Saffron extract and its use for the prevention of mood disorders related to depression

Abstract

The present invention relates to a powdered composition obtained from saffron stigmas characterized in that it comprises from 0.03% to 1% dry weight of safranal; and at least 3.48% dry weight of crocins that encompasses the various trans-crocin-4 (major isomer), trans-crocin-3, trans-crocin-2, cis-crocin-4, trans-crocin-2, trans-crocin-1 isomers and any of their mixtures.

Claims

1. A method for treatment of low mood, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an extract of saffron stigmas analyzed by HPLC, the composition comprising: a) between 0.03% and 1% dry weight of safranal, and b) at least 3.48% dry weight of crocins, wherein said crocins comprise trans-crocin-4, trans-crocin-3, trans-crocin-2, cis-crocin-4, trans-crocin-2, and/or trans-crocin-1.

2. The method according to claim 1, wherein the composition is administered orally.

3. The method according to claim 1, wherein the composition is administered in a dosage greater than 15 mg/day.

4. The method according to claim 1, wherein the composition is administered in a daily dosage of 28 mg/day.

Description

BRIEF DESCRIPTION OF THE CONTENT OF THE DRAWINGS

(1) FIG. 1. Overlaid HPLC chromatograms of the main components of the saffron extract of the invention. P: Picrocrocin; t-c-4: trans-Crocin-4; s: Safranal.

(2) FIG. 2. Changes in the mean POMS values showing five negative subcategories (T: tension; D: depression; A: anger; F: fatigue; C: confusion) and one positive (V: vigour). POMS MCS: POMS Mean Change Score; Plb: Placebo.

(3) FIG. 3. Changes in the POMS values showing the values of the Total Mood Disturbance, TMD MCS analysis: Total Mood Disturbance Mean Change Score; Plb Placebo.

(4) FIG. 4. Changes in the mean PANAS values for evaluating Positive Affect and Negative Affect. PANAS MCS: PANAS Mean Change Score; Plb Placebo; PA: Positive Affect; NA: Negative Affect.

(5) FIG. 5. Changes in the mean DASS values for evaluating depression (D), anxiety (Ax) and stress (S). DASS MCS: DASS Mean Change Score; Plb: placebo.

EXAMPLES

(6) The invention is illustrated below by the trials conducted by the inventors, which demonstrate the composition and the activity of the composition of the invention.

Example 1Determining the Concentration of the Bioactive Compounds in the Composition of the Invention

(7) Firstly, saffron stigmas were selected based on the quality thereof (saffron from crops in Castilla-La Mancha, Spain) and macerated in water at a temperature lower than 70 C. and stirred. Then, a more exhaustive concentration was performed without exceeding a temperature of 70 C., and a raw dry extract was obtained, which was subsequently diluted with dextrin until obtaining a minimum concentration in safranal and crocins of 0.03% and 3.48% on a dry basis respectively.

(8) The quantitative analysis of the bioactive compounds of the saffron extract thus obtained is performed in a 1260 Infinity Series HPLC system coupled to a Diode Array detector (Agilent Technologies, Palo Alto, Calif., USA). The chromatographic separation was carried out by a C18 column (250 mm/4.6 mm/5 m; ACE-5 PFP, Scotland) at 25 C. and a flow rate of the mobile phase of 1 mL/min. The mobile phase was made up of a component A) water:acetonitrile (85:15, v/v) and component B) methanol, with a linear gradient of 10 to 100% of methanol in 60 min. Both the samples of the extract of the present invention and the reference standards were diluted in a methanol:water solution (50:50, v/v). The elution of the picrocrocin, safranal and crocins was monitored at absorbance values of 250, 310 and 440 nm, respectively (Caballero-Ortega et al., 2007). The quantification of these compounds was carried out by external calibration curves of p-nitroaniline (Sigma Aldrich, St. Louis, Mo., USA) to quantify the picrocrocin, safranal (Sigma Aldrich, St. Louis, Mo., USA) to quantify the safranal, and the trans-crocin-4 isomer (Phytolab, Hamburg, Germany) to quantify the different crocin isomers.

