Saffron the “Red Gold” and Its CNS Activity: A Challenge for Future Applications in Nutraceuticals

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Introduction
Crocus sativus L. is a perennial stemless herb of the Iridaceae family that is cultivated in various parts of the world.In Europe, France, Italy, Spain, Greece, and Turkey are known for safron production.Other very important producers are Iran, Azerbaijan, and very big countries such as India and China.Te plant reaches approximately 10-25 cm in height and it is, in general, developed from a corm (bulbous tuberous structure) replicated every year.Te fower consists of six tepals, three stamens, and a style, which culminates with three red-branched stigmas.Te spice that is indicated as "safron" is obtained from the dried stigma of Crocus sativus L. and is one of the most highly valued agricultural products that is used for cooking purposes to give favor, color, and aroma to food [1][2][3][4][5].Safron is known as "red gold" because of its high price; its value can be $40-80 per gram.Tis cost is mainly caused by manual operations for plucking safron fowers during harvesting, requesting manual work [6].Further reasons for the high cost are the time-consuming cultivation and the need for a large number of human resources.Also, the high amount of plant material needed can be approximately calculated as 80 kg of C. sativus fowers; nearly 500,000 stigmas are required to produce 1 kg of dried safron [6].Te cultivation of Crocus species for culinary and medicinal utilization has been performed since ancient times, and a long tradition is present in Greece and Iran.Due to commercial exchanges, the use of this spice was spread worldwide being difused into the Mediterranean region, Eastern Europe, South Asia, and China [2].Tere are many traditional applications of C. sativus as it is used in food and cosmetics as well as in several traditional medicine preparations.In Persian traditional medicine, safron was used as a tonic, and many preparations have indications, especially for protecting the vascular and nervous systems.Safron is a famous spice and a remedy largely described in the traditional medicine of several countries.Many medicinal uses are reported in textbooks in the traditional receipts of herbal medicine.Te main area of use is to treat spasms, menstrual and other pains, and digestive ailments to improve liver function [7].Safron is a valuable ingredient in diferent remedies for the treatment of depression, insomnia, measles, and dysentery.[2].Furthermore, this species has been indicated for insomnia as well as a stimulant, aphrodisiac, antidepressant, and supportive treatment of cancer, thus suggesting that it can have several efects on the central nervous system (CNS).
In folk medicine, safron is claimed not only for diferent applications ranging from eupeptic to carminative to stomachic but also as an antispasmodic, anticatarrhal, and expectorant.Other reported uses suggest its application as a sedative, diaphoretic, stimulant, and aphrodisiac showing large difusion in many countries [1,2,6].Te extract obtained from safron and its active phytoconstituents have been considered for their potential usefulness as anticonvulsants, antidepressants, and anti-infammatory agents [5].Safron's claimed efects can be related to its complex group of constituents ranging from the aldehydic compound safranal to the carotenoids related to crocetin and crocins as the most known.Some preclinical studies have evidenced some possible antidepressant efects of crocin and crocetin, two of the major pigments of safron [5,8,9].Clinical pilot studies evidenced the antidepressant efects of safron extracts, but standardization and chemical composition can be a challenge.Furthermore, more extensive studies are needed to establish possible efcacy in this area [8].
In recent years, the role of oxidative stress (OS) has been highly considered especially related to CNS diseases and mostly to neurodegenerative diseases (NDDs).
In NDDs, as in many other human diseases, the OS can be defned as an imbalance between the ROS production and antioxidant capacity of the cell.One of the essential ATP sources is oxidative phosphorylation, which occurs in mitochondria and generates free radicals, reactive nitrogen species (RNS), and carbon-and sulfur-centered radicals.OS induces damages in many of the important biomolecules such as nucleic acid and protein.Furthermore, OS induces lipid oxidation and contributes to the formation of advanced glycation end products.Te strong role of OS is also recognised in glial cell activation, apoptosis, and proteasome dysfunction and can be one of the causes of mitochondrial dysfunction.Other mechanisms related to the efect of OS on CNS tissue can be the appearance of defects in the ubiquitinproteasome system and the induction of oligomerization of alpha-synuclein (αSyn) or beta-amyloid (Aβ) involved in chronic CNS diseases.Te OS can cause induction of cytokine and increase of the infammatory response and can be related to the damage of the blood-brain barrier.All these mechanisms can strongly afict the function of CNS also considering the peculiarity of this tissue that makes it particularly vulnerable to oxidative damage.In fact, the brain is characterized by high energy consumption and metabolic demand, and small imbalances can generate tissue damage and induce neuroinfammation.CNS metabolisms consume nearly 20% of the oxygen of the body in normal conditions, and for this reason, there is a large mitochondrial activity with physiologically signifcant production of radical species of oxygen (ROS).A further point is that the brain and CNS tissue have a high content of lipids, which can obviously be subjected to the peroxidation induced by ROS, and metals.Finally, the low antioxidant defense of neurons and their reduced regenerative capacity should be considered to fully understand the high susceptibility of this tissue to OS damage [10].
Te structural and functional perturbations in the brain that are associated with depression structure can be related to OS. Preclinical and clinical studies have shown that increased ROS generation and consumption of antioxidative defenses are key points in the altered brain structure, and this is considered the basis for the "oxidative stress hypothesis of depressive disorders," and the excess of OS has emerged as a major cause of the depressive disorder [11].In this regard, a signifcant role in the management of depression can be suggested for all those compounds as herbs and natural products known to possess a substantial antioxidant capacity.Due to its peculiar chemical composition, safron and several of its antioxidant constituents may result as promising agents useful for the management of CNS disease and, for example, as an antidepressant.Furthermore, thanks to their antioxidant activity as well as their possible action on mitochondria, safron constituents can be considered signifcant in further studies.Finally, due to their importance in many CNS diseases of oxidative stress and management of cellular function, particular emphasis will be dedicated to potential targets of safron constituents on mitochondria.
Due to its extensive use as a spice and the recent advances in research, safron can be considered a valuable natural product useful for developing nutraceuticals and/or remedies focused on the CNS.In this review, we will summarise the most recent fndings related to safron and its possible uses for CNS considering the improvement in product obtaining, standardization, and trials related to bioavailability as well as any clinical or pilot study showing potential usefulness.
In a recent review, nutritional and health-benefcial properties of safron (Crocus sativus L.) were taken into account showing the literature data related to its marker compounds, the bioavailability and bioaccessibility of the putative active compounds, and the bioactivity of safron phytochemicals summarising some of the therapeutic efect of this spice useful to counteract diferent ailments [6].Te authors considered all the parts of the botanical species focusing also on the possible uses and exploitation of other fower parts.In this review, we will focus mainly on the literature published in the last fve years (2017-2023).In the frst part of the manuscript, the most studied bioactivities of safron in CNS diseases are reported, and in the second part, clinical applications of safron are discussed.We also discuss the potential usefulness of safron for CNS-related ailments, focusing on a perspective of nutritional supplements and nutraceutical intervention.

