Anxiety and stress are closely related psychological conditions that frequently co-occur and influence one another. Chronic stress can lead to the development of anxiety disorders, the dysregulation of the body’s stress response system, and alterations in hormones and neurochemicals, significantly affecting mood or sleep patterns. Furthermore, chronic stress can impair sleep by causing difficulties in falling asleep, maintaining sleep, or achieving restorative rest. In turn, poor rest or sleep deprivation results in daytime fatigue, irritability, and cognitive impairment during the day, which can amplify the perception of stressors. Therefore, it is critical to interrupt this negative feedback loop (stress–mood–sleep cycle) to restore overall well-being.

This study investigated the effect of VAN, standardized to 10% total vanilloids, on anxiety, emotional well-being, and sleep quality using the m-HAMA, POMS, and LSEQ questionnaires in a healthy adult population with moderate anxiety indicative of chronic stress. After three days of daily supplementation compared to the placebo, VAN doses ranging from 50 to 150 mg significantly improved the subjective perception of anxiety, mood, and stress, as well as sleep onset and quality, with no reported side effects. These results indicate that VAN may help restore the stress–mood–sleep cycle.

To the best of our knowledge, this is the first study to show that the A. melegueta seed extract has a beneficial effect on anxiety, stress management, mood, and sleep. The extract’s anxiolytic properties manifest rapidly, making it a promising supplement for mild anxiety, particularly due to its effectiveness within a short time frame and its absence of adverse effects. These biological effects highlight VAN’s potential to support the emotional well-being of individuals exposed to demanding lifestyles, including workers in high-pressure jobs, women going through perimenopause, and other populations dealing with daily challenges that may impact their emotional state.

Most of the health benefits attributed to A. melegueta have been associated with its anti-inflammatory and analgesic properties, primarily through the inhibition of Cyclooxygenase-2 (COX-2), leading to a concomitant decrease in prostaglandin synthesis [6,19]. This mechanism may help explain the positive effects of this spice in alleviating chronic inflammatory diseases [20]. In addition, a previous clinical trial demonstrated that 12 weeks of A. melegueta seed extract intake increased energy expenditure through the activation of brown adipose tissue (BAT) and a decrease in fat reduction [21,22]. In an animal model, the oral administration of 6-paradol significantly reduced visceral and subcutaneous fat depots, whereas no such effect was observed with 6-gingerol or 6-shogaol, suggesting that 6-paradol regulates several obesity-related genes in an AMPK-independent manner [23].

Notably, these three bioactive compounds—6-paradol, 6-gingerol, and 6-shogaol—are vanilloids, which are pungent components with activity on the TRPV1, similar to that of capsaicin. Animal studies have demonstrated that the oral administration of capsaicin can activate the TRPV1 expressed in sensory nerves within the gastrointestinal tract, increasing sympathetic nerve activity, which innervates the BAT. This results in a rapid rise in BAT temperature, increased whole-body energy expenditure (EE), and a reduction in body fat mass [24]. This connection suggests that the effects of vanilloids through TRPV1 activation could also be responsible for the increases in whole-body EE and reductions in body fat observed in clinical trials.

The in vitro results presented in this research show that VAN activates the TRPV1 in the same way as capsaicin, which correlates with the presence of vanilloid compounds present in VAN that are also responsible for the characteristic pungent taste of this species. Beyond the potential role of TRP1V in fat tissue, the therapeutic potential of TRP1V activation has primarily focused on the development of receptor antagonists as a source of analgesia [25]. However, the over-activation of the TRP1V can lead to desensitization, which may reduce tissue damage and inflammation [26]. This opens up the possibility of exploring VAN’s role as an analgesic in physiological situations that generate inflammation, although this was not the focus of the present study.