(9) FIG. 1 shows the overlaid chromatogram, acquired for wavelengths at 250, 310 and 440 nm of the saffron extract of the invention, in the aforementioned chromatographic conditions. At 250 nm, a peak may be seen with a retention time (rt) of 7.6 minutes, which corresponds to picrocrocin, whilst at 310 nm, another peak may be seen with a rt of 33.9 minutes, which corresponds to safranal. In the chromatogram recorded at 440 nm, now in the visible spectrum, there are different crocin isomers, the greatest peak corresponding to the trans-crocin-4 isomer (minute 22.3).

(10) To confirm the identity of each peak, an analysis of the masses of each molecule found in the extract of the present invention was also carried out. In order to do so, an Agilent series 1100 system (Palo Alto, Calif., USA) was used connected to a quadrupole mass spectrometer detector (Hewlett-Packard, series 1100 MSD) with an electrospray ionization source (ESI), which worked both positively and negatively. The mass range was 50 to 1500 u, the drying gas flow was 10 L/minutes, drying gas temperature was 340 C., nebulizer pressure was 40 psig, vaporizer temperature was 150 C., capillary voltage was 2000V, and charging voltage was 2000V.

(11) Table 1, below, shows the individual analysis of the main crocin isomers, found in the saffron extract of the present invention at 440 nm, along with the main bioactive compounds found at 250 nm (picrocrocin and kaempferol diglucoside) and 310 nm (safranal). All the UV-VIS spectra of the crocin isomers had maximums close to 250, 310 and 440 nm in common, and the main quasi-ions and fragments analyzed correspond to those found by Lech et al., (2009) in strands of saffron, where the ESI+ mode was the most effective in determining each one of these carotenoids. Picrocrocin shows a maximum at 251 nm, safranal also shows a single maximum at 313 nm, while kaempferol diglucoside has a spectrum characteristic of this flavonoid with maximums at 265 and 346 nm. The rest of the crocin isomers found in the saffron extract of the present invention showed the same spectrum characteristic of this carotenoid, as shown in Table 1, with maximums around 257, 330 and 440 nm.

(12) Commercially available reference standards were also used to identify trans-crocin-4 and safranal, from which solutions were prepared and subjected to the same analysis conditions as those used for the extract certificated by HPLC, object of the present invention.

(13) TABLE-US-00001 TABLE 1 Compounds identified in the chromatogram of FIG. 1, with ESI MS detection in positive ion (ESI+) and negative ion (ESI) mode. RT (min- Maximum UV-Vis utes) Compound (nm) ESI+ (m/z) ESI (m/z) 7.6 Picrocrocin 251 511.2; 365.1; 1480.8; 353.2; 337.1; 667.2; 329.1; 185.1; 517.1; 159.9; 151.1; 385.0; 123.0; 81.3 181.2; 153.1 11.5 Kaempferol 266; 347 633.2; 347.1 609.0; 284.0 diglucoside 16.7 262; 325; 442; 467 17.8 223; 270; 429; 438 18.0 223; 270; 327; 419; 440 19.3 261; 441; 465 22.1 trans-crocin-4 261; 323; 441; 464 999.4; 675.2; 1336.2; 511.2; 347.1 651.2; 326.8; 283.1 22.7 261; 440 999.2; 742.8; 23.8 261; 323; 439; 462 717.0; 510.9; 347.0 24.2 260; 439; 461 26.0 262; 323; 440; 463 26.7 trans-crocin-3 261; 323; 440; 463 837.4 27.9 261; 438; 462 29.8 260; 437; 462 30.7 trans-crocin-2 261; 328; 440; 464 675.2; 593.0; 513.2; 351.1; 130.1; 102.2; 74.1 31.2 460 32.3 243; 305; 420; 441 32.6 243; 305; 420; 441 33.9 Safranal 313 36.2 cis-crocin-4 225; 262; 325; 433; 999.4; 821.3; 651.2 456 668.3; 611.0; 391.0; 347.0 37.6 trans-crocin-2 260; 322; 433; 458 675.2; 541.1 651.2; 327.1; 311.1; 283.1 38.7 224; 262; 225; 432; 455 41.1 trans-crocin-1 224; 259; 318; 433; 768.8; 748.8; 458 575.2; 513.2; 473.2; 351.1

