresinol displayed weaker PAM activity, with moderate cooperativity (αβ = 3.42 for adenosine and αβ = 0.79 for CPA) and limited efficacy (τB = 0.93 for adenosine and τB = 1.66 for CPA). The allosteric modulation observed in valerian extract Ze 911 suggests a mechanism of action in which valerenic acid and pinoresinol enhance receptor activation through allosteric interactions, potentially amplifying the effects of endogenous adenosine. By targeting A1ARs’ allosteric sites, valerian extract Ze 911 offers increased therapeutic selectivity and reduced off-target effects, emphasizing its potential for managing sleep disorders.

Cucurbita pepo (CP) seeds are traditionally used to alleviate lower urinary tract symptoms associated with benign prostatic hyperplasia and overactive bladder. While these effects are often attributed to lipophilic constituents, recent studies have highlighted the therapeutic potential of oil-free hydroethanolic extracts. However, their composition remains insufficiently characterized, considering the species’ significant phenotypic and phytochemical variability. This study aimed to characterize the phytochemical profile of hydrophilic hydroethanolic seed extracts from ten CP cultivars originating from different European regions, with a focus on compositional variability. The elemental composition, along with primary and secondary metabolites, was analyzed using established spectroscopic and chromatographic methods. The extracts showed considerable variation in protein (45.39 to 114.58 mg/g dw) and free amino acid content (46.51 to 111.10 mg/g dw), as well as differences in elemental composition. Principal component analysis revealed distinct clustering patterns, with several samples displaying metabolite profiles comparable to the Cucurbita pepo var. styriaca variety currently recommended by the European Pharmacopoeia (Ph. Eur.) and the Committee on Herbal Medicinal Products (HMPC). These findings open the possibility of using other CP varieties as alternative sources for extract preparation and offer novel insights into the composition of less explored hydrophilic extracts derived from CP seeds.

Actaea racemosa L. (syn. Cimicifuga racemosa (L.) Nutt., black cohosh) rhizomes contain triterpene glycosides (TTGs) that serve as principal quality markers in the European Pharmacopoeia (Ph. Eur.) monograph. However, discrepancies between chromatographic profiles of the plant material and the herbal reference standard (HRS) dry extract complicate quality assessment. This study elucidated the chemical origin of these discrepancies and evaluated whether the extract-based HRS can represent the analytical fingerprint of the plant material. Thirteen TTGs were identified and characterized within the official Ph. Eur. HPLC–ELSD method using centrifugal partition chromatography, semi-preparative UPLC, HRMS, GC–MS and NMR spectroscopy, employing reference standards and isolated compounds. Three malonylated TTGs (8a, 9a, 10a) were detected in wild and cultivated rhizomes but were absent from the A. racemosa dry extract HRS and other dry extracts. Quantitative analysis revealed that cultivated plants contained an insufficient amount of TTGs; however, after extraction, both the amount and the profile aligned with the Ph. Eur. requirements. This behavior is explained by the degradation and rearrangement pathways of the TTGs. Overall, these findings clarify the origin of chemical variability between plant and extract fingerprints and support the inclusion of malonylated TTGs in the quantitative determination of A. racemosa triterpene glycosides in the Ph. Eur.

Triterpene glycosides (TTGs) are characteristic constituents of Cimicifuga racemosa extract Ze 450 and may contribute to its pharmacological effects in the treatment of menopausal symptoms. This exploratory study investigated their gastrointestinal stability, transformation during incubation with human fecal microbiota, and potential effects on microbial community composition and short-chain fatty acid (SCFA) production. The stability of 23-epi-26-deoxyactein was assessed during simulated oral and gastric digestion using the INFOGEST protocol. Microbiota-associated biotransformation of actein, 23-epi-26-deoxyactein, cimigenol-3-β-D-xyloside, and cimigenol-3-α-L-arabinoside was subsequently examined during 48 h anaerobic ex vivo cultivation with fecal microbiota from eight female donors. 23-Epi-26-deoxyactein remained largely stable during simulated oral and gastric digestion. During fecal incubation, all investigated TTGs were depleted and the corresponding aglycones were detected. Quantification of the 23-epi-26-deoxyactein aglycone revealed marked donor-dependent differences, indicating substantial interindividual variability in TTG biotransformation. Individual TTGs had little effect on microbial community composition or SCFA profiles, whereas Ze 450 induced a small but significant shift in overall community structure without substantially affecting SCFA production. These findings demonstrate extensive microbiota-mediated TTG deglycosylation and highlight marked interindividual variability in TTG metabolism, providing a rationale for further investigation of microbiome-dependent differences in the pharmacological fate of Ze 450.

Hydroethanolic Cucurbita pepo seed extracts are traditionally used for alleviating lower urinary tract symptoms (LUTS), yet their mechanisms remain unclear. Adenosine, a purine nucleoside involved in neuromodulation and smooth muscle relaxation, was recently identified in C. pepo seeds. Since A₁ adenosine receptors (A₁AR) suppress parasympathetic bladder overactivity by inhibiting acetylcholine (ACh) release, we investigated to which extent purines from pumpkin seed extracts contribute to A₁AR activation. Complementary antioxidant capacity was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Three hydrophilic seed extracts containing different adenosine levels (0.60–1.18 mg/g dw) were evaluated for agonist activity using a cAMP inhibition assay. The most active extract showed an EC₅₀ of 40.22 µg/mL. Selective removal of adenosine shifted the dose–response curve rightward, while further elimination of an adenosine derivative increased the EC₅₀ to 212.10 µg/mL, confirming adenosine as the principal active compound. Guanosine and inosine did not exhibit A₁AR agonist or allosteric effects. All samples exhibited measurable but weak antioxidant activity (IC₅₀ = 1.02–4.19 mg/mL), consistent with their low total phenolic content. These findings underscore the importance of accounting for naturally occurring agonists in plant extracts to avoid overestimating receptor-mediated effects in vitro which are not translatable in vivo.