Current Knowledge of the Antidepressant Activity of Chemical Compounds from Crocus sativus L.

Psychotropic effect of Crocus sativus L. (family Iridaceae) biologically active chemical compounds are quite well documented and they can therefore be used in addition to the conventional pharmacological treatment of depression. This systematic review on antidepressant compounds in saffron crocus and their mechanisms of action and side effects is based on publications released between 1995–2022 and data indexed in 15 databases under the following search terms: antidepressant effect, central nervous system, Crocus sativus, cognitive impairement, crocin, crocetin, depression, dopamine, dopaminergic and serotonergic systems, picrocrocin, phytotherapy, neurotransmitters, safranal, saffron, serotonin, and biologically active compounds. The comparative analysis of the publications was based on 414 original research papers. The investigated literature indicates the effectiveness and safety of aqueous and alcoholic extracts and biologically active chemical compounds (alkaloids, anthocyanins, carotenoids, flavonoid, phenolic, saponins, and terpenoids) isolated from various organs (corms, leaves, flower petal, and stigmas) in adjuvant treatment of depression and anxiety. Monoamine reuptake inhibition, N-methyl-d-aspartate (NMDA) receptor antagonism, and gamma-aminobutyric acid (GABA)-α agonism are the main proposed mechanism of the antidepressant action. The antidepressant and neuroprotective effect of extract components is associated with their anti-inflammatory and antioxidant activity. The mechanism of their action, interactions with conventional drugs and other herbal preparations and the safety of use are not fully understood; therefore, further detailed research in this field is necessary. The presented results regarding the application of C. sativus in phytotherapy are promising in terms of the use of herbal preparations to support the treatment of depression. This is particularly important given the steady increase in the incidence of this disease worldwide and social effects.


Symptoms of Depression
Major depressive disorder (MDD), known also as depression (lat. depressio 'deepness' from deprimere 'overwhelm'), is a chronic, recurrent, and potentially life-threatening mental disorder characterised by at least two weeks of omnipresent low mood. It is usually accompanied with persistent feeling of sadness, anhedonia, pain without a clear cause, difficulties in thinking and concentration, loss of interest in doing anything, psychomotor retardation, fatigue, spending time sleeping, feelings of worthlessness or inappropriate guilt, and recurrent thoughts of death. These symptoms cause distress or impairment in social life and are not an effect of the influence of other medical conditions [1][2][3][4][5][6][7][8]. The Table 1. Raw material of selected plant species from various families with antidepressant properties.

Application of C. sativus in Herbal Medicine and Industry and Therapeutic Activity
Crocus sativus is used in Asian and, in particular, Indian (Ayurveda) and Persian (Islamic) traditional medicine (ITM) as a sedative agent to strengthen the body against such stresses as trauma and anxiety, an anticonvulsant and memory enhancer, and a remedy for alleviation of chronic fatigue, depression, and inflammation [71,135]. This therapeutic activity of Crocus, known since the 6th century BC, has been confirmed in the most recent basic research conducted on animals (rodents) and in human clinical studies [134,[175][176][177][178][179][180][181][182][183]. Currently, there is a search for new methods of treatment based on the use of phytochemicals contained in herbal raw materials with significant efficacy in relieving the symptoms of depression confirmed by meta-analyses and clinical trials [75,90,111]. The numerous side effects of antidepressants as well as the attitudes of many patients preferring herbal rather than conventional drugs support the assessment of the impact of saffron crocus stigmas on depression patients [70,71].
In vitro studies have confirmed the antigenotoxic and anticytotoxic effects of active substances isolated from C. sativus [245]. This should be emphasised, as other aspects of the pleiotropic activity of some cytokines and a wide spectrum of the impact of the transcription factor called the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is present in almost all animal type cells [275][276][277][278].
As reported by Wang et al. [226], the antidepressant properties of stigma aqueous extracts are related to the presence of crocin 1, but further studies regarding the precise site and mechanism of the anti-depressive action of chemical compounds isolated from petroleum ether and dichloromethane fractions of C. sativus corms are required. Karimi et al. [175] have found that flavonoids and anthocyanins are the main constituents involved in the antidepressant action of C. sativus extracts.
Considering the multidirectional phytotherapeutic effect of C. sativus, this paper is a review of available literature data and presents the current information about the effectiveness of bioactive chemical compounds contained in this species and the mechanisms of their action in the supportive therapy of depression, with emphasis on the safety of application of these substances. The thesis of the antidepressant effectiveness was verified by an analysis of the results of the latest basic research conducted in animal models, and human clinical trials. Additionally, the study highlights the difficulties and limitations in laboratory analyses and clinical studies of the antidepressant effects of the phytochemicals and indicates further perspectives of research on their use and potential methods for control of treatment in relation to the disease pathogenesis.
Since depression is a serious growing global health problem with social and economic consequences, intensified investigations are being carried out to search for biologically active chemical compounds of plant origin, which will prove effective in supporting the treatment of this disease. Crocus sativus L. is a species known for its healing properties and widely used in folk medicine to alleviate the symptoms of many diseases. The thesis on antidepressant effectiveness was verified by analysis of the results of the latest basic research in cell cultures, animal models and human clinical trials. Table 3. Therapeutic effects of selected biologically active chemical compounds from the classes of anthocyanins, terpenoids, and saponins extracted from C. sativus corms and flowers.   Table 4. Therapeutic effects of selected C. sativus biologically active chemical compounds from the class of phenolic compounds and essential oils.

Classes of Biologically Active Chemical Compounds
Biologically Active Chemical Compounds Organ Therapeutic Effects Reference Polyphenol pyrogallol stigma antioxidant [171] Phenolic acid benzoic acid derivatives gallic acid p-hydroxybenzoic acid corm [283] salicylic acid gentisic acid syringic acid cinnamic acid derivatives caffeic acid p-coumaric acid t-ferulic acid cinnamic acid

Carotenoids Therapeutic Effects Reference
Crocin inhibited xylene-induced swelling of mouse ear and increased capillary permeability and writhing induced by acetic acid in mice; at 50 mg/kg, it inhibited carrageenan-and fresh egg white-induced oedema of the hind paw in rats. It inhibited sheep red blood cells (SRBC)-induced footpad reaction and inhibited picryl chloride-induced contact dermatitis [285] cytotoxic effect on human and animal adenocarcinoma cells (HT-29 and DHD/K12-PROb cells) [286] a prolonged blood coagulation time in mice and markedly inhibited dose-dependent thrombin-and ADP-induced blood platelet aggregation in rabbits (in vivo); an inhibitory effect on thrombus formation in rats with arteriovenous shunt and relieved respiratory distress due to pulmonary thrombosis in mice induced by ADP and AA [287] cardiovascular protective effects; the cardioprotective effects of crocin may be attributed to the attenuation of [Ca 2+ ] through inhibition of ICa-L in rat cardiomyocytes as well as negative inotropic effects on myocardial contractility [288] it affected tubulin polymerisation and structure, increased the microtubule nucleation rate, induced conformational changes in tubulin, and affected several cell processes through interaction with tubulin proteins or microtubules [289] Crocetin vasomodulatory effects in hypertension, improvement of endothelium-dependent acetylcholine relaxations via endothelial nitric oxide, improvement of acetylcholine-induced vascular relaxation in hypertension [290] Crocetin interaction of carotenoids with topoisomerase II, an enzyme involved in cellular DNA-protein interaction, immunomodulatory activity on T Helper Cell Type 1 (Th)1 and Th2, anticancer properties [

