Effect of saffron supplementation on Parkinson’s disease: a study protocol for a triple-blind randomised controlled clinical trial

Background and rationale

Parkinson’s disease (PD) is a long-term neurodegenerative disease with a second-degree prevalence among neurodegenerative diseases worldwide.1 Additionally, its prevalence is at a growing rate, such that a total of 12 million people will suffer from PD by 2050.2 PD usually emerges after 50 years. Nevertheless, some risk factors might trigger the PD incidence in younger people.3 Men suffer from PD twice as often as women due to the protective features of female sex hormones, exposing different risk factors in male and female, and healthcare system disparities.4 5 Although in the first decade after PD onset, the mortality rate does not change significantly, in the following decades, it doubles.6

PD has both motor symptoms (MS) and non-motor symptoms (NMS).7 Although the NMS depends on the severity of PD, its onset is earlier than MS. The NMS would be manifested by mental health disorders such as depression, anxiety, distress, cognitive impairment, sleep disorders, apathy, hallucinations and psychosis. Additionally, constipation, fatigue, incontinence, low blood pressure (orthostatic hypotension), pain, sexual dysfunction, sweating and impaired temperature are other symptoms of NMS. MS emerged by tremors, rigidity (stiffness), slowness of movement, falls and dizziness, freezing, dystonia and muscle cramps. Therefore, the quality of life is drastically affected by PD severity.8

Dopamine degeneration (in the substantia nigra pars compacta) is the leading cause of the incidence of PD. PD has two main characteristics, including the degeneration of the dopaminergic neurons in specific brain areas (substantia nigra) and the accumulation of α-synuclein. Many factors are involved in the pathogenesis of PD, including oxidative stress, inflammation and mitochondrial dysfunction. Dopaminergic neurons are more sensitive to oxidative stress due to their low antioxidant content. Additionally, the dopamine, unsaturated fatty acids and iron contents of dopaminergic neurons are susceptible to oxidation.9 10

Moreover, oxidative stress impairs mitochondrial function, decreases the energy production for the brain and indirectly increases the apoptosis process, especially in dopaminergic neurons.11 The oxidative stress would lead to the secretion of inflammatory cytokines such as tumour necrosis factor-alpha, which facilitate the secretion of caspase 1, 3 enzymes and neuron degeneration. Additionally, oxidative stress would facilitate the process of protein carbonylation and aggregate the protein carbonyl, consequently leading to inflammation and apoptosis in neurons. Hence, it is worth controlling the sources of free radicals such as foods, air, stress and even the intestinal microbiome, because intestinal permeability is a potential source of inflammation and neurodegenerative disease.12 Additionally, using the antioxidant and anti-inflammation plants would alleviate the inflammatory and oxidative pathways, prevent the onset of PD and control its progression.13

Although there are some drugs to control the MS and NMS of PD, they have several side effects. Moreover, they do not control the inflammation and oxidative stress, the leading causes of PD progression.14 On the other hand, there is a growing interest in herbal medicine as a safe and cheap adjunctive therapy to suppress the inflammation and oxidative stress and prevent the PD progression. Therefore, finding a natural substance to control the oxidative stress and inflammation, the leading causes of PD, is worthy.

Saffron (Crocus sativus L) consists of four major bioactive compounds, including Crocin, Crocetin, Picrocrocin and Safranal, which are involved in the sensory characteristics of saffron (colour, taste and aroma, respectively). The protective effects of saffron on different diseases are due to its antioxidant and anti-inflammatory features. Additionally, saffron has positive effects on the digestive system and intestinal permeability by modulating the intestinal microbiome. On the other hand, evidence documented the pharmacological effects of saffron on neurodegenerative diseases.15 Moreover, the therapeutic effects of saffron on psychological, sleep quality and other related diseases were indicated by previous studies. Such that, a systematic review and meta-analysis indicated that 6–8 weeks of saffron supplementation could alleviate depression and anxiety.16 Another meta-analysis documented that sleep disorders would be cured by a mean dosage of 100 mg/d saffron.17 Additionally, another systematic review and meta-analysis indicated that patients with Alzheimer’s disease might experience better cognitive function by saffron supplementation.18 Moreover, experimental studies indicated protective effects of saffron supplementation on animal models of PD.19 20 Furthermore, a recently published randomised controlled clinical trial investigated the effect of 60 mg/d crocin on movement disorders and oxidative DNA damage.21 However, only movement disorders were significantly improved after 8 weeks intervention. Another recently published study examined the effect of 30 mg/d saffron supplementation or placebo on 52 patients with PD during 8 weeks.22 Although significant changes were observed in the depressive symptoms, there were no significant changes in MS, following saffron supplementation.

Regarding the limited data on this subject, it seems necessary to assess the effect of optimal dosage of saffron during appropriate time on MS, NMS, inflammation, oxidative stress and other PD outcomes on a higher sample size of patients with PD. Therefore, we aimed to perform a triple-blind randomised clinical trial to assess the effect of 100 mg/d saffron supplementation on several PD outcomes in patients with PD (n=92) for 12 weeks.

Objectives

The current study will investigate the effect of saffron supplementation on serum values of C-reactive protein (CRP), total antioxidant capacity (TAC), malondialdehyde (MDA), glutathione (GSH), zonulin, the activity of catalase enzyme (CAT), PD stage, symptoms of PD (MS, NMS), quality of life, mental health, sleep quality, cognitive status, anthropometric indices, blood pressure (BP), gastrointestinal symptoms, appetite and fatigue, in patients with PD in a context of a triple-blind randomised controlled clinical trial.

Patient and public involvement

The patients and members of the public will not be involved in the study design, conduct, reporting or dissemination plans of the current study.

The patients will be informed of the aim and process of this study. After that, the volunteers will sign an informed consent. The patients and their caregivers will be instructed about how to take supplements. Additionally, they will be asked to record the patient’s physical activity (PA) on the weeks of the second and eighth of intervention. Furthermore, in order to gather the dietary intakes of patients through a food recall, the patients or their caregivers will be asked about the dietary intakes on the third, sixth, ninth and 12th weeks during the intervention. Moreover, the other questionnaires will be completed through an interview at the beginning (before intervention) and end of intervention. The process of blood collection will also be conducted before and after the intervention. In addition, phone calls to patients or their caregivers will be used to increase the adherence of patients during the intervention. As the end-stage patients (Hoehn and Yahr score=5) might have severe cognitive impairment and other disabilities, they will not be included in the study. However, the other patients (Hoehn and Yahr score <5) might also suffer from various degrees of cognitive impairment. Therefore, for the patients with cognitive impairment (Abbreviated Mental Test, AMT score less than 8), the patients and their caregiver or adult child will also be involved in the study to evaluate the compliance, probable side effects and outcomes.

Trial design and setting

A triple-blind, randomised, parallel, two-arm, placebo-controlled superiority clinical trial with the allocation ratio of 1:1 will be performed.

The participants will be selected from patients with PD, who are referred to the neurologist’s clinic of Al-Zahra and Khorshid Hospitals in Isfahan city, Iran.

Eligibility criteria

Intervention and comparator

In the current RCT, tablets containing 100 mg of saffron or placebo will be given to patients for 12 weeks. Human studies