(14) The sections in bold correspond to quasi-molecular ions (999.4; 837.4; 675.2; 633.2; 609.0; 513.2; 511.2; 353.2 m/z) or ionic fragments (151.1; 123.0 m/z), which coincide with the work carried out by Lech et al., (2009).

(15) Due to the fact that safranal and crocins are the active substances related to mood improvement, these were quantified together as the sum of the safranal concentrations and the different crocin isomers in g/100 g of dry matter (%), according to the chromatograms obtained from the previously described HPLC analysis. Calibration curves were used, representing area units compared to concentration (mg/L), in order to quantify them, which were obtained through the analysis of the commercial reference substances safranal and the trans-crocin-4 at 310 and 440 nm respectively. The concentration of these compounds in the saffron extract of the present invention was calculated using the formula (1), taking into account the humidity of said extract and the purity of the reference substances used.

(16) $\begin{array}{cc}\left(\%\right)=\frac{\left(A_{310\mathrm{nm}}-a_{310\mathrm{nm}}\right)\text{/}{m}_{310\mathrm{nm}}+\left(A_{440\mathrm{nm}}-a_{440\mathrm{nm}}\right)\text{/}{m}_{440\mathrm{nm}}}{C}100& \left(1\right)\end{array}$

(17) Where:

(18) In the saffron extract of the present invention:

(19) A.sub.310nm is the peak area of safranal at 310 nm.

(20) A.sub.440nm is the sum of the areas of the peaks corresponding to the different crocin isomers at 440 nm.

(21) C is the concentration of the extract (mg/L)

(22) In the reference substances:

(23) a.sub.310nm is the ordinate in the origin of the calibration curve of the safranal standard

(24) a.sub.440nm is the ordinate in the origin of the calibration curve of the trans-crocin-4 standard

(25) m.sub.310nm is the slope of the calibration curve of the safranal standard

(26) m.sub.440nm is the slope of the calibration curve of the trans-crocin-4 pattern

(27) Among the active substances quantified in the above formula (1), crocins are the major compounds. Safranal is found in a smaller proportion, but its presence is essential in the saffron extract in order to obtain the desired effect in the prevention of mood disorders related to depression.

Example 2HPLC Analysis of Commercial Saffron Extracts, Certificated in Safranal According to the ISO3632 Standard

(28) Five commercial saffron extracts were analyzed by HPLC. All the samples indicated that they contain 2% by weight of safranal through the methodology used according to the ISO3632. The HPLC analysis was carried out in the same way as in Example 1 for the saffron extracts of the invention (Table 2).

(29) TABLE-US-00002 TABLE 2 HPLC analysis according to the method described in Example 1 of different commercial samples (A-E) of saffron extracts certificated at 2% in safranal according to the ISO3632 Standard. Commercial Samples HPLC Analysis 2% safranal Safranal Crocins * (ISO3632) (%) (%) (%) A 0.007 0.619 0.626 B 0.006 0.384 0.390 C 0.000 1.929 1.929 D 0.007 2.450 2.457 E 0.006 0.443 0.450 *Sum of Safranal and Crocins analyzed by HPLC according to formula (1)

(30) As can be observed in Table 2, all the samples are standardized to 2% safranal by dry weight according to the ISO3632, but the chromatographic analysis shows that the safranal content is considerably below this amount, even this active substance is completely absent in one of the analyzed extracts (Sample C). Moreover, the crocin concentration varies greatly, despite the fact that all the samples were standardized to 2% in safranal through the ISO3632 standard.