Methodology
This review is a presentation of possible treatment methods available across the range of herbal medicines that are relevant to the pathogenesis of depression, with the indication of ways of treatment control with clinical tests used by authors of the cited papers and medical imaging of brain functions for the future scientific purposes. This publication is based on a search in scientific databases of literature reports covering the contemporary research on antidepressant bioactive substances from Crocus sativus L.  The content of safranal in saffron crocus stigmas is in the range of 0.1-0.6% d.w. [302][303][304], however, other authors have reported that the content of this compound ranges from 1.07 to 6.15% d.w. [305]. In turn, there are also reports showing that the content of safranal in red stigma samples were 49.64 and 50.29%, while in threads with yellow styles it was 50.42%, 57.02% and 61.31% [306]. The concentration of crocin was estimated at 20-30% of the total dry matter of the spice [154,303,307], but some study results revealed a much wider range of this compound, i.e., 0.85-32.4% [305]. As reported by Zhang and coworkers [308], the content of crocin varied significantly among saffron populations from seven different production areas, i.e., Nepal, Greece, Morocco, Spain, Iran, and China (Jiande, Chongming), and ranged from 80.59 to 230.36 mg/g. Zeka et al. [309] reported that dried petals contained 0.6% of crocin. As suggested by Acar et al. [302] the crocin content of commercial saffron dried in a freeze dryer and dried naturally under the sun was 900 and 600 mg/g, respectively. Azarabadi and Özdemir [306] found that crocin amount was higher in red stigmas samples (66.67 mg/g) than in yellow stigmas samples (51.66 mg/g). The content of crocetin esters represents 16-30% of saffron stigma [310] Crocin is largely absent from petal extracts [311].
The picrocrocin content found in dried stigmas ranged from 0.8 to 26.6% [312,313]. Some authors propose a slightly narrower range of limits for the content of this compound i.e., 0.79-12.94% [305,314], 7-20% [315] and 5-7 mg/g d.w. [303]. The reasons for such a large discrepancy of limits in the content of safranal, crocin, crocin, crocetin and picrocorocin should be sought for in the different drying methods, and storage and extraction conditions of saffron, which degrade these compounds significantly; the degree of degradation depends on temperature, humidity, light irradiation and other compounds in the environment [305].
Othman and co-workers [316] found markedly various crocin and crocetin content in saffron crocus stigmas from different geographical origins. Iranian saffron was characterised by substantially higher amount of crocin content than Turkish and Kashmiri saffron (11,414.67 and 311.63 µg/g d.m. of crocin, respectively). In turn crocetin was detectable in Iranian and Turkish (1054.73 and 186.64 µg/g d.m. of crocetin, respectively) but not in Kashmiri saffron. These differences were suggested to be related to various environmental factors, e.g., climatic conditions, agricultural practices, and stigma separation, as well as storing and drying processes [316].
The information about physicochemical properties of the saffron crocus bioactive compounds, which are important in the preparation of medicinal formulations were presented in the Table 6. The most important stigma constituents include antioxidative carotenoids (with the water-soluble crocin and its derivatives responsible for the colour: zeaxanthin βcarotenes, lycopene), anthocyanins (delphinidin), terpenes (fat-soluble safranal responsible for the odour and aroma and its monoterpene glycoside precursor picrocrocin responsible for the special bitter flavour), polysaccharides, amino acids, proteins, starch, mineral matter, gums, and other chemical compounds [191,201,214,228,317,318].
α-Crocin (systematic IUPAC name: 8, 8-diapo-8, 8-carotenoic acid), which is primarily responsible for the golden yellow-orange colour of the stigma, is a trans-crocetin di-(β-D-) ester. Crocin, underlying the aroma of saffron, is a digentiobiose ester of crocetin. Crocins are hydrophilic carotenoids that are either monoglycosyl or di-glycosyl polyene esters of crocetin. In contrast, crocetin is a hydrophobic and thus oil-soluble conjugated polyene dicarboxylic acid. However, the product of esterification of crocetin with two water-soluble gentiobioses (sugars) is soluble in water [300,317,319].
It is believed that the glycolysed carotenoid crocetin-a natural apocarotenoid dicarboxylic acid-is the most pharmacologically active constituent of stigma extracts, besides the carboxylic carotenoid crocin. Saffron extracts and crocetin had a clear binding capacity at the phencyclidine (PCP) binding side of the N-methyl-D-aspartate receptor (NMDA receptor; NMDAR) and at the σ 1 (sigma-1) receptor, while the crocins and picrocrocin were not effective, which give the biochemical support for the pharmacological effect of saffron including depression treatment [188,191,[294][295][296]320].
Hosseinzadeh et al. [321] postulated that the flavonol kaempferol was responsible for the antidepressant effect of C. sativus petals. Kaempferol 3-O-β-sophoroside-7-O-βglucoside is the most important flavonol in saffron. Its relative content ranges from 37% to 63% of total flavonoids, and its absolute content values vary between 1.47 and 2.58 equivalent milligrams of rutin g −1 . Kaempferol 3-O-β-sophoroside is the next major flavonol in order of importance, related to the concentration in saffon. Its relative content ranges from 16% to 47% of total flavonoids with absolute content values ranging from 0.61 to 3.12 equivalent milligrams of rutin g −1 [166]. This flavonol was extracted from saffron floral bioresidues that were mainly made up of tepals, and an extract yield of 2.3 mg g −1 dry weight was obtained. Its content in tepals ranges from 0.69 to 12.60 equivalent milligrams of kaempferol 3-O-β-glucoside g 1 dry weight [169,309]. According to other literature data, the content of kaempferol-3-O-sophoroside in saffron crocus tepals was 62.19-99.48 mg/g [322]. Another flavonol found in saffron is kaempferol 3,7,4 -tri-O-β-glucoside. Its relative content ranges from 16% to 22% of total flavonoids, and its absolute content values ranges  [166]. In the stamen, the number of flavonoids was lower than in the tepal. The amount of kaempferol-3-O-glucoside, as the most abundant compound, ranged between 1.72-7.44 mg/g [322]. Structures of saffron crocus kaempferol 3-O-β-sophoroside-7-O-β-glucoside and kaempferol 3-O-β-sophoroside are presented in Figure 2.