In a preclinical study, the administration of the A. melegueta seed extract to conscious, freely moving rats showed EEG modulation with prominent alpha-wave activity, indicative of the potentiation of serotoninergic signaling, similar to several antidepressants [16]. Serotonin modulation affects anxiety, depression, stress, memory, sleep, and thermal regulation and has been targeted by serotonergic antidepressants. The 5HTR1A activity is involved in these processes [27]. The 5HTR1A are widely distributed throughout the limbic system, cortical regions, and dorsal and median raphe nuclei and are expressed both pre- and post-synaptically. Both receptor elimination (knock-out models) and overexpression have been shown to lead to anxiety-like behaviors, suggesting a complex relationship between 5HTR1A activity and anxiety [28]. Several studies suggest that the activation of this receptor facilitates adaptation to stress [29], and animal models and clinical trials have demonstrated that both 5HTR1A agonists and partial agonists show promise in treating generalized anxiety disorder and depression [30].

The prominent alpha-wave activity reported in the electropharmacogram of rats administered the A. melegueta extract [16] is similar to that seen in rats treated with antidepressant drugs, underscoring the potential relationship between the A. melegueta extract and its functional application. The preliminary in vitro studies presented here show that VAN is capable of partially interacting with 5HTR1A. However, the neurophysiological relevance of this receptor modulation requires confirmation through specific functional in vivo tests. Although this partial modulation might not fully explain the alpha-wave activity observed in the in vivo EEG studies, previous research has reported functional crosstalk between the endocannabinoid and the serotoninergic system, involving various direct and indirect mechanisms across multiple brain regions (for review, see reference [31]). These interactions may underpin the parallel effects observed between the endocannabinoid and serotonin systems in the regulation of body temperature [32], feeding behavior [33], sleep and arousal [34], and emotional processes [35].

In recent years, anxiolytic treatments have increasingly focused on targeting the hCB1R, which is widely distributed across several brain regions, as well as in the peripheral tissues and organs, and is involved in regulating the balance between excitatory and inhibitory signals at the neuronal level [36]. However, the widespread distribution of hCB1R increases the likelihood that any therapeutic benefits may be accompanied by unwanted side effects. The endogenous hCB1R agonist anandamide (AEA), like 2-arachidonylglycerol (2-AG), is synthesized on demand and degraded by FAAH. Exposure to acute stress typically causes a rapid reduction in the tissue levels of AEA, which may be mediated, at least in part, by an increase in FAAH activity within the amygdala, hippocampus, or medial prefrontal cortex.

The effects of stress on AEA levels appear to occur rapidly and may precede the activation of the HPA axis, implying that the underlying mechanism takes place before glucocorticoid release [37]. FAAH inhibitors are considered promising pharmacological targets for reversing the stress-induced anxiety state [38] with fewer side effects [39]. The in vitro results presented in this publication showed that VAN inhibits FAAH in a similar manner to URB597, which has been demonstrated to exert anxiolytic and antidepressant effects both in animals, models, and humans [40]. The inhibition of the FAAH enzyme suggests that VAN has a direct anxiolytic effect by restoring the buffering capacity of endocannabinoid signaling in the stress response (Figure 8). This mechanism may also explain the mood-elevating effects observed during the trial, as AEA is considered a mediator of emotional homeostasis [41].

In addition, VAN exerts the agonist modulation of hCB2R, which may contribute to its neuroprotective effect in situations of cognitive stress that can lead to neuroinflammation. hCB2R, present mostly in microglia and immune cells, is upregulated during tissue damage or neuroinflammation associated with psychiatric disorders, such as depression or anxiety. Its activation inhibits the release of proinflammatory mediators, resulting in a neuroprotective effect under neuropsychiatric conditions [42]. Moreover, findings from animal models suggest that hCB2R overexpression may mitigate behavioral responses to chronic stress [43]. It is hypothesized that, in conditions of cognitive dysregulation, the increase in immune system cells within the CNS, particularly the microglia, may account for the observed elevation in hCB2R, which may underlie the behavioral effects of hCB2R modulation [44].