Methods
Te literature search was performed using Scopus, PubMed, ScienceDirect, ACS publication, and MEDLINE databases, as well as Wiley Online Library for published articles in the English language, mostly referring to the period from 2015 to 2023.Te search terms included crocus sativus, crocus constituents, CNS system disease, antioxidant, depression, nutraceutical, and food supplements.More relevant papers were selected, and relevant reviews of the last 4 years were considered to prepare manuscripts focusing on diferent aims compared to the previously published reviews.

Main Text
Te efcacy in the prevention and protection of CNS of crocus preparation is strictly related to the chemical constituents that can be determined in the plant materials.Crocus stigmas are rich in diferent classes of secondary metabolites that are responsible for the color and the organoleptic properties of this spice [6,12,13].Tere are several papers considering the safron composition, and literature indicates that more than 150 volatile and nonvolatile compounds have been reported belonging to several chemical classes as proteins, amino acids, carbohydrates, minerals, vitamins, and pigments [14].A review summarising the contents of bioactive constituents of the plants of the species crocus was recently published [15].Details on chemical constituents of safron are well described in other papers [4,16] and are summarized in the supplementary materials included in the present review in which we report structures, classes of compounds, and their contents.

Bioactivity of Safron Related to CNS Diseases.
Several studies have considered the potential application of safron and its constituents as a treatment for CNS-related diseases.A few diferent experimental models and approaches have been proposed, but in the last years, research focused mostly on Alzheimer's disease and Parkinson's disease.Several authors have recently published reviews on the topic focusing on the safron or its constituents [17,18].Recent paper reported potential mechanisms related to the possible efects of safron or safron constituents on some CNS diseases and the most relevant ones are summarized in Table 1.
Te summarized paper reported in Table 1 presents some examples of the signifcant efects of safron in the feld of the most signifcant CNS diseases considering both neurodegenerative pathologies and depression and anxiety.
Furthermore, several papers considered the possibility of clinical applications, and some of the general uses are summarized in Table 2, where the fndings related to some of the relevant CNS diseases are reported.
Tus, the literature on safron revealed that there is a relatively large amount of published literature related to its possible use in CNS-related diseases and most of the proposed references indicate the efcacy and safety of the used remedies (Figure 1).
C. sativus L. possesses several pharmacological properties mainly attributed to its biologically active substances which are part of the class of terpenoids and include crocin, safranal, and picrocrocin.Increasing scientifc evidence considers C. sativus L. as a medicinal plant with antioxidant, antiatherosclerotic, anti-infammatory, hypotensive, hypolipidemic, antidiabetic, antidepressant, anxiolytic, and hypnotic characteristics [5,9,13,32].As demonstrated in several animal studies, the constituents of C. sativus L. are relatively safe considering the data available on acute, subacute, subchronic, and chronic exposure.Te data also indicate that safron and its metabolites present limited toxicity [1,2,6].Te major constituents of safron, safranal, crocetin, and crocin in animal models with various disorders associated with oxidative stress system, immune system imbalance, and infammation have demonstrated antioxidant, immunomodulatory, and anti-infammatory effects, respectively.Te efects of this plant have also been indicated by in vitro models, which confrmed the antiinfammatory, immunomodulatory, and antioxidant efects [2,6,13,33,34].Aluminum (Al) exposure is one of the environmental risk factors possibly related to the pathogenesis of neurodegenerative disease.Safron extract was studied as a protective agent against aluminum maltolate (Almal)-induced oxidative stress and apoptosis in PC12 cells.Te results showed a dose-dependent activity [35].
Considering the CNS-directed efects, recent studies have suggested the correlation between oxidative stress and depression [11].In particular, some ideas underpinning this connection should be highlighted.First, due to its high oxygen consumption and higher lipid content, the brain is vulnerable to OS.Second, OS is a cause of neurodegeneration, and its involvement in the pathogenesis of a major depressive disorder is well established.OS and proinfammatory signaling have emerged as mainstays in the pathogenesis of MDD (major depressive disorder).Tus, targeting these aberrant alterations with suitable antioxidants could be an efective strategy to manage MDD [11].In this regard, the studies supporting the antioxidant efects of safron can be taken into account for CNS pathologies.As reported in a comprehensive review [14], the efects of the main constituents of C. sativus on oxidative, infammatory, and immune responses in neurodegenerative, respiratory, coronary, gastrointestinal, hematological, and urinary system diseases have been studied, also related to the biochemical pathways involved.Te potent antioxidant activity of C. sativus stigma is mainly due to the presence of safranal, crocin, and crocetin.C. sativus and its constituents eliminate Journal of Food Biochemistry  1477 studies published until November 2020 were considered and 24 met the inclusion criteria for the review.Only 4 studies indicated that the efects of safron on cognitive impairment were not diferent from those produced by donepezil and memantine and that it had a better safety profle.So further studies are needed to establish the possible role of safron in AD [24] Mild to moderate depression clinical Safron was efective for treating mild to moderate depression and had comparable efcacy to synthetic antidepressants [25] Multiple sclerosis review Safron may prove benefcial in improving antioxidant defense and oxidative stress in patients with MS; however, the evidence appears scattered, heterogeneous, and inadequate, so trials of better design and MS-specifc outcomes are required [26] Adults with poor sleep, sleep quality, cortisol, and melatonin concentrations Supplementation with standardized extract (Afron ® ) 14 mg or 28 mg.Te authors reported sleep-enhancing efects of 28 days with safron supplementation in adults [27] C. sativus for insomnia Although there is limited evidence of a very low to moderate quality, C. sativus may beneft people with insomnia [28] Adjunctive therapy in adults with attention-defcit/hyperactivity disorder Safron combination therapy with Ritalin can efectively improve symptoms of patients with ADHD [29] Management of premenstrual dysphoric disorder 15 mg of safron for 2 weeks in the luteal phase of two menstruation cycles.
Journal of Food Biochemistry ROS, stabilize cell membranes, and decrease the peroxidation of unsaturated membrane lipids [14].Te antioxidant properties of safron constituents have been extensively studied and considered in many reviews [7,13,32,33,[36][37][38], and all data indicate that crocins and favonoids are very well established as antioxidants and their importance in diferent biochemical pathways involved in the antioxidant response of organism has been evidenced.An intranasal formulation containing violet oil, safron oil, and lettuce seed oil was studied for the efects on insomnia on ffty patients with primary chronic insomnia.Te authors concluded by afrming that the intranasal use of the multiherbal preparation can be used to improve chronic insomnia and to reduce the dose of conventional hypnotic medications in insomniac patients [31].