Antidepressant Effect of C. sativus L.
Extracts of C. sativus and their active biologically chemical substances have been shown to exert beneficial effects on the activity of the central nervous system. Therefore, they can potentially be used as adjuvant agents in treatment of mental disorders, including depression [204,233,311,[332][333][334][335][336]. Literature data have demonstrated in a number of in vitro, in vivo, basic and clinical trials that dried C. sativus stigmas and petals as well as their active ingredients exhibit strong antidepressant properties similar to those of the current conventional antidepressant medications from the class of the selective serotonin re-uptake inhibitors (SSRIs), including citalopram [337], fluoxetine (Prozac) [338][339][340][341][342], and sertraline [343], as well as the tricyclic antidepressant imipramine [176,344] and the benzodiazepine diazepam [345,346]. Tables 7-10 summarise the results of preclinical studies, conducted in animal models and human clinical trials, on the antidepressant effect of extracts and bioactive chemical compounds from the saffron crocus. [230] Saffron stigma aqueous extract and its constituents, crocin and safranal 10 mL/kg normal saline as vehicle (g 1; negative control for extract and crocin); paraffin as vehicle (g 2; negative control for safranal); diazepam at a dose of 3 mg/kg (g 3; reference group); stigma extracts at doses of 56, 80, 320, and 560 mg/kg (g IV; V; VI, and VII, respectively); crocin at doses of 50, 200, and 600 mg/kg (g VIII; IX and X. respectively); safranal at doses of 0.05, 0.15, and 0. 35   saffron water extract and safranal reduced metabolic and behavioural signs of acute stress without the involvement of the amygdala. As opposite to intra-amygdala-treated groups stress did not elevate the corticosterone plasma in groups that received extract or safranal intraperitoneally. Anorexia was reduced only in groups that received the extract or safranal intraperitoneally (50 s). Intraperitoneal but not intra-amygdala administration of saffron extract and safranal counteracted stress-induced increase in sniffing, rearing, locomotion, and coping time. [347] Crocin vehicle (0.9% NaCl) + vehicle (g. 1); vehicle + crocins 30 mg/kg (g. 2); vehicle + crocins 50 mg/kg (g. 3); The non-selective serotonin(5-HT) receptor agonist mCPP 0.6 mg/kg + vehicle (g. 4); mCPP 0.6 mg/kg + crocins 30 mg/kg (g.5); and mCPP 0.6 mg/kg + crocins 50 mg/kg (g. 6). Each treatment grup consisted of 8 rats.
Male adult (3 months old), 250-300 g male Wistar Albino rats The number and duration of grooming events were recorded for 20 min.
Crocins attendued the mCPP-induced OCD (obsessive-compulsive disorder)-like behaviour (excessive self-grooming) by an antagonistic action at the non selective serotonin (5-HT) 5-HT 2C receptor site. The pharmacological mechanism(s) that might account for the effect of crocins on compulsive behaviour has yet to be determined. Active constituents of C. sativus L. crocins might play a role in compulsive behaviour that often encompasses anxiety and depressive symptoms and support a functional interaction between crocins and the serotonergic system.
[348] The level of brain neurotransmitters was assaeyd thirty minutes after drug and/or extract injection; this time is considered to be sufficient for extract action Active compounds of aqueous saffron extract triggers significant production of neutrotransmitters in brain, which is related to the effect of the extract on depression rehabilitation. Aqueous extracts of saffron (stigma) enhanced release of brain dopamine and glutamate in rats without affecting serotonin or norepinephrine concentration. These results provide a cellular basis for reports concerning the antidepressant properties of saffron extract in humans and animals. To clarify this issue the additional experiments focused on the change in dopamine concentration in brainspecific regions or serotonin concentration in the raphe nuclei are required. [349] Saffron (Crocus sativus L.) stigmas Saffron (200,400 and 800 mg/kg) administered alone or with concomitant administration of submaximal dose of imipramine (7.5 mg/kg) and compared with standard dose of imipramine (15 mg/kg) and normal saline (5 mL/kg) as neutral control. Route of adminstration: intraperitoneal injection (i.p.) A total of 48 animals (n = 48) were used for each behavioural test (FST and TST), 6 animals in each of 8 group.
20-30 g. male 3-4 months old, healthy and with normal behaviour and activity swiss albino mice The experiment was conducted 30 min after injecting the drug. The total duration of immobility in FST and TST was recorded during 4 and 6 min, respectively.
Saffron markedly reduced immobility time. Immobility time of combination of saffron with submaximal dose (7.5 mg/kg) of imipramine was significantly reduced on comparison with control and it was comparable to standard dose of imipramine (15 mg/kg) in both FST and TST. Crocus sativus L. stigmas showed significant, comparable to that of imipramine, antidepressant-like activity on its own and also added to the action of a submaximal dose of imipramine. Saffron can be considered as potential antidepressant.
[295] Crocin attenuates some neurochemical and behavioural subacute exposure malathion-induced depressive-like behaviour, in particular in the FST test. Crocin ameliorated maltion-induced brain oxidative damages via antioxidant effects, which were manifested by the increased malondialdehyde (MDA) and decreased glutathione (GSH) level in cerebral corthex and hippocampus. The neuroprotective effect of crocin may be in part due to its effect on brain-derived neurotrophic factor (BDNF). Crocin and imipramine prevented the decreasing effect of malathion on the protein level of BDNF in hippocampus. Based on the increase in P-CREB protein level together with insignificant increase in the levels of VGF, CREB, and BDNF proteins after stigma extract administration the antidepressant effect of saffron in the cerebellum is related to the enhanced phosphorylation of CREB. The slight increase in protein level of the activated form of CREB indicated that the antidepressant activity of crocin is partially mediated to CREB. Other factors than BDNF and VGF neuropeptides may alter following long term crocin treatment in the cerebellum. [352,353]  Group 1 was used as the negative control. Group 2 was used to assess for anxiolytic and/or antidepressant effects of crocin. Group 3 was used as the positive anxiolytic control. Group 4 was used to determine in the crocin group the effects attributed to the benzodiazepine binding site of the GABA A (γ-aminobutyric acid type A) receptor; the flumazenil was given 10 min before the crocin. Group 5 was used to assess for any interactions between midazolam and crocin. Route of adminstration: intraperitoneal injection (i.p.)
All drugs were administered 30 min before the first test. The elevated plus-maze (EPM) and forced swim test (FST) test lasted 5 min each. 24 h before FST the 15 min habituation session was conducted.
Crocin attenuated the anxiolytic effects of midazolam, but did not affect psychomotor activity (elevated plus-maze EPM test). The orced swim test (FST) showed a significant increase in mean mobile time in the midazolam plus crocin group, suggesting a decrease in behavioural despair because of the interaction between crocin and midazolam. Potential limitations of this study include the one-time administration of the medications instead of the recommended two or three pretest adminstration, as well as the use of much lower doses of crocin (50 mg/kg) compared with other studies (150, 300, and 600 mg/kg-see reference Wang et al. [226] Crocetin ameliorated the chronic resistant stress-induced depressive-like behaviour by decreasing oxidative damage in the brain. Crocetin treatment reduced the immobility time in FST and increased the number of crossing in OFT test in the chronic restraint stress rats. Crocetin also reverted the levels of MDA and GSH and the activities of antioxidant enzymes (catalase CAT, superoxide dismutase SOD, glutathione peroxidase GPx and glutathione reductase GR) to the normal levels in the stressed groups. Crocetin may be an effective agent in the progression of alternative medicines for ameliorating stress-induced depression.
This active constituent of saffron might inhibit behavioural modifications through alternating endocrine, oxidative, and nervous systems in rodent submitted to the long term stress.
[355] Crocin attenuates lipopolysaccharide (LPS)-induced anxiety, depressive-like behaviours and neuroinflammation through suppressing the NF-kB and NLRP3 inflanosome signalling pathway and promoting the M1 (neurotoxic) to M2 (neuroprotective) phenotypic conversion of microglia. This bioactive saffron constituent inhibited LPS-induced production of NO, TNF-α, IL-1β and ROS in BV-2 microglial cells as well as markedly declined the expression of oxygen and nitrogen metabolite-metabolising enzyme iNOS, NF-κB p65 and M1 marker CD16/32 but elevated the expression of M2 macrophage marker CD206 in the BV-2 cell line with decreased LPS-induced anxiety and depressive-like behaviours manifested by improved locomotor activity, reduced sucrose intake, and decreased immobility time in FST and TST. Expression of NLRP3, ASC and caspase-1 by the administration of LPS was neutralised with reductions in levels of IL-1β, IL-18 and TNF-α in the hippocampus.
Physical activity and crocin prevented the detrimental symptoms of adolescent stress induced anxiety or depressive-like symptoms and dendritic morphology remodeling in prefrontal cortex in adult male rats. Plasma corticosterone levels increased at 40, but not 60 days old in stressed rats. Stressed rats exhibited enhanced anxiety levels and depression-like behaviours in adulthood accompanied by a decline in apical dendritic length in both infralimbic and prelimbic regions and dendritic branches in infralimbic region of the prefrontal cortex. Treatment with crocin, exposure to wheel running activity, and the combined interventions alleviated both behavioural and morphological deficits induced by adolescent stress.
These treatments exerted positive neuronal morphological effects in the prefrontal cortex in non-stressed animals. Exercise as a non-pharmacological intervention and crocin treatment during the pre-pubertal period can protect against adolescent stress-induced behavioural and morphological abnormalities in adulthood. [357] In a chronic unpredictable mild stress (CUMS) mouse in vivo model used to assessment of depression-like behaviour in OFT, TST, FST, SPT, and NSF tests the mice were assigned randomly to four groups (n = 10 each): control (sodium chloride 0.9%), CUMS, CUMS plus crocin 30 mg/kg (intragastric administration, i.g.), and CUMS plus fluoxetine 20 mg/kg (intraperitoneal injection, i.p.). were acclimatised for a week before the CUMS procedures were initiated-crocin and fluoxetine were administered once daily until the CUMS paradigm end.
Crocin significantly alleviated CUMS induced depression-like behaviours, reversed the decrease of body weight and elevation of serum CORT, and protected PC12 cells against CORT-induced injury by increasing the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and thereby enhanced the photophosporylation of its downstream ERK and CREB signalling pathways. It is thus conceivable that PACAP will be an important target for antidepressant treatment. [358] Crocin-I crocin-I (20 and 40 mg/kg for 2 weeks, 4 weeks) administered orally after induction of depression with 20 mg/kg corticosterone by subcutaneous injection in mice 8 week-old male C57BL/6J mice 2 weeks crocin-I exerted severe antidepressant effects in a model of chronic corticosterone (CORT)-induced depression, as evidenced by the attenuation of depression-like behaviours in the OFT, FST, and TST which was due to the suppression of neuroinflammation (IL-1β) and oxidative stress in the hippocampus. The oral administration of crocin-I (40 mg/kg) decreased the CORT-induced nicotinamide accumulation in the liver to improve the synthesis of NAD+, thereby stimulating the activity of SIRT3 deacetylase to elevate the activity of antioxidants such as superoxide dismutase 2 and glutathione reductase. Crocin-I reduced the levels of oxidative damage markers (ROS and MDA) to rescue impaired mitochondrial function caused by CORT treatment, which was represented by the electron transport chain and oxidative phosphorylation normality, and thus rescued ATP production to the level of that in wild-type mice. This results provide new information on the mechanism of action of crocin-I on depression-like behaviour and oxidative stress in perceived conditions-stressed individuals.
[359] Crocin Crocin (50 mg/kg; group 1), anti-inflammatory medicine Dexamethasone (Dex; 2 mg/kg; group 2-positive control), or the activator of Phosphatidylinositol 3-kinase (PI3K) Insulin-like growth factor 1 (IGF-1; 2 mg/kg; group 3) administered to mices randomly exposed to cigarette smoke for 7 weeks to induce chronic obstructive pulmonary disease (COPD) depression model; cigarette smoke-exposed group No. 4; fresh air-exposed control group No. 5. Each of five experimental group consisted of 8 individuals (n = 8). To establish of cigarette smoke COPD model mice were exposed to cigarette smoke of 5 3R4F Kentucky reference cigarettes (without filter, University of Kentucky, Lexington, KY, USA), one after another, four times a day (total of 20 cigarettes per day). Animals were alternately exposed to the smoke for 30 min with a smoke-free interval for 30 min. The procedure lasted for 7 consecutive weeks Route of administration: vehicle, Dex (2 mg/kg) or IGF-1 (2 mg/kg) via a single i.p.
injection or 50 mg/kg of crocin orally 1 h before exposure to cigarette smoke once a day 7-8 week male C57BL/6 mice Behavioural test were determined at 24 h after the last cigarette smoke exposure. The total duration of the OFT, FST, and TST was 6, 5, and 4 min, respectively. SPT-1% sucrose solution was given for 1 h after 12 h period with no water and food Crocin alone or with concomitant administration of Dex or IGF-1 improved cigarette smoke-induced depression-related behaviours. This bioactive compound of saffron markedly reveresd body weight loss, sucrose preference, and elevation of immobile time in TST and FST as well as improved exploratory behaviour and general activity in OFT. Crocin markedly inhibited the number of inflammatory cells (macrophages, neutrophils, and lymphocytes), suppressed the infiltration of peribronchial inflammatory cells, and strongly reduced the concentration of proinflammatory cytokines in hippocampus in bronchoalveolar lavage (BAL) fluid and lung tissue. Crocin blunted cigarette smoke-induced IκB phosphorylation and degradation, and NF-κBp65 nuclear translocation. IGF-1, an activator of PI3K, abrogated the effect of crocin against cigarette smoke-induced activation of the NF-κB pathway. Crocin protected against cigarette smoke-induced COPD with comorbid depression via the inhibition of the inflammatory response via PI3K/Akt-mediated Nuclear factor-κB (NF-κB) signalling. Crocin exhibits therapeutic potential in inflammatory lung disease with comorbid depression.
[360] Kaempferol Treatment and stress procedure: After 1 week of adaptation, all C57 mice were divided randomly into five groups (n = 10/group): control (non-stressed), chronic social defeat stress CSDS, CSDS + 10 mg/kg kaempferol, CSDS + 20 mg/kg kaempferol, CSDS + 10 mg/kg fluoxetine. Chronic social defeat stress procedure: adult C57 mice were functioned as intruders and the aggressive CD1 were retired breeders. During the 10 days the C57 mice were exposed to attack othe aggressive CD1 mice for up to 10 min each/day. The stressed C57 mices displayed submissive behaviours including fleeing, trembling, immobility, crouching and upright posture (8-10 min). On the day 11, SPT, social interaction test and TST were used to screen the successful models, which were delivered by drugs. Selected stressed mice were divided into various groups receiving fluoxetine, a morpholine-containing LY294002 that is a strong phosphoinositide 3-kinases (PI3Ks) inhibitor used to investigate the role of AKT/β-catenin signaling in the antidepressant effects of kaempferol, and vehicle for 28 days. The behavioural tests were preformed from day 29 to day 34 and at the day 35 the mices were sacrificed. All drug were administered via direct intraperitoneal injection (i.p.) inbred C57BL/6J (C57) 8 week-old mice and 8 week-old CD-1 outbred mouse line derived from the original colony of Swiss mice Duration of the experiment: 35 days. The SPT lasting for 4 days was carried out. The social interaction test consisted of "target absent" and "target present" trial and the each trial lasted for 5 min. In the "target absent" trial, each mouse explored in an open-field apparatus with fixed plastic enclosure freely and the interaction zone was predefined. In the "target present" trial, each mouse was returned to the same open-field apparatus with an unfamiliar CD1 mouse. TST-recorded for 5 min and reflected the depressive state Kaempferol and fluoxetine therapy conspicuously ameliorated behavioural dysfunction in depression as well as attenuated the malonaldehyde (MDA) and protein carbonylation contents, increased oxidative stress markers (superoxide dismutase SOD, glutathione peroxidase GPx, catalase CAT, glutathione s-transferase GST), reduced the concentrations of proinflammatory markers (IL-1β and TNF-α), inhibited CD-11b mRNA levels in the prefrontal cortex of CSDS mice, as well as enhanced AKT/β-catenin pathway. LY294002 appeared to partly inhibit kaempferol-mediated protective effects in the CSDS mice. Antidepressive effects of kaempferol are mediated by reduction of oxidative stress, proinflammatory cytokines and up-regulation of AKT/β-catenin cascade activity in the prefrontal cortex of CSDS mice. Kaempferol might be a promising, effective, and safe food-medicine agent for depression treatment.