(31) These data reveal the need to carry out further specific quantitative analyses such as the HPLC methodology described above to evaluate the active substances in saffron extracts.

Example 3Clinical Trial

(32) In another preferred modality, a parallel, placebo-controlled, double-blind clinical trial was conducted in healthy participants, with the aim of determining the effective dosage of the active substances present in the extract of the present invention, which have an effect on the prevention of mood disorders related to depression.

(33) Methodology

(34) The following example describes the composition of the tablets that the participants included in the clinical trial received. As may be seen in Table 3, the new saffron extract was transmitted in two dosages, one of 28 mg/day and another of 22 mg/day, divided into two daily administrations, which along with the placebo group, formed the three treatment groups to be studied.

(35) TABLE-US-00003 TABLE 3 Composition of the saffron extract object of the present invention and of the placebo, present in the tablets used in the clinical trial. Daily Dosage (in tablets) Formula offered Placebo in the clinical trial 2 14 mg 2 11 mg 2 0 mg Extract of the invention 14.0 11.0 0.0 Excipients Red iron oxide 0.0 0.0 0.1 Microcrystalline cellulose 187.0 190.0 176.9 Calcium hydrogen phosphate 144.0 144.0 144.0 Carrot extract 0.0 0.0 22.0 Povidone 5.0 5.0 5.0 Croscarmellose sodium 9.0 9.0 9.0 Colloidal anhydrous silica 3.0 3.0 4.0 Magnesium stearate 3.0 3.0 4.0 Coating Hypromellose 9.0 9.0 9.0 Macrogol 8000 3.7 3.7 3.7 Red iron oxide 2.3 2.3 2.3 Carnauba wax q.s. q.s. q.s. q.s.: quantum sufficit

(36) In this study, the effectiveness of the saffron extract object of the present invention for improving general mood, reducing feelings of stress, anxiety, fatigue, and increasing vitality and sleep quality in healthy adults was studied.

(37) In the clinical trial, 128 individuals (healthy men and women aged between 18 and 77, self-diagnosed, with subsequent confirmation by doctors, with low mood but not depression) took part in order to study the effect of the new saffron extract during one month of treatment. The participants were divided into three groups to which the saffron extract of the present invention was supplied in tablet form with a dosage of 28 mg/day, 22 mg/day, or a placebo treatment, respectively, as may be seen in Table 4. Each subject received precise instructions by the doctors to take two tablets per day for four weeks (one table with lunch and another with dinner).

(38) TABLE-US-00004 TABLE 4 Demographic data on the participants at the beginning of the clinical trial. Treatment groups Total 28 mg/day 22 mg/day Placebo Demographic data (n = 121) (n = 41) (n = 42) (n = 38) Age Mean (SD) 39.1 (13.77) 40.4 (12.71) 36.7 (14.59) 40.38 (13.97) Range of variation 18-77 21-68 18-77 23-68 Sex (Number, %) Women 75 (62.0%) 26 (63.4%) 26 (61.9%) 23 (60.5%) Men 46 (38.0%) 15 (36.6%) 16 (38.1%) 15 (39.5%) Status (Number, %) Married 74 (61.2%) 25 (61.0%) 27 (64.3%) 22 (57.9%) Single 47 (38.8%) 16 (39.0%) 15 (35.7%) 16 (42.1%) Work (Number, %) Employed/student 103 (85.1%) 34 (82.9%) 37 (88.1%) 32 (84.2%) Unemployed/retired 18 (14.9%) 7 (17.1%) 5 (11.9%) 6 (15.8%) Weight 76.34 (17.22) 75.89 (16.48) 77.54 (18.20) 75.56 (17.39) Mean kg (SD) Body Mass Index 26.42 (6.33) 26.74 (5.90) 27.01 (7.91) 25.38 (4.77) Mean (SD) Smoker (Number, %) Yes 17 (14.0%) 8 (19.5%) 6 (14.3%) 3 (7.9%) No 104 (86.0%) 33 (80.5%) 36 (85.7%) 35 (92.1%) Alcohol (Number, %) Less than 3 per week 44 (36.4%) 14 (36.1%) 12 (28.6%) 18 (47.4%) More than 3 per week 77 (63.6%) 27 (65.9%) 30 (71.4%) 20 (52.6%) Exercise per week Yes (Number, %) 85 (70.2%) 29 (70.7%) 27 (64.3%) 29 (76.3%) No (Number, %) 36 (29.8%) 12 (29.3%) 15 (35.7%) 9 (23.7%) No significant differences were seen between groups at the start of the treatment (p > 0.01, two-tailed) SD: Standard deviation