Mechanism of Actions Supporting Safron Activities on CNS.
Several papers reported specifc mechanisms of action of safron extracts and their constituents.Due to the peculiarity of the nervous tissue, the specifc cellular model was developed to recreate in vitro the most similar pathophysiological condition of specifc neuron groups.Te neuroprotective mechanism of safron was related to its direct action on the P2X7 ionotropic receptor (P2X7R), and its alteration is related to retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration.Safron protects photoreceptors from ATPinduced cytotoxicity.Two fractions isolated from safron stigmas, picrocrocin/kaempferol and crocin, were used for parallel experiments on 661W (immortalized cone photoreceptor cell line derived from the retinal tumor of a mouse) and human embryonic kidney 293 cells expressing P2X7R.Te frst cellular model was derived from retinal tumors of a transgenic mouse line and showed biochemical properties and cellular properties of photoreceptors, while the P2X7 receptor was proposed as a potential therapeutic target in central nervous system (CNS) diseases.High levels of extracellular ATP in the retina could be the cause of retinal neurodegeneration and safron demonstrated a direct action on this receptor and, in both cellular models, the presence of crocin guaranteed a high level of protection from stress induced by the ATP extension [39].
Another cellular model was developed to study the fbrillation propensity of α-synuclein, a presynaptic neuronal protein, that can trigger Parkinson's disease (PD).Specifcally, the E46K mutation of the α-synuclein gene has been linked to autosomal dominant early-onset PD.Crocin was studied to evaluate its ability to reduce the formation of the amyloid fbril of α-synuclein E46K.Crocin dose dependently inhibited the amyloid formation by incubating α-synuclein E46K as demonstrated by the reduction of the fuorescence intensity of thiofavin-T.Static light scattering also demonstrated that crocin caused a signifcant reduction in the aggregation of E46K α-synuclein [40].
PC12 cells are a type of catecholaminergic cells that synthesize, store, and release norepinephrine and dopamine and are used as a model to study apoptosis in neuronal cells.PC12 cells were examined to evaluate the efects of crocin on serum/glucose-deprived cultures versus α-tocopherol used as a reference antioxidant.Te culture of glucose-deprived PC12 cells had a signifcant change in morphology and caused peroxidation of membrane lipids and decreased  intracellular superoxide dismutase (SOD) activity.Te oxidative stress induced the transfer of phosphatidylserine (PS) residues across the outer membrane, and these can be used as an early marker of apoptotic induction.Treatment with crocin kept the cell morphology more intact, even compared to the action of α-tocopherol.Crocin signifcantly reduced membrane-level lipid peroxidation and restored SOD activity.Crocin, therefore, appears to have a fundamental role in the modulation of antioxidant efects thanks to its SODrestoring activity.Crocin also suppressed caspase-8 activation caused by serum/glucose deprivation in a concentration-dependent manner (0.1-10 μM).Crocin did not inhibit caspase-8 activity in cell lysates and its inhibitory efect may be indirectly caused by its antioxidant activity [41].Tus, the antioxidant efect of crocin is not only a direct radical scavenger but also can help SOD restoration.
Te efects of safron components on the activity of Nmethyl-D-aspartate (NMDA), which plays an important role in regulating glutamate levels in the brain, have been analyzed.Some of these fndings were summarized in a recent review focused on safron and Alzheimer's.Crocin antagonizes the inhibitory efect of ethanol on NMDA receptormediated responses in the dentate gyrus of rat hippocampal slices [24].
Te efects of C. sativus extract and its main active ingredients were evaluated in vitro on the blood-brain barrier (BBB) function and integrity and on Aβ-related pathology.In addition, the extract was studied in 5XFAD, a mouse model for AD to evaluate its possible usefulness.In vitro results showed that the C. sativus extract increased the maintenance of membrane stability of a cell-based BBB model and improved Aβ transport.Safron inhibited tau aggregation with an IC 50 of 100 μg/mL [20].