Biological Object Duration of Study Main Results Reference
Crocin-I crocin-I (40 mg/kg) administered orally for six weeks to mices exposed to 4 week chronic restraint stress CRS).
Crocin-I alleviated CRS-induced depression in mice. This effect was accompanied by reduction of lipopolysaccharide (LPS), Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels in serum and TNF-α expression in the hippocampus, and the increase in the hippocampal brain-derived neurotrophic factor (BDNF). As revealed 16 s rRNA sequencing, crocin-I mitigated the gut microbiota dysbiosis in depressed mice as represented by the decreased abundance of Proteobacteria and Bacteroidetes, Sutterella spp. and Ruminococcus spp. and increased abundances of Firmicutes and Lactobacillus spp. Gas chromatography-mass spectrometry revealed that crocin-I reversed the decreased levels of short-chain fatty acids (SCFAs) in faeces of depressed mice as well as improved the impaired intestinal barrier by increasing expression of Occludin and Claudin-1, which contributed to the decreased LPS leakage.

18-24 g, 8-10 weeks old male Balb/cJ albino mice
The behavioural tests were carried out on the 18th day and biochemical assays on the 20 day Crocin treatment alleviated the MPTP-induced depressive-like behaviour assessed in FST in via protection of the dopaminergic (DA) projection neurons in the ventral tegmental area (VTA) through activating mammalian target of rapamycin (mTOR), and improving the neural synaptic plasticity of medial prefrontal cortex (mPFC).
[363] Animals were randomly assigned to one of the three of the experimental groups: oral affron ® , intraperiotoneally administered affron ® (ip) and vehicle control-normal saline i.p. (10 rats per group). In the oral group, a single dose of affron ® (200 mg/kg in a volume of 2 mL/kg) was administered via intragastric route at the beginning of the experiment, and then for the next 20 days, standardised stigma extract was dissolved in in drink tap water. In the intraperitoneal group rats received a daily dose of affron ® (50 mg/kg). Behavioural tests were performed on the first day of the experiment in order to assess the acute effects of the treatment, and also after the chronic treatment.