(39) The study was revised and approved by the Queensland Clinical Research Ethics Committee and registered in the Australian New Zealand Clinical Trials Registry (approval number HREC2014002), protected by current legislation, in the National Health and Medical Research Council; Australia). The participants were evaluated by doctors following the inclusion and exclusion criteria approved by the Ethics Committee of the present clinical trial.

(40) After a first interview with the doctor, people diagnosed with a mood disorder such as MDD (Major Depressive Disorder), bipolar disorder or any other type of disorder that may have given positive results for depression in the Beck Depression Inventory (Beck et al., 1988) were excluded. People suffering from insomnia or working night shifts and were unable to have a normal night's sleep, or suffering from severe premenstrual syndrome with mood changes and/or pain that may mask the results during the study period, as well as people suffering from any neurological disorders or taking nutritional diet supplements, including herbs, that may affect mood, such as St. John's wort, tryptophan, S-adenosyl methionine, 5-hydroxytryptophan, melatonin and -aminobutyric acid, were excluded. People that were taking a saffron supplement or could not exclude saffron or food containing saffron from their diet were also excluded. Furthermore, candidates that showed signs of any type of known hypersensitivity to medicaments or nutritional supplements, foods or if they were receiving heparin, warfarin, dalteparin, enoxaparin or other similar anticoagulant therapy, that had been diagnosed with hypertension or were being treated with antihypertensive drugs, with kidney or liver failure, or any other type of illness, alcohol problems or drug use were excluded. Individuals who were participating or had participated in another clinical trial in the last 30 days were also excluded.

(41) After analyzing 137 potential participants under the exclusion criteria, 128 healthy adults between the ages of 18-77 were randomly assigned into three groups. Seven participants abandoned the study, the study reaching the end of the experimental stage with 121 active participants, 28 mg/day (n=41), 22 mg/day (n=42), and the placebo (n=38).

(42) The effect of the saffron extract of the invention on mood was evaluated by using the following indicators, which are extensively described in medical literature and clinical psychology: POMS (Profile of Mood States), PANAS (The Positive and Negative Affect Schedule) and DASS (Depression Anxiety Stress States). Sleep was controlled by the PSQI index (Pittburgh Sleep Quality Index).

(43) The POMS (Profile of Mood States. McNair et al., 1971) analysis, used in the clinical study to evaluate initial mood, consists of 65 items, with adjectives that describe an emotion (for example, sad, angry, happy), on a five-point scale for each one, where 0=not at all; 1=a little; 2=moderately; 3=quite a bit; and 4=extremely (except for relaxed and efficient items, which were recorded inversely). The participants were asked how they felt at that moment, and the answers were grouped into six subcategories; 5 negative (tension, depression, anger, fatigue and confusion) and 1 positive (vigor). For each participant, the Total Mood Disturbance (TMD, which includes Tension+Depression+Anger+Fatigue+ConfusionVigor) was calculated, projecting a general vision of the mood of the individual. The score variation from the beginning (baseline) to week 4 of the study was calculated for each subscale, with the aim of reducing the variance within the groups (Davidson et al., 2002; Edwards and Haythornthwaite, 2004). The score variations during the treatment with respect to TMD ranged from 232 to 200 (a positive result indicated an increase in depression, while a negative value indicated a decrease in depression).