Infammation.
Crocins can be considered potent antiinfammatory compounds, and many works reported their activity in this regard.Some of the most important mechanisms are related to the inhibition of cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2) and the blocking of prostaglandin-2 (PGE2) production by inhibiting the nuclear translocation of NF-κB p50 and p65 subunits [5].Te bioactive compounds crocin, crocetin, and safranal show encouraging efects in the prevention of diseases related to oxidative stress, such as cardiovascular and respiratory diseases, cancer, and neurological disorders [12].
Neurodegenerative diseases are characterized by the presence of infammatory markers including IL-1β, IL-6, IL-8, TNF-α, and NF-κΒ [42].A study of cognitive defcits after an intrahippocampal injection of amyloid-beta (Aβ1-40) in rats showed the benefcial efects of safranal treatment by decreasing the levels of infammatory markers [19].Hippocampal Aβ1-40 lesions increase the levels of deleterious free radicals and mitochondrial dysfunction.After safranal treatment, mitochondria reestablish the membrane potential suggesting a benefcial efect of safron against mitochondrial dysfunction [19].Te activity of crocins was studied in HT22 cells and Alzheimer's disease mouse model.In particular, positive efects were observed in l-Glu-damaged HT22 cells; moreover, the ability of crocin to improve memory abilities and cognitive functions was verifed in AlCl3 and d-gal-induced AD mice [43].
Te protective efect of safranal on toxicity and oxidative damage induced by beta-amyloid (Aβ) and hydrogen peroxide was studied in PC12 cells as a model of Alzheimer's cell damage.When PC12 cells are exposed to amyloid-beta, safranal treatment reduces amyloid-beta-induced apoptosis via the P13K/AKT and MAPK/ERK pathways [43], thus, demonstrating safranal protection against free radicals produced by H 2 O 2 .[43].Studies revealed that lead exposure could be a risk factor in the development of Parkinson's disease, and for this reason, the possible therapeutic potential of C. sativus was evaluated.In detail, C. sativus (50 mg/kg BW) was administered by oral gavage to counteract the neurotoxic efect of Pb (25 mg/kg BW i.p) for three consecutive days on the dopaminergic system and locomotor performance in Meriones shawi.Animals treated with safron presented protection, and their locomotor activity was restored signifcantly compared to the control group [43].
Chronic restrictions can induce depressive behaviors in mice.Te efects of safron on depression as well as its neuroprotective and pharmacological efects on the intestinal function of crocetin in mice exposed to chronic restraint stress were studied [43].Administration of a crocetin-containing extract attenuated the levels of MKP-1, proBDNF, alanine transaminase, and aspartate transaminase and increased serum dopamine and CREB levels.Histopathological analysis showed that crocetin attenuated hippocampal damage in stressed mice and demonstrated a protective efect in neuronal cells.Immunofuorescent and WB analyses showed increased expression levels of ERK1/2 and CREB and inhibited expression levels of MKP-1 and proBDNF in the hippocampus.After treatment with crocetin, the intestinal ecosystem partially recovered to the level of the control group.Tis work concludes that crocetin has potential neuroprotective properties and ameliorates the efects of stress-related brain damage by regulating MKP-1-ERK1/2-CREB signaling and the intestinal ecosystem [43].
Several in vitro studies and many in vivo studies describe safron as an important antioxidant agent and its efcacy seems to be attributed to the synergy of its bioactive ingredients.

Mitochondria and Safron.
Mitochondria play different key roles in the cell.Among their multiple duties, their primary function is ATP production through oxidative phosphorylation.Even though mitochondrial density in neurons is not the highest, the brain consumes ten times more glucose and oxygen than other tissues; thus, it is not surprising that mitochondrial dysfunctions are linked with several neurological pathologies.Furthermore, it is worth mentioning that, on the one hand, mitochondria play a role in the catabolism of carbon-rich fuel molecules; on the other hand, they control the intrinsic apoptotic pathway.Tus, mitochondria act as yin and yang, simultaneously coordinating cell growth and cell death pathways [44].
Mitochondria are the primary source of reactive oxygen species (ROS) formation in the form of hydrogen peroxide and superoxide anion; indeed, the function of the mitochondrial Journal of Food Biochemistry transport chain is coupled with ROS production [45].In terms of mitochondrial ROS overproduction, the increased oxidative stress could trigger mitochondrial damage resulting in the initiation of apoptosis in cells, especially in nonmitotic and long-living cells, such as neurons.In addition, enhanced oxidative stress and decreased antioxidant capacity are linked with several CNS disorders, such as neurodegenerative diseases; thus, the crucial role of endogenous or exogenous antioxidant molecules is not up for discussion.
Herein, we will discuss the role of safron in ameliorating mitochondrial activity by decreasing neuronal cell death and enhancing antioxidant defenses in diferent pathological conditions.
In the last two decades, several works focused on Parkinson's disease (PD), a well-established neurodegenerative condition of the CNS characterized by uncontrollable movements and balance and coordination difculties, which are caused by extensive cell death of neurons located in the substantia nigra pars compacta.It has been demonstrated that crocetin treatment enhances the antioxidant capacity decreasing neuronal cell death in the substantia nigra of a rat model for PD [46].Notably, the brain transcriptome of a PD mouse model treated with safron extract shows increased expression of genes linked with neuroprotective activity; several mitochondrial genes linked with antioxidant defense were found [22].Moving to an easier in vivo model, such as Drosophila melanogaster, rotenone-induced neurotoxicity, which specifcally triggers mitochondrial dysfunction, is reduced in a fy model of PD by safron methanolic extract treatment.Interestingly, safron treatment rescues locomotor activity in rotenone-treated fies [47].Successively, similar experiments were carried out in a mouse model of neurotoxin-induced PD; also in this case, daily crocin treatment is sufcient to attenuate the rotenone-induced neurotoxic efect in the striatum.In particular, crocintreated mice show decreased anxiety and increased motor coordination and exploratory behavior.Mainly, enhanced levels of mitochondrial enzymes were detected in the striatum, supporting safron's positive efect on mitochondrial functionality [48].Te ability of safron to sustain mitochondrial activity was also confrmed in a study on depression.In detail, crocin-I preserves oxidative phosphorylation and ATP production by attenuating oxidative markers in the hippocampus of the chronic corticosterone-induced depression mouse model [49].
Finally, a few studies underlined the crucial role of safron in apoptosis regulation.It has been demonstrated that crocin exhibits antiapoptotic behavior modulating the expression of Bcl-2 family proteins in neuronally diferentiated PC12 cells [50].Crocetin can also counteract photoreceptor degeneration and retinal dysfunction through caspase-3 and caspase-9 inhibition [51].Importantly, this peculiar antiapoptotic activity of safron was also demonstrated in two diferent in vivo models of Alzheimer's disease, another common neurodegenerative disorder; specifcally, hippocampal and intraperitoneal crocin administration is sufcient to inhibit beta-amyloid-induced apoptosis [21,52].
Tese data indicate that mitochondrial activity is involved in the protective efect of safron on cell death and oxidative damage in the CNS (Figure 2).However, even if safron is used in Chinese traditional medicine and it well established its antioxidant capacity, little is known about the mitochondrial molecular mechanisms involved in this scenario.Tus, more efort should be made to unveil specifc mitochondrial pathways regulated by safron.