300-350 g. adult male Wistar rats
Duration of the experiment: 3 weeks. Acute condition-rats were tested a first time in the EPS and FST 30 and 60 min., respectively after the first administration of affron ® . The animals were assayed in SPT test, used as complementary test for anhedonia, on the 17th day of the experiment 30 min after affron ® administration.
On the 21st day of the experiment, the animals were assessed for the second time at the EPM and 30 min later at FST-chronic treatment.
Oral affron ® improved anxious/depressive state of rats-enhanced consumption of a sweet solution, as well as increased certain escape responses FST, but was equally as ineffective either orally administered or by the intraperitoneal anxiety-related behaviour using elevated plus-maze test (EPM). The evidence of the antianhedonic, and mild antidepressant actions of a 50 mg/kg acute i.p. dose and a 200 mg/kg oral dose of a standardised saffron extract of affron ® , when administered acutely or repeatedly, orally, has been provided. These results open new fields for the possible application of affron ® to prevent negative emotional states or as a co-adjuvant therapy in the treatment of depression.
[364] Crocin Treatment and stress procedure: female mice were randomly assigned into stressed and nonstressed groups; the stressed mice received a chronic mild stress procedure, i.e., 6 h restraint stress in a 50 mL centrifuge tube daily, combined with overnight illumination twice a week for three weeks. Females were mated with naïve males. Stressed females developed depression-like behaviour postpartum. The offspring of prepregnancy stressed nonstressed (naïve) females were defined as prenatal stress mice (PNS) and control group. The two groups of offspring were housed with dams until three weeks postnatally. PNS and control group were tested for depression-like behaviours at postnatal day (PND) 28 (juvenile) and 60 (adulthood). Drug administration: Ketamine (30 mg/kg, i.p.) or crocin (10, 20, 40 mg/kg, intragastrically) or saline control was administered i.p. 24 h before the behaviour tests. In the time-course test, crocin (40 mg/kg) was administered 0.5, 2, 24, and 72 h prior to the behavioural tests, respectively. JMV2959 (12 mg/kg, i.p.)-an antagonist of growth hormone secretagogue receptor type 1a (GHS-R1a) and LY294002 (50 mg/kg, i.p.)-a highly selective inhibitor of phosphatidylinositol 3 (PI3) kinase (PI3k) were administered 30 min before crocin (40 mg/kg). Growth hormone secretagogue receptor (GHSR) and phosphoinositide 3-kinase (PI3K) inhibitors were used to test their effects in antidepressant-like effect of crocin.
18−24 g., six to eight weeks old, female and male Balb/cJ mice Duration of the experiment: 3 weeks. OFT was used to assess the locomotor as well as the exploratory behaviour in an open area and the locomor activity was recorded for five minutes FST immobility time was measured for 4 min. SPT 2% sucrose solution were given for 1 h after 18 h period of water and food deprivation; novelty suppressed feeding (NSF) test latency to feeding was measured for 5 min Rapid and prolonged antidepressant-like effect of crocin associated with GHSR-mediated hippocampal plasticity-related proteins in prenatal stress exposed mice was demonstrated. Crocin activated the hippocampal GHSR-PI3K signalling and induced a rapid and enduring antidepressant effect, which is similar to the effect of ghrelin-a 28-amino-acid peptide feeding peptide recognised as an endogenous ligand for the growth hormone secretagogue receptor 1a (GHSR-1a) and identified as an important mediator in the pathology of mood disorders. The study demonstrated the adverse effect of prenatal stress as evidenced by significant depressive-like behaviours in mice. Crocin produced a fast and long-lasting antidepressant-like effect in PNS mice and restored the impaired the expression of hippocampal synaptic plasticity-associated proteins via modulation of GHSR-PI3K signaling. This signalling pathway contributes to the antidepressant properties of crocin, as the inhibition of both GHSR and PI3K abolished its unique effect. The GHSR inhibitor JMV2959 was blocked by crocin. These findings illuminated the promising antidepressant-effect of crocin as a novel antidepressant agent.
[365] Crocetin ameliorated chronic restraint stress-induced depressive-like behaviours in ICR mice (TST and OFT). This bioactive compound of saffron stigma markedly attenuated the elevated levels of the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), the precursor of brain-derived neurotrophic factor (proBDNF), alanine and aspartate transaminase as well as increased the serum level of dopamine and phosphorylated cAMP response element-binding (CREB) (enzyme-linked immunosorbent assay ELISA kits; Immunoenzymatic test ELISA). Histopathological analysis showed that crocetin suppressed hippocampus injury in restraint stress mice by protecting neuronal cells. Immunofluorescent and Western-bolt assays revealed elevated expression levels of ERK1/2, CREB and inhibited expression levels of MKP-1, proBDNF in the hippocampus. High-throughput sequencing showed that the composition of intestinal microbiota of the crocetin group partially recovered and was quite similar to the control group. Therefore crocetin showed neuroprotective properties and reduced the effects of chronic stress-associated barin damage by regulating the MKP-1-ERK1/2-CREB signalling and intestinal ecosystem.
[366] Saffron extract during induction of morphine dependence did not affect anxiety and depression-like behaviours, but markedly decreased the severity of withdrawal signs. Saffron extract during morphine withdrawal resulted in the percentage increase (or ratio) of open/total arm entries, higher levels of sucrose preference, a lower morphine preference ratio as well as decrease in locomotor activity and an increase in the cerebrospinal fluid serotonin levels in rats challenged to morphine. Saffron extract may exert a protective effect against morphine-induced behavioural sensitisation in rats, probably through increasing serotonin levels. [367] saffron extract Safr'Inside™ acute (n = 10) and chronic (n = 10) treatment (6.25 mg/kg per os) or its vehicle (water; n = 10) orally administered in the morning. The dose of saffron extract was calculated based on the ratio given by the United States Food and Drug Administration to reflect, for a mouse, the equivalent of the effective dose classically administrated to humans, namely 30 mg/day. For acute experiments, the solutions were administered 30 min before the behavioural assessment. For the chronic experiment, mice received one gavage per day for 4 weeks, the last being done 3 h before the behavioural test.
Acute condition-mice were tested in the 6 min FST a first time 30 min after the first administration of saffron extract or water. Duration of swimming, climbing, and immobility was determined during the last 4 min of the test. Then, after 3 weeks of daily treatment, rodents were tested in a light-dark test (LDT) and again in the FST 1 week later-chronic conditions.
Saffron extract mitigated depressive-like behaviour in the FST through neurobiological modifications, particularly through an increase in serotonergic and dopaminergic neurotransmission, suggesting that Safr'Inside™ may share common targets with conventional pharmacological antidepressants. Further studies are needed to deeply understand how Safr'Inside can modulate the activity of these systems, as well as their causal role in the observed effects, and to test the contribution of other known pathophysiological bases of mood disorders. Safr'Inside administration does not reduce anxiety-like behaviour, as assessed in chronically treated mice exposed to a classical and pharmacologically validated rodent test of anxiety, the LDT. [368] Explanations: ASC-apoptosis-associated speck-like protein containing a caspase recruitment domain; BALB/c mice-albino, laboratory bred strain of the house mouse; BDNF-brainderived neurotrophic factor; CREB-response element-binding protein; DA-dopamine; DMSO-dimethyl sulfoxide (vehicle); EPS-Elevated Plus Maze Test; ERK-extracellular regulated protein kinases; FST-Forced Swimming Test; GSH-glutathione; IL-1β-Interleukin-1β; iNOS-Inducible nitric oxide synthase; MDA-malondialdehyde (MDA); NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells); NF-κBp65-NF-κB nuclear transcription factor subunit p65; NLRP3-The Nod-Like Receptor (NLR) family pyrin domain-containing protein 3; NO-nitric oxide; OFT-Open-Field Test; PC12-a cell line derived from a transplantable rat pheochromocytoma which catecholamine type cells synthesise, store and release norepinephrine and dopamine; TST-Tail Suspension Test; TNF-α-tumor necrosis factor-α; SPT-sucrose preference test; NSF-novelty-supressed feeding test; BDNF-brain-derived neurotrophic factor; VGF-non-acronymic neuropeptide. The petroleum ether fraction and dichloromethane fraction of corms at doses of 150, 300, and 600 mg/kg as well as the aqueous stigmas extract exerted antidepressive effects in the behavioural models (FST, TST, OFT). Antidepressant-like properties of aqueous stigma extracts are due to crocin 1, and by means of a gas chromatography-mass spectrometry technique, twelve compounds of the petroleum ether fraction were identified. Therefore, the low polarity parts of C. sativus corms should be considered as a new plant material for curing depression, and further studies regarding antidepressive-like activities of chemical compounds isolated from the two fractions and mechanism of action are highly recommended. [226]. Paw Edema Test) and the coagulation system. These pro-health effects are related to intrinsic active compounds, mainly carotenoids and flavonoids, found in the highest amounts in stigma and petals, respectively. Further epidemiological investigations, laboratory research, and clinical trials are needed to isolate the pharmacologically active molecules that contribute to the therapeutic effects and to explicate the possible mechanism of action and effect of the plant on various critical illnesses and medicinal formulations. [205] Explanations: g.-group; FST-Forced Swimming Test; OFT-Open-Field Test; TST-Tail Suspension Test.  using only a fixed dose of saffron; the small number of participants and short period of follow up should be considered, and further research in this area in particular comparison with an active agent such as fluoxetine is needed.
[369]    too-small scale of the trial and single dose of saffron. The minor sample size and the temporary follow up is suggested in further survey.
[232]  too small sample size and a slightly short duration in addition to ethical constraints impeding the assessment of the effects of saffron alone-without sertraline or any other prescribed medication on GAD.
[217] based on the Female Sexual Function Index (FSFI) and HDRS saffron is recognised to be safe and effective agent in alleviation of some of fluoxetine-induced sexual problems including arousal, lubrication, and pain in women.
Short study duration limited the interpretation of the present study regarding long-term effects of saffron on sexual dysfunction. Different dosages of saffron should be investigated in future [375]    No significant effect of the time×treatment interaction on the HDRS score between saffron and fluoxetine group.
Therefore saffron is a safe alternative medication for improving depressive symptoms of postpartum depression lack of a placebo group, a small number of participants and short period of follow-up [341] significant medical illness (gestational diabetes, pre-eclampsia); pregnance; present or past history of drug or alcohol abuse, smoking, alcohol, or illicit drug use during pregnancy; treatment with any medications affecting mood; anticoagulants therapy; current psychotropic medication; absence of a control groups, a double-blind placebo-controlled study is needed to confirm these results [378]    the small number of participants and the short period of follow-up. Further research with a longer study period, an active agent such as venlafaxine and a higher sample size to consider patients with different biological and racial backgrounds is needed.
[381]  improvement of anxiety and depressive symptoms in teenagers with mild-to-moderate symptoms after stigma administration from the perspective of the adolescents. Youth in self-reports declared greater improvements in overall internalising, separation anxiety, social phobia and depression, however, these beneficial effects were not corroborated by their parents.
self-reporting nature of both the screening and testing, limited duration of the study, single treatment dose [234]  physically healthy volunteers both of sex aged 18-65 years, with persistent depression, currently (at least eight weeks) treated with a stable dose of single pharmaceutical antidepressant who continued to suffer from mild-to-moderate depressive symptoms as assessed by a score greater than six on the Montgomery-Åsberg Depression Rating Scale (MADRS) (nine-items). Of the 160 participants enrolled, 139 provided usable data. current or 12 month history of any psychiatric disorder other than mild-to-moderate depression or anxiety; in self-harm behaviours and/or serious suicidal ideation treatment with any pharmaceutical medication, apart from a single pharmaceutical antidepressant, oral contraceptives and the occasional use (no more than fortnightly) of analgesics (e.g., ibuprofen, paracetamol); currently taking saffron or other herbal supplements; a current or history of a clinically significant, chronic medical condition including cardiovascular disease, organic brain disorder, seizure, diabetes, severe obesity, or use of illicit drugs; pregnant or breastfeeding women, 8 week randomised double-blind placebocontrolled trial Based on the clinician-rated Montgomery-Åsberg Depression Rating Scale (MADRS) affron ® markedly reduced depressive symtoms compared to placebo with decreases of 41 and 21%, respectively, but the decrease in the scores of self-rated MADRS was comparable in both of experimental groups.
As it was assessed with Antidepressant Side-Effect Checklist (ASEC) and Short Form-36 Health Survey (SF-36), saffron was associated with a greater reduction in adverse effects of antidepressants, although this was non-significant after covarying for baseline values.
Quality of life improved in both groups with no significant between-group differences.
The longer duration period of the trial and dose-escalation studies for treatment non-responders will be useful to examine the efficacy and safety of higher than studied affron ® dose. Support for the saffron antidepressant efficacy should be demonstrated via clinician-rather than self-administered assessment. In order to clarify the saffron antidepressant mechanisms the objective measures of change including changes in cortisol, neurotrophins and inflammatory and oxidative stress markers or changes in neurological activity through the measurement of EEG activity and cognitive testing may be helpful. [383]