(44) As a secondary method, PANAS (Watson and Clark, 1994) was used, which is an indicator consisting of 20 items; 10 positive words (for example, excited, proud, alert . . . ) and 10 negative words (for example, hostile, irritable, afraid, etc.), scored on a 5-point scale that is the same as POMS. Participants were asked how they felt compared to the previous week and the answers were grouped into two subscales (Positive Affect, PA, and Negative Affect, NA). The score variation range for PA and NA was from 40 to 40.

(45) Another secondary analysis method used was that which corresponds to the Depression Anxiety Stress States or DASS-21 (Lovibond and Lovibond, 1995), which consists of 21 items divided into three subscales that correspond to depression, anxiety and stress (each one with 7 self-report items). Participants were asked how they felt during the previous week and each item was scored from 0 to 3, where 0=never; 1=sometimes; 2=often; and 3=almost always.

(46) The PSQI or Pittsburgh Sleep Quality Index (Buysse et al., 1989) is designed to measure sleep quality through 19 self-classification questions and five questions proposed by people who have frequent and direct contact with the study participant, whenever it was possible.

(47) The statistical analyses were carried out through the SPSS 23.0 program with a level of significance <0.05. The different score variations of each of the parameters studied, related to mood and sleep, were calculated for each of the participants included in the clinical trial (Davidson et al., 2002; Edwards and Haythornthwaite, 2004).

(48) Results

(49) POMS

(50) FIG. 2 shows the results obtained for each of the emotions analyzed during the treatment period in the three study groups (treatment with saffron extract in dosages of 28 mg/day or 22 mg/day, and placebo). Gabriel's Post Hoc Test showed a significant improvement in all the POMS subscales of the treatment with saffron extract in the change scores for the group treated with 28 mg/day of the saffron extract of the present invention.

(51) For the case of Tension, a significant improvement was noted at the end of the treatment period, F (2.113)=3.82; p=0.025. Gabriel's Post Hoc Test revealed that the group taking 28 mg/day of the saffron extract was four-fold less tense than the placebo group (improvement of 1.06 to 4.00, FIG. 2). The same happened for the Depression subscale with F (2.113)=9.46, p<0.001, =0.36 (large effect); the group treated with 28 mg/day of the saffron extract significantly improved, being sixfold less depressed at the end of the treatment compared to the placebo group as can be seen in FIG. 2 (improvement of 1.33 to 8.43).

(52) The same trend was seen for the subscales Anger, with values F (2.112)=4.39, p=0,010, =0.26 (intermediate effect); Fatigue, with F (2.113)=4.92, p=0.009, =0.25 (intermediate effect) and Confusion, with F (2.113)=7.81, p=0.001, =0.32 (intermediate effect). The treatment with 28 mg/day of the saffron extract showed a significant fivefold reduction compared to the placebo group, anger (from 1.14 to 5.05), fatigue (from 1.11 to 5.00) and confusion (from 0.83 to 4.35; FIG. 2).

(53) The POMS vigor subscale showed a significant improvement on an inverse scale, with a value of F (2.112)=5.25, p=0.007, =0.26 (intermediate effect). The group treated with 28 mg/day of the saffron extract experienced a significant increase in vigor at the end of the treatment, improving by tenfold the vigor scale compared to the placebo group (from 0.39 to +4.00; FIG. 2).