Clinical Applications of Safron (Crocus sativus L.) and Its
Constituents in CNS Disorders.Safron and its active biomolecules such as crocin, crocetin, and safranal have shown several clinical applications in CNS disorders.Safron extract has been studied in more than twenty antidepressant clinical trials versus placebo or standard therapy, showing efectiveness as an antidepressant drug.An international task force of Te World Federation of Societies of Biological Psychiatry (WFSBP) and the Canadian Network for Mood and Anxiety Disorders (CANMAT), involving 31 clinicians and leading academics from 15 countries, indicated safron extract as a complementary treatment in depression [53].

Depression and Anxiety.
Depression and anxiety are two of the prevalent mood disorders and the most relevant causes of disability worldwide [57].Although several drugs have been approved for these conditions, their use is often associated with severe side efects or inefcacy.In addition to medical treatment, in recent years, plant-derived products are providing interesting applications both alone in the early phase of the diseases and in association with psychiatric drugs through a network pharmacology approach [58,59].Herbal medicines, food supplements, and phytotherapy approaches provide a plethora of bioactive molecules and entire phytocomplexes potentially useful for mood disorders [60,61].Safron and its main bioactive components crocin and safranal are promising candidates.Safron has demonstrated antidepressant efects in clinical studies alone or associated with other natural compounds such as curcumin [25,62].
In a 12-week double-blind, placebo-controlled trial, the efect of safron on anxiety and depression was evaluated.Sixty adults with depression and anxiety randomly received a 50 mg safron capsule or placebo twice daily for 12 weeks.BDI and BAI scores were assessed at baseline and at 6 and 12 weeks.Te results showed signifcant antidepressive and anxiolytic efects of safron extract compared to placebo at 12 weeks with rare side efects [76].
Safron's antidepressant and anxiolytic efects were also evaluated in 68 teenagers aged 12-16 years with mild to moderate anxiety or depression symptoms, in an 8-week, randomized, double-blind, placebo-controlled study.
Teenagers in the safron (Afron ® , 14 mg b.i.d) group re- ported relevant improvements in depression, overall internalizing symptoms, social phobia, and separation anxiety analyzed with the Revised Child Anxiety and Depression Scale (RCADS).Te authors highlighted the limitations of the study with the use of a self-report questionnaire, a single dose, and the parent's result showing no diferences [77].
Interestingly, a recent study aimed to measure in untrained young males safron's efect combined with six weeks of resistance training (RT) on neurotransmitters and biomarkers related to depression and happiness levels.Safron (150 mg) was given immediately after each RT session and at the same time on nontraining days.Te results showed a signifcant increase in 2-arachidonoylglycerol (2-AG), anandamide (AEA), dopamine, and β-endorphin in the safron group, while serotonin levels and happiness scores increased in both groups as well as muscular endurance [78].
Since ancient times, on Santorini island, safron was used as a remedy for depression and mood changes in women during the menstrual cycle, menopause, or postpartum [79].In the last decade, many clinical trials have investigated safron use for treating these women's conditions [30,[79][80][81][82].In a recent study, sixty women presenting postmenopausal hot fashes received either safron (30 mg/day; 15 mg twice per day) or a placebo for 6 weeks.Te authors reported a signifcant improvement in hot fashes and depressive symptoms measured with the Hot Flash-Related Daily Interference Scale (HFRDIS) and the Hamilton Depression Rating Scale (HDRS).Safron use results as an efective and safe treatment in improving postmenopausal symptoms in healthy women [79].
Two main clinical trials have shown safron efectiveness in mild to moderate postpartum depressive disorder.One double-blind, randomized, and placebo-controlled trial was performed on sixty new mothers with a maximum score of 29 on the Beck Depression Inventory-Second Edition (BDI-II).Patients received safron (15 mg/b.i.d) or placebo treatment.Safron treatment showed a more signifcant impact on the BDI-II scores (96% remission) than the placebo (43% remission) [80].A second double-blind clinical trial compared the efectiveness and safety of safron (15 mg capsule) or fuoxetine (20 mg capsule) in women aged 18-45 years with mild to moderate postpartum depression with an HDRS score of ≤18.Safron supplementation was a safe and efective alternative treatment for improving depressive symptoms of postpartum depression [68].
Safron's antidepressive and antianxiety properties were confrmed by three relevant, recent meta-analyses.According to the frst one, safron showed signifcant antidepressive activity, more than placebo, and was noninferior to standard antidepressant drugs [66].Te second meta-analysis in 2020, included twelve studies that compared the HAM-D or BDI scores, remission, response rate, and adverse efects between safron and placebo or safron and antidepressant drugs.Overall, results indicated a better improvement of depressive symptoms with safron compared to placebo and the same efectiveness with antidepressant drugs [25].
In 2022, a relevant umbrella meta-analysis, including 7 meta-analyses, was carried out to fully understand the evidence of safron's efectiveness in depression.Safron use signifcantly decreased the BDI scores, with no change in the HAM-D scores and mixed scores (HAM-D/BDI/DASS) [83].Overall, safron might alleviate depression symptoms, but it cannot be a single therapeutic option to treat depression [64].Based on the abovementioned meta-analysis and clinical trials, a minimum of 4-6 weeks of safron treatment is required for antidepressant and antianxiety efects, with a daily dose ranging between 28 and 150 mg.Safron might represent an alternative to standard antidepressants in the treatment of early symptoms of depression and anxiety.In combination with standard therapy, it seems to be a promising approach to treating mild to moderate depression.However, multicenter trials with longer treatment duration, larger sample sizes, and crossover, as the third phase of clinical trials, are necessary for a better understanding of safron efectiveness in depression and anxiety.Furthermore, the use of standardized preparations with a precise amount of active constituents will be of help in understanding the relevance of safron extracts in this therapeutic area.