Limitations of Studies and Recommendations for Future Investigations
Reference women intending to fall pregnant; subjects reporting a greater than ten-year continuous use of antidepressant medication with no remission in depressive symptoms greater than six months over this period. Eligibility was initially assessed via the completion of a questionnaire that screened for current medication use, suicidal ideation, self-harm behaviours, history of medical/psychiatric disorders, alcohol, nicotine and other drug use, supplement and vitamin intake, and pregnancy/breastfeeding status.
Due to the conflicting results, further research is needed to clarify the clinical benefits of saffron as an adjunctive treatment for adults with persistent depressive symptoms despite antidepressant drug treatment. The efficacy of adjunctive saffron use on specific antidepressant types and classes should be investigated.
Recruitment of the participants solely via social media promotion might have skewed the examined population. Multi-measure approaches i.e., diaries, questionnaires and pharmacokinetic measurements, not only participant self-reporting of remaining tablet numbers, should be used for adherence to pill intake. Crocin severely decreased Beck Depression Inventory score and Beck Anxiety Inventory score. It also reduced fasting glucose, insulin levels and resistance, triglycerides, very low-density lipoprotein as well as total cholesterol levels, but markedly increased insulin sensitivity. Crocin intake was associated with a reduction in high-sensitivity C-reactive protein and MDA, as well as a rise in total antioxidant capacity levels. Administration of crocin supplements to patients undergoing an MMT program had ameliorating effects on mental health scales, and improved their metabolic, and genetic parameters.
Too short-term of an intervention; further studies focused on the cognitive functions, craving, and withdrawal syndrome in subjects under a methadone maintenance treatment (MMT) program are necessary; the effects of crocin administration on urinary or/and serum crocin should be evaluated.
[384] Eighty-six, 40-60 year-old, perimenopausal women experiencing menopausal complaints with a total score of greater than 16 on the Greene Climacteric Scale (GCS), an intact uterus and ovaries and a body mass index (BMI) between 18 and 35 kg/m 2 ; patients were medication-free for at least 3 months (apart from the contraceptive pill and/or once weekly use of analgesics) and had no plan to commence new treatments over the study period. A total of 39 subjects from saffron group and 37 subjects from placebo group completed the trial.
smokers; consumption more than 14 standard drinks of alcohol per week; current or illicit drug abuse within the last 12 months; suffering from medical conditions including but not limited to: diabetes, hyper/hypotension, cardiovascular disease, a gastrointestinal disease requiring regular use of medications, gallbladder disease/gallstones/biliary disease, endocrine disease, psychiatric disorder (excluding mild-to-moderate anxiety), or neurological disease (Parkinson's or Alzheimer's disease, intracranial haemorrhage, head or brain injury); women who had any significant surgeries over the last year, or women taking saffron or other supplements that may affect menopausal symptoms.
two-arm, parallel-group, 12 week, randomised, double-blind, placebocontrolled trial Affron ® markedly improved psychological symptoms in perimenopausal women reducing depressive and anxiety sydromes. Data from the Greene Climacteric Scale (GCS) revealed a significantly greater reduction in the GCS psychological score, characterised by a 33% reduction in anxiety and a 32% reduction in depression scores. Saffron to a greater extent than placebo reduced the PANAS negative affect score (Positive and Negative Affect Schedule). However, vasomotor or other somatic symptoms within intervention and control groups were not markedly different. Given the positive, mood-enhancing findings, further investigations into the benefits of various saffron doses in more clearly-defined populations, As no formal medical assessment comprising an evaluation of hormone concentrations and a comprehensive examination of confounding medical, lifestyle, and dietary factors was undertaken some women in other reproductive stages might have been recruited in this study. The effects of saffron in women with a formally diagnosed depression or anxiety-related disorder, and with varying levels of severity should be studied. The co-administration of saffron with pharmacological antidepressants in perimenopausal women with more severe specific climacteric symptoms currently taking antidepressants and/or on hormone replacement therapy should be evaluated.
[385]. Saffron and its ingredients had been effective in reducing depression assessed with using self-report Beck depression inventory (BDI-II).
Saffron with its active ingredients (crocin and safranal) by serotonin and dopamine secretion in the brain help in reducing depression among recovered consumers of methamphetamine living with HIV/AIDS. the duration of intervention, sample size and a tested saffron extract dose was similar to other studies that might aim to find a dose-response effect. Further studies with different doses of saffron extract, different durations of intervention and larger sample sizes are required.
[386]    [390] Saffron to a similar extent as fluoxetine reduced the premenstrual syndrome symptoms such as abdominal bloating, depression, and mood swing, and could even better relieve the breast and abdominal pain than fluoxetine. Therefore saffron could be effective in reducing the symptoms and cause fewer side effects than chemical drugs.
using only a fixed dose of saffron and short period of follow-up.
[391]       Although the results of clinical trials clearly suggest that saffron reduces the severity of depression based on Hamilton Depression Rating Scale (HAM-D) and Beck's Depression Inventory (BDI) scores, the optimum dose and duration of treatment is still unclear [75].
Saffron and its bioactive constituents (crocetin esters, picrocrocin, and safranal) may be considered as a potential adjuvant in the form of anti-depressants in the future drug formulations. Recently, they seem to be a suitable candidate for the management of anxiety, depression, neuropsychiatric disorders and the other long-term effects including subacute and chronic abnormalities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection such as fatigue, dyspnoea, cognitive problems, sleep abnormalities, and deterioration in the quality of life. Detailed research on dosage, methods of administration and others needs to be undertaken to explore the potential of saffron in managing the health issues arising due to the COVID-19 pandemic [397,398]. Moreover, crocin appears to reduce the COVID-19-related cytokine cascade and downregulate angiotensin-converting enzyme 2 (ACE2) gene expression. Lastly, in silico studies suggest that saffron's astragalin and crocin could have inhibitory actions on the SARS-CoV-2 protease and spike protein, respectively. However, future appropriate randomised clinical trials using biomarkers as surrogates to assess inflammatory status should be designed in order to assess the clinical efficacy of saffron and allow its use as an adjunct treatment modality, particularly in resource-poor settings where access to drugs may be limited [399]. Soheilipur and co-workers claim [400] that the oral use of a single-dose of 40 mg saffron extract is effective in alleviating anxiety among the candidates for coronary angiography (CA), while lippia extract (capsule 40 mg; Lippia citriodora Kunth) and saffron-lippia (20 mg:20 mg) extract combination had no significant effects on their anxiety.
Saffron application is recognised as a promising natural and safe nutritional strategy to improve sleep duration and quality. The investigations carried out by Shahdadi et al. [401] revealed that daily (between 12 noon and 2 pm) intake of a 300 mg saffron capsule after lunch for a week was effective in reducing anxiety and improving the quality of sleep among diabetic patients. Six weeks of saffron extract supplementation (15 mg/day) to the subjects presenting with mild-to-moderate sleep disorders associated with anxiety led to an increased time in bed assessed by actigraphy, to an improved ease of getting to sleep as evaluated by the LSEQ (the Leeds sleep evaluation) questionnaire, and to an improved sleep quality, sleep latency, sleep duration, and global scores evaluated by the PSQI questionnaire (Pittsburgh Sleep Quality Index) [402]. Standardised saffron extract (affron ® ; 14 mg twice daily per 28-days) improved sleep quality in adults with self-reported sleep problems. The beneficial effect of saffron was manifested by improvements in ISI total score (The Insomnia Severity Index), RSQ total score (the Restorative Sleep Questionnaire), and PSD (Pittsburgh Sleep Diary) sleep quality ratings [403]. Further investigations concerning four weeks of treatment with affron ® (14 mg, or 28 mg 1 h before bed) revealed improvements in sleep quality ratings assessed with Pittsburgh Sleep Diary, mood ratings after awakening (Profile of Mood States), the ISQ total score (Insomnia Symptom Questionnaire), and ISQ insomnia classifications without affecting the score of the Restorative Sleep Questionnaire and the Functional Outcomes of Sleep Questionnaire. Moreover, saffron supplementation was associated with increases in evening melatonin concentrations but did not affect evening cortisol. Sleep improvements were similar for the two saffron doses with no reported significant adverse effects. [404]. Results of the studies on the effect of crocetin on on sleep quality in healthy adult participants with mild sleep complaint assessed showed that supplementation with this bioactive compound contributes to sleep maintenance, leading to improved subjective sleep quality. This beneficial effect of two intervention periods of 14 days each, separated by a 14 day wash-out period, was manifested with an increase in an objective sleep parameters (delta power) measured using single-channel electroencephalography and improvements in the subjective sleep parameters sleepiness on rising and feeling refreshed assessed with using the Oguri-Shirakawa-Azumi Sleep Inventory, Middle-age and Aged version (OSA-MA).There were no significant differences in the other sleep parameters, including sleep latency, sleep efficiency, total sleep time, and wake after sleep onset [405].
A single study indicated that saffron odor was effective in treating menstrual distress by relieving the symptoms of premenstrual syndrome (PMS) and alleviating dysmenorrhea (menstrual pain) as well as helping to control irregular menstruation. As Fukui and coworkers claim [406], healthy woman with a normal sense of smell exposed to saffron aroma for 20 min experienced a decrease in salivatory cortisol and increase in 17-β estradiol level in both the follicular and luteal phases, which was accompanied with a decrease in anxiety measured using the State-Trait Anxiety Inventory (STAI). It was the first evidence of beneficial psychological and neuroendocrinological effects of saffron odour.