This is the first study to demonstrate that the water–ethanolic extract of A. melegueta seed can inhibit enzymes and bind to receptors associated with the eCBs while providing clinical evidence for stress relief and mood enhancement. The immediate effects on sleep quality and sleep onset appear to result from the alleviation of daily stress. While the present study does not provide in vitro evidence to suggest that these sleep effects are mediated by the GABAergic or serotonergic systems, the modulation of eCB components by VAN may contribute to the breaking of the negative stress–mood–sleep cycle.

Several popular botanicals, through different mechanisms of action, have been shown to reduce anxiety and stress and improve mood, including Crocus sativus (saffron), Piper methysticum (kava kava), Ginkgo biloba (ginkgo), Lavandula angustifolia (Lavanda), and Panax ginseng (ginseng), although with limited evaluation of their effects on sleep [45]. In contrast, other plants used in several Western traditional systems of medicine, such as sleep aids, have been the subject of preclinical and human research as sedative agents in recent years to combat sleep disturbances. Among them, Valeriana officinalis (Valerian), Passiflora edulis (passionflower), Matricaria chamomilla (chamomilla), and Melisa officinalis (lemon balm) have shown the best potential for sleep support, primarily by modulating GABAergic neurotransmission in the brain [46,47]. However, although several clinical studies have shown remarkable positive results and generally favorable safety profiles, the quality of evidence differs substantially, and several concerns exist regarding the study design, purity grade, standardization, and potential negative herbal–drug interactions, especially with benzodiazepine (BZD) anxiolytic medication [48,49]. In addition, the roots of Withania somnifera (ashwagandha) are one of the most well-known and recognized ayurvedic ingredients for managing chronic stress, with significant improvements observed in symptoms of stress, anxiety, insomnia, or depression in human clinical trials. Noteworthy, its functional effects are mediated, almost in part, by the GABAergic system [48,49,50,51] and typically manifest after several weeks of consumption—at least 8 weeks—and at relatively high doses of 600 mg [52]. This contrasts with the present study, where significant improvements in sleep quality were observed with VAN from the first day, as well as improvements in stress and mood management after only three days at a lower dose (100–200 mg of extract) and apparently through a GABAergic-independent mechanism with potentially lower risks of anxiolytic drugs interactions.

From a safety viewpoint, vanilloid-rich extracts or seed preparations from A. melegueta are generally well tolerated in the medium-to-long term. In fact, except for one old uncontrolled clinical trial which showed that consumption of 0.35 g of A. melegueta seed resulted in blurred and double vision [53], all studies in humans evaluating the side effects of the supplementation of A. melegueta seeds for 4 to 12 weeks have shown no side effects at doses ranging from 30 to 500 mg/day of supercritical CO₂ or ethanol extracts [21,22,54,55]. In addition, several clinical studies conducted with Zingiber officinale (ginger) rhizome powder or extracts, of which the main bioactive is vanilloids, with a duration of 2 to 28 weeks have reported no apparent side effects at doses between 1.2 and 2 g/Day [56,57,58].

Our study presents some limitations. First, this pilot trial included a small number of participants; hence, the extrapolation of the results to other populations should be made with caution. Second, the 3-day trial period was too short to provide data on long-term functional effects and/or the long-term safety risk associated with VAN. Third, the carry-over effects associated with the crossover design cannot be completely excluded. However, the counterbalance design, the progression strategy employed to exclude sequence effects, and the fast metabolization of VAN’s vanilloids linked to the 1-week washout period resulted in a negligible carry-over effect in the crossover study.

Future clinical studies should focus on a larger, more diverse population, an extended intervention duration, and parallel-group design to confirm these findings and evaluate VAN’s long-term safety in the organism. In addition, the use of selected rodent models to further investigate VAN’s mechanism of action at the CNS level and its potential synergistic effects with other botanical ingredients could shed light on its efficacy and applicability as a functional nutraceutical.