Sleep Disorders.
Sleep disorders are common complaints afecting human performance and may be experienced at diferent ages, in many cases becoming a chronic condition impacting patient's daily life.Sleep disorders can also be a symptom of other disorders, for example, depression, and frequently consist of repeated awakenings during the nighttime, prolonged wakefulness during the sleep period, long sleep latency, and transient arousals with reduced sleep quality.With pharmacological therapies, herbal remedies are commonly used as complementary and alternative insomnia treatments.In particular, safron, crocin and safranal, has shown relevant hypnotic efects due to the inhibition of the wakefulness-promoting neurons in the tuberomammillary nucleus and the simultaneous activation of the sleep-promoting neurons in the ventrolateral preoptic nucleus.Diferent clinical trials and two recent meta-analyses have highlighted safron's hypnotic properties [27,28,[84][85][86] and one clinical trial for safron's bioactive molecule crocetin [87].In a 28-day, 3-arm, parallel-group, double-blind, randomized controlled trial, the efect of 14 mg, or 28 mg of a standardized safron extract (Afron ® ) 1 h before bed, on 120 adults with unsatisfactory sleep versus placebo was measured using the Insomnia Symptom Questionnaire (ISQ), the Pittsburgh Sleep Diary, Restorative Sleep Questionnaire, Profle of Mood States, the Functional Outcomes of Sleep Questionnaire, and evening salivary cortisol and melatonin levels [27].Safron supplementation induced relevant improvements in sleep quality ratings, ISQ total score, and ISQ-insomnia classifcations and mood ratings after awakening with no diferences in the other questionnaires and outcomes.Moreover, safron supplementation induced an increase in evening melatonin concentrations without afecting cortisol levels.No adverse side efects were reported [27].
A recent meta-analysis including eight articles with a total of 431 participants has shown a reduction of insomnia and increased sleep quality and duration with the safron treatment compared to placebo groups [28].Another metaanalysis run by Lian et al. in 2022 [84] compared the efect of diferent safron dosages on sleep quality among healthy adults, patients with insomnia or type 2 diabetes, and patients under methadone maintenance treatment.Safron supplementation (100 mg/day) improved sleep quality in the subgroup analysis compared to placebo according to the Pittsburgh Sleep Quality Index (PSQI), the Restorative Sleep Questionnaire (RSQ), and the Insomnia Severity Index (ISI).
Overall, even though evidence of a moderate-to-high quality is still limited, safron supplementation benefts people with insomnia and may reduce the use of sedative drugs, dependency, and withdrawal symptoms.Furthermore, RCT and meta-analysis with larger sample size and long duration are required to consolidate safron benefts in diagnosed insomnia and other psychogenic and demographic characteristics and to unveil its potential sleepenhancing mechanisms of action.

Attention-Defcit/Hyperactivity Disorder (ADHD).
ADHD is a common neuropsychiatric disorder in childhood and adolescence.More than 30% of patients do not respond appropriately to standard therapy or cannot tolerate its side efects.Tus, alternative treatments, such as herbal medicine, are under investigation [88].Safron extract has been evaluated in at least fve clinical trials for its efectiveness in ADHD compared to standard therapy methylphenidate (MPH) or in combination with MPH [55].
A randomized double-blind 6-week study considered 54 children (aged 6-17 years) with a diagnosis of ADHD.Patients received either MPH (20-30 mg/die) or safron (20-30 mg/die) randomly.Results revealed no signifcant changes in teacher and parent ADHD Rating Scale scores between the safron group and the MPH group, suggesting that short-term safron supplementation can have the same efcacy as MPH treatment [89].
Two studies evaluated the efectiveness of MPH in combination with safron (20-30 mg/day) in children with ADHD: in the frst study, 70 children aged between 6 and 16 years diagnosed with ADHD were treated with a combination therapy of MPH and safron and resulted in more efectiveness compared to separate treatments [90].In a second randomized, double-blind, placebo-controlled clinical trial, ffty-six patients with ADHD received Ritalin ® (30 mg/day) plus placebo or Ritalin ® plus safron (15 mg twice daily) for six weeks.When safron was added to therapy with Ritalin, symptoms were efectively improved [29].
A recent clinical trial investigated the efect of psychoeducation and safron (30 mg/day) versus psychoeducation and extended-release MPH in 63 children aged 7-17 with ADHD for 3 months.Children and their parents could choose their treatment group.Results showed comparable efcacy of safron to that of MPH.Interestingly, safron was more efective in hyperactivity symptoms, while MPH was more efective in inattention symptoms [91].
Despite safron seeming a promising natural medicine for ADHD, further RCT and meta-analysis with larger size samples and long duration are required.