Mechanism of Antidepressant Action
The mechanism of the in vitro and in vivo antidepressant action of C. sativus stigmas is attributed to e.g., crocin, which inhibits monoamine (noradrenaline and dopamine) reuptake, and safranal, which inhibits serotonin reuptake, and to their action towards GABAergic (gamma-aminobutyric acid) receptors and neurotrophic effects, e.g., through activation of BDNF (brain-derived neurotrophic factor). Stigmas of C. sativus (called saffron) have been demonstrated to contain an antagonist of postsynaptic NMDA (N-methyl-Daspartate) receptors [71,407,408]. It has been proven that C. sativus modulates the levels of neurotransmitters, especially serotonin, in the brain by inhibiting serotonin reuptake, thereby retaining serotonin in the brain longer [409] (Figure 3). Azumi Sleep Inventory, Middle-age and Aged version (OSA-MA).There were no significant differences in the other sleep parameters, including sleep latency, sleep efficiency, total sleep time, and wake after sleep onset [405]. A single study indicated that saffron odor was effective in treating menstrual distress by relieving the symptoms of premenstrual syndrome (PMS) and alleviating dysmenorrhea (menstrual pain) as well as helping to control irregular menstruation. As Fukui and co-workers claim [406], healthy woman with a normal sense of smell exposed to saffron aroma for 20 min experienced a decrease in salivatory cortisol and increase in 17-β estradiol level in both the follicular and luteal phases, which was accompanied with a decrease in anxiety measured using the State-Trait Anxiety Inventory (STAI). It was the first evidence of beneficial psychological and neuroendocrinological effects of saffron odour.