(54) FIG. 3 represents the average variation corresponding to the Total Mood Disturbance (TMD) analysis for each of the study groups during the month of treatment with the saffron extract of the present invention F (2.111)=9.94, p<0.001, =0.37 (large effect). Specifically, the group treated with 28 mg/day of the saffron extract significantly reduced its TMD level, approximately sixfold compared to the placebo group (from 5.37 to 30.83; FIG. 3).

(55) PANAS

(56) FIG. 4 shows the average change values for the PANAS analysis, which although they are not significant, show a clear trend for the Positive Affect (or Positive Mood) and Negative Affect (or Negative Mood), indicating an improvement in mood in the groups treated with both the 22 mg/day and 28 mg/day dosages with the saffron extract of the present invention, with respect to the placebo group, after one month of treatment.

(57) The variance analysis (ANOVA) showed a significant difference in the treatment, between the study groups in the scores related to Negative Affect, F (2.111)=6.97, p=0.001, =0.31 (intermediate effect). The variation of the Negative Affect value in the group treated with 28 mg/day of the saffron extract, significantly improved by threefold compared to the placebo group (from 2.40 to 6.63), p=0.001, d=0.42 (intermediate effect, according to Cohen's conventions).

(58) DASS

(59) The ANOVA treatment was also carried out with the DASS test in order to analyze the variations observed in the subscales: depression, anxiety and stress among the different treatment groups.

(60) For the change scores in the specific case of the DASS depression subscale, a significant effect was noted with F (2.118)=12.96, p<0.001, =0.41 (large effect). As can be seen in FIG. 5, the group treated with 28 mg/day of the saffron extract of the present invention improved significantly with respect to the placebo group, reducing fourfold the symptoms related to a depressive condition (values from 1.42 to 5.61). A dosage dependent effect was also observed, obtaining a significant improvement in participants treated with 28 mg/day compared to those treated with 22 mg/day of the saffron extract, which halved the symptoms related to depression (values from 2.87 to 5.61).

(61) For the anxiety subscale, the treatment also showed a significant improvement, F (2.118)=4.33, p=0.01, =0.23 (intermediate effect). According to Gabriel's Post Hoc Test, there was a significant reduction in anxiety of almost three-fold in the group treated with 28 mg/day of the saffron extract compared to the placebo group, after one month of treatment (improvement of 1.23 to 3.22; FIG. 5).

(62) In the case of the DASS stress subscale, a significant change was also observed after treatment with the saffron extract, F (2.118)=14.29, p<0.001, =0.42 (large effect). The group treated with 28 mg/day of the present extract significantly improved the stress condition with respect to the rest of the groups studied. Specifically, the signs of stress at the end of the treatment were reduced by twofold and fourfold, compared to the group treated with 22 mg/day (reduction from 3.11 to 6.12) and to the placebo group (reduction from 1.49 to 6.12), respectively.

(63) Similarly to the effect of the treatment on the Mood component, the effect of the saffron extract of the present invention was also noteworthy in the component corresponding to Sleep quality, which was evaluated through the Pittsburgh index (PSQI). After carrying out the ANOVA statistical analysis on score variations in the overall PSQI, a significant improvement was observed in the sleep of the group treated with 28 mg/day in the overall PSQI change score, F (2.106)=4.27, p=0.016, compared to the placebo group.

CONCLUSION

(64) The results obtained in the clinical trial expand the scientific literature since it is the first such trial that has been carried out with healthy people. Furthermore, it shows that the saffron extract of the invention obtained at industrial scale, analyzed through HPLC, does not have any secondary or undesirable effect on the organism, but does show significant positive effects in all the psychological studies carried out to analyze mood at the end of the treatment (POMS-TMD, PANAS, DASS and PSQI).

(65) With regards to the dosage, it was possible to observe a clear dosage-dependent relationship between the two concentrations that were studied, which means that this study is one of the first to identify a clinically appropriate and empirically justified dosage schedule. The effects of the saffron extract, object of the present patent specification, on mood were consistent in both sexes, and they have been achieved without adverse effects on the performance or safety parameters.

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