Multiple Sclerosis. Multiple sclerosis (MS
) is a debilitating autoimmune disease resulting in progressive demyelination and neurodegeneration.Excessive oxidative stress, infammation, and DNA damage have been observed 10 Journal of Food Biochemistry in MS patients together with fatigue and mental and psychological discomfort.Due to its anti-infammatory and antioxidant capacity and antidepressive and cognitiveenhancing properties, safron supplementation has recently been investigated as a complementary adjunct supplementation in multiple sclerosis (MS).
A prepost study evaluated the consumption of 7.5 cc every 8h for two months of safron syrup to relieve the fatigue of 30 MS patients according to the fatigue severity scale (FSS).Results showed promising antifatigue efects of simple safron syrup in patients with MS with no signifcant side efects [92].
Patients with MS often possess a higher level of oxidative stress, infammatory markers, and DNA damage than healthy subjects [93,94].In one study, the efect of oral crocin on oxidative stress in MS patients in addition to standard treatment for 4 weeks was tested.Te crocin group (15 mg twice daily) resulted in a signifcant increase in total antioxidant capacity levels, saliva total thiol groups, and catalase activity and a marked decrease in lipid peroxidation level.According to the study, crocin can reduce several oxidative stress factors in MS patients [95].Another study aimed to evaluate the efect of crocin on oxidative damage, infammatory markers, and DNA damage in the blood of MS patients.40 patients were randomly divided into two groups where the intervention groups received two crocin capsules per day for 28 days.A signifcant reduction in DNA damage, lipid peroxidation, tumor necrosis factor-alpha, and interleukin-17 and a signifcant increase in the total antioxidant capacity were measured in the serum of MS patients treated with crocin, suggesting a benefcial efect of crocin in MS patients [96].
In MS patients, matrix metalloproteinase-9 (MMP-9) facilitates T-cell migration to the CNS, while tissue inhibitors of metalloproteinase-1 (TIMP-1) decrease MMP-9 activity [97].Safron supplementation in relapsing-remitting MS patients for 12 months noticeably decreased MMP-9 serum levels in patients with MS and signifcantly increased the levels of TIMP-1.No diferences were observed with the placebo [98].
A recent systematic review of randomized controlled trials (RCTs) comparing safron supplementation to placebo, or other interventions, in patients with an MS diagnosis, concluded that safron supplementation might improve antioxidant defense and reduce excessive oxidative stress in MS patients.Te strength of the evidence is limited, scattered, and inadequate, and further better-designed trials with MS-specifc outcomes are required [26].

Te Use of Safron as Nutraceutical for Preventive and
Protective Activities on CNS.As seen so far, diferent preclinical and clinical trials have revealed the potential usefulness of safron in diferent CNS pathological manifestations.As shown in Table 3, doses used in many experiments with patients are relatively low, ranging from 15 to 150 mg/die.Literature indicates that at those doses that are considered therapeutic, no signifcant toxicity is reported [2,6,33].Te large amount of preclinical and clinical data showing the potential usefulness and low toxicity suggest the potential use of safron in large part of population ranging from children to elderly people.Te relevant role of safron constituents on oxidative stress and mitochondrial metabolism underlines the basis of its possible application as a preventive and protecting agent especially related to CNS due to its sensitivity to oxidative stress.Several studies have shown a signifcant role of safron as an active agent in Parkinson's disease [5,22] and in some models of Alzheimer's disease [20,24,43], and thus, it can be presumable that its supplementation in healthy subjects can be protective to control ROS-related degenerative processes, supporting its role as a nutraceutical.
Due to the need to administrate sufcient doses of bioactive safron constituents, attention should be paid towards levels of safron compounds and their bioavailability when used in products.Nowadays, standardization and analysis of diferent classes of safron constituents can be performed with great attention and confdence using several analytical approaches.Nevertheless, in the market products, safron can be present both with optimal levels of phytoconstituents or without any precise determination.Terefore, to establish the quality of safron supplements, the chemical constituents should be kept in mind.
Many research studies have been performed in the area of safron considering the various extraction procedures ranging from the more traditional going to the more sustainable and technologically driven [99,100].More recent papers underlined the importance of plant material selection and the standardization of the active constituents in the starting materials to ensure the accurate amount in the fnal formulated product [101].Up to now, limited information is available related to the product used in the food supplement or nutraceutical area, and more research studies are needed both in the analytical approach and in the whole processing of the product to ensure accurate labeling.Up to now, the published literature allows us to consider the average doses that we reported in Table 3 and in the further chapters as schematized in Figure 3. Te new challenge for the future application of safron will be the gap-flling between the observed clinical efects and the "active" constituents that can be extracted or formulated in various forms.