Mechanism of Antidepressant Action
The mechanism of the in vitro and in vivo antidepressant action of C. sativus stigmas is attributed to e.g., crocin, which inhibits monoamine (noradrenaline and dopamine) reuptake, and safranal, which inhibits serotonin reuptake, and to their action towards GA-BAergic (gamma-aminobutyric acid) receptors and neurotrophic effects, e.g., through activation of BDNF (brain-derived neurotrophic factor). Stigmas of C. sativus (called saffron) have been demonstrated to contain an antagonist of postsynaptic NMDA (N-methyl-Daspartate) receptors [71,407,408]. It has been proven that C. sativus modulates the levels of neurotransmitters, especially serotonin, in the brain by inhibiting serotonin reuptake, thereby retaining serotonin in the brain longer [409] (Figure 3).  [216,228,231,293,345,364,374,375,410].
In other studies, increased levels of CREB, BDNF, and VGF in the hippocampus were found [229,294]. There is strong evidence that VGF and BDNF are involved in depressive disorders and transcription thereof is CREB-dependent. The neuropeptide VGF enhances hippocampal synaptic activity and is involved in energy balance and homeostasis regulation. In turn, BDNF, which is widely expressed in the mammalian brain, is implicated in the survival of neurons during hippocampal development, neural regeneration, synaptic transmission, synaptic plasticity, and neurogenesis [269,270].As reported by Asrari et al. [353], there is mediation of the P-CREB protein in cerebellum, which is consistent with the  [216,228,231,293,345,364,374,375,410].
In other studies, increased levels of CREB, BDNF, and VGF in the hippocampus were found [229,294]. There is strong evidence that VGF and BDNF are involved in depressive disorders and transcription thereof is CREB-dependent. The neuropeptide VGF enhances hippocampal synaptic activity and is involved in energy balance and homeostasis regulation. In turn, BDNF, which is widely expressed in the mammalian brain, is implicated in the survival of neurons during hippocampal development, neural regeneration, synaptic transmission, synaptic plasticity, and neurogenesis [269,270]. As reported by Asrari et al. [353], there is mediation of the P-CREB protein in cerebellum, which is consistent with the increased expression of this protein in the cerebellum described by Ghasemi et al. [229]. There is evidence that the cerebellum not only plays a role in motor function and coordination of movement, but also contributes to an important role in emotion and cognition processing. To sum up among many different proposed mechanisms explaining C. sativus stigma and petals effectiveness in the depression treatment, the most important is the one involving the anti-inflammatory and antioxidant effects, followed by the action on neurotransmitters in favour of the hypothesis of their deficiency in depression. Neurotrophic factors, particularly BDNF, are also of interest since they are involved, even if indirectly, in the regulation of neurotransmitters such as 5-HT, DA, glutamate and GABA and in various types of signalling such as CREB [411,412].
Studies of multiple genes have yielded some positive results regarding the usefulness of genotyping cytochrome P450 enzymes (CYP450) in the treatment of depression in groups of patients, but the choice of medications for a specific patient is not still established [413,414]. The results of investigations conducted on male Wistar albino rats receiving safranal (4, 20, and 100 mg/kg/day) or intraperitoneal injections of crocin (4, 20, and 100 mg/kg/day) indicate that both these compounds increase the total protein content and determine the metabolic activity of liver microsomal CYP450 isoforms (CYP3A, CYP2C11, CYP2B, and CYP2A) [410]. It was found that, in general, crocin markedly reduced and safranal significantly enhanced the metabolic activity of all the CYP enzymes mentioned above, except for changes in CYP2A activity induced by safranal. Therefore, the authors claim that crocin and safranal could increase the risk of interactions with co-administered substances metabolised by cytochrome P450 enzymes.

Challenges for Further Research
The scientific knowledge of the beneficial or negative impact of herbal treatment of depression is incomplete. Further investigations should focus on: (1) adequate methods of extraction of selected biologically active compounds and practical pharmaceutical applications thereof; (2) promotion of trust in phytotherapy and the use of biotechnological procedures to ensure the biodiversity of the product; (3) the use of genetic technologies to obtain good quality and high concentrations of effective phytochemicals that can be used in the future to support treatment of depression as progress in herbal psychopharmacology; (4) standard medical therapies based on herbal products, including changes in the regulations, standardisation, and financing of research on selected phytochemicals with anti-depressant effects; (5) insightful and more detailed analyses of natural compounds in terms of the basic mechanisms involved in the anti-depressant actions and justifying the application of selected plant species in the therapeutic practice of depression, taking into account antidepressant properties of these plants that have already been confirmed by scientists; (6) thorough clinical trials of selected phytochemicals-effective substances in depression treatment facilitating production of antidepressant drugs and antioxidants from these substances; (7) confirmation of the safety and efficacy of action in the treatment of depression, which will support the decision to use these compounds (as in the case of pharmaceutical drugs).

Conclusions
Crocus sativus, commonly known as saffron crocus, is native to the Western and Eastern Asia and Southern Europe. For centuries, it has been used in traditional Asian medicine as an agent for healing various health problems, including infections, pain, inflammation, chronic fatigue, insomnia, memory impairment, mood and personality disorders (anxiety, depression), and other mental illnesses. The medicinal activity of C. sativus extracts in alleviation of inflammation and central nervous system disorders, including depression, has been confirmed in the most recent basic animal (rodent) studies and human clinical trials. A number of in vitro, in vivo, and clinical trials have demonstrated that both dried stigmas and petals of C. sativus (water and alcohol extracts) as well as their ingredients are safe and effective antidepressants. Saffron stigma, bulbs and petals and its bioactive compounds may be considered as a potential adjuvant in the form of anti-depressant in future drug formulations. Their efficacy is similar to current antidepressant medications such as fluoxetine, imipramine, and citalopram, but fewer side effects are reported. The ac-tive compounds of aqueous and alcoholic crocus extracts exhibiting antidepressant activity include unique hydrophilic crocin carotenoids, i.e., monoglycosyl or di-glycosyl esters of crocetin, hydrophobic crocetin, and terpenoid safranal. The following mechanisms of the antidepressant action of C. sativus components are proposed: (1) inhibition of monoamine (dopamine, norepinephrine, serotonin) reuptake, (2) N-methyl-D-aspartate (NMDA) receptor antagonism, and (3) gamma-aminobutyric acid (GABA)-α agonism. Crocin acts via inhibition of dopamine and norepinephrine uptake, while safranal acts via serotonin. The antidepressant and neuroprotective effect of C. sativus extracts and their components is associated with anti-inflammatory and antioxidant activity. This activity is manifested by e.g., mood improvement, alleviation of anxiety symptoms, beneficial effects on learning and remembering, and a positive influence on the emotional sphere. However, due to many limitations presented in the papers cited in this protocol, there is a need for conducting further experiments to confirm the current results on the effectiveness of the antidepressant activity of C. sativus extract and its components and to elucidate the mechanisms of their action fully. Research reported by many authors has documented the application of herbal formulations in treatment of depression, insomnia, and anxiety, but detailed research on dosage, methods of administration and others needs to be undertaken to explore their potential in managing the health issues Although phytochemicals are natural substances and should therefore be safe, side effects have been noted due to contamination of preparation or drug interactions.