Bioavailability of Main Safron Constituents.
Te efcacy of a nutraceutical product containing safron and its specifc activity on the CNS system imply the absorption distribution and metabolism of the phytochemicals involved in the bioactivities.Te study of the bioavailability of natural products is extremely challenging because plant extracts are composed by diferent chemicals and can be contained in diferent matrices.Plant-derived products are chemically diferent and present distinct solubilities.In addition, they can be modifed by host and microbial metabolism, and their plasmatic levels can be altered by the presence of other compounds such as saponins, polysaccharides, or proteins.Safron apocarotenoids have been studied, and some papers described pharmacokinetics and bioaccessibility.In 2018, a review summarized the main information about the Journal of Food Biochemistry Journal of Food Biochemistry pharmacokinetic properties of safron and its constituents [102].More recently, other authors evaluated the bioaccessibility of safron constituents or at least their plasma levels after the administration of commercial extracts [16,32,36,103].Some papers considered the carotenoids of safron and their pharmacokinetics or absorption after oral administration.Results have been reviewed by Hosseini et al. in 2018 [102] and more recently by Song et al. [104].Crocin and its derivatives contain ester linkage with sugars and crocin-I had a strong water solubility and weak lipophilicity and was not easily absorbed by oral administration [104].Furthermore, after oral administration, at least a part of this ester is cleaved [36,104] by the acidic medium of gastric fuid or by enzymatic activities present in the gastrointestinal tract.Crocin bioavailability has been extensively studied measuring the crocetin plasmatic levels due to this modifcation [102].Tanks to its lower molecular weight and the absence of sugar residues, crocetin is more easily absorbed than crocin.Indeed, crocetin is rapidly eliminated from plasma.Most of the studies published so far related to the bioaccessibility and bioavailability of safron constituents were mostly conducted in in vitro experiments, cell models, and some cases animal tests [36,104].Crocetin penetrated the intestinal tract through passive transcellular transport without specifc carriers as transcrocetin [104].Studies performed in animal models indicated that after oral dosing of crocins or crocetin, only crocetin can be measured in the rat and mouse plasma in the form of free glucuronide, monoglucuronide, and diglucuronide conjugates [2,36,104].Tus, crocins are highly metabolized and converted into smaller and more lipophilic crocetin.Furthermore, when crocin is administered intravenously, lower crocetin concentration is observed in plasma, suggesting that crocin is converted into crocetin mainly in the gastrointestinal tract [102].In 2018, the penetration of trans-4 crocin in mice brains after intraperitoneal injection was reported [105] confrming that the transformation of crocin into crocetin occurs only in the GI tract.A schematic representation of the crocetin absorption is represented in Figure 4.
Recently, a lyophilized extract mostly containing transcrocetin was used to assess the pharmacokinetics of crocetin and its metabolite after the administration of a single dose (i.v. and oral) of safron extract at 60 mg/kg of body weight to male mice (C57Bl/6J).Te authors reported that the kinetic can be described by a one-compartment PK distribution model and crocetin's oral absorption can be described by frst-order kinetic constant.
Serum and tissue noncompartmental pharmacokinetic analysis revealed a relative oral bioavailability for total crocetin that was calculated to be 1.17 times the administered crocin.Tis is caused by the modifcation of the diferent crocin derivatives into crocetin.Furthermore, the authors observed extensive crocetin distribution to the liver and kidneys [32].Te pharmacokinetics of these compounds are complicated because the crocin present in aqueous safron extract is subjected to a rapid hydrolysis in the gastrointestinal tract leading to crocetin formation.Tis latter compound is extensively absorbed as demonstrated by the measured serum and tissue crocetin and crocetin levels Te total crocetin serum AUC values were 1.5-2.5 times higher than those measured for crocetin refecting crocetin glucuronidation [32].
A key point for the evaluation of the safron constituent's bioavailability is to use standardized extracts.In recent years, some works have proposed pharmacokinetic studies on specifc extracts.Afron ® , a standardized extract, has been used to study the efect of in vitro digestion on the main bioactive components of safron extract tablets and to evaluate the pharmacokinetic parameters in humans after the administration of the product to thirteen healthy volunteers [36].Te extract contains 3.6% of total crocins, 3.2% of picrocrocin, 0.13% of kaempferol diglucoside, and 0.04% of safranal.Te in vitro digestion induced an increase in the safranal content (with 200% bioaccessibility) and a decrease in the picrocrocin content (81% bioaccessibility) due to the hydrolytic processes and a reduction in kaempferol diglucoside (70% bioaccessibility) [36].In the plasma of volunteers receiving 56 mg or 84 mg of afron, crocins, picrocrocin, and safranal was not detected.Crocetin was detected, and its concentration in plasma grew up to 60 and 90 min, respectively, and then gradually decreased [36].Te same authors reported that a standardized extract containing crocetin induces the same Tmax of crocin when compared to previous studies in which purifed crocin was administered.In addition, crocins, thanks to the sugar moieties, are probably more bioavailable to enterocytes for later hydrolysis and absorption than the arrival of crocetin itself [36].
Other papers described safron's constituent bioavailability after an oral administration to 10 healthy volunteers receiving 300 mg of the safron extract.Te extract used was a standardized product containing crocins (mainly trans-4-GG, trans-3-Gg; cis-4-GG, and trans-2-G) >3%, safranal >0.2%, picrocrocin derivatives (mainly picrocrocin and HTCC) >1%, and kaempferol derivatives (mainly kaempferol-3-sophoroside-7-glucoside and kaempferol-3sophoroside) >0.1%, measured by the UHPLC method.Crocetin was used as a validated marker of safron extract absorption.Te absorption profle showed that the maximum crocetin concentration was reached 90 min after ingestion [103].No information was reported related to other constituents of the extract.Up to now, studies considering the pharmacokinetics of diferent compounds are still lacking, and further research is needed to clarify the role of favonoids and other constituents beyond crocins able to infuence the bioavailability of active safron compounds.

Conclusion
Safron, the "red gold," is a spice used and known in several parts of the world and is largely present in cooking traditions as well as used as a traditional remedy.Many studies have considered this spice for its potential bioactivities showing relevant antioxidant and anti-infammatory activities using diferent in vitro models showing potential usefulness for the CNS system, especially considering the crucial role of oxidative stress in diferent pathological conditions of the brain.Due to the low toxicity of safron and its constituents, there is a renewed attention to this spice as a potential nutraceutical ingredient targeted on CNS.In particular, several studies have shown signifcant efects in depression, ADHD, multiple sclerosis, and sleep disorders.
Te new challenge for a more efective and safe use of this nutraceutical will be a more deep investigation on the diferent chemical constituents and their role in the observed efects, thus the need of accurate standardization of the product.Furthermore, the role of such products as protective remedies is able to reduce at least in some parts of the population the onset of pathological conditions such as insomnia or depression ofering a nutritional or nutraceutical valuable approach.

Figure 1 :
Figure 1: Safron as a possible nutraceutical for CNS-related diseases due to its efcacy and safety profle.

Figure 2 :
Figure 2: Chart of CNS disorders investigated with supplementation of safron, doses used in clinical trials, and positive aspects of the treatment.

Figure 3 :
Figure3: ROS is considered signaling molecules and dangerous elements that afect normal cellular homeostasis.Terefore, a balance between their actions as regulatory molecules of signaling pathways and damaging signals should be maintained to sustain cellular homeostasis.However, during stress conditions, the equilibrium shifts toward increased oxidative stress.With its antioxidant capacity, safron can restore normal cellular homeostasis.

Figure 4 :
Figure 4: Schematic representation of the formation of crocetin in the GI tract and its absorption.

Table 1 :
A summary of the most recent studies considering specifc efects of crocus on CNS diseases.

Table 2 :
Main paper and fndings considering possible clinical applications of safron on CNS diseases.

Table 3 :
Studies involving the use of safron or safron extracts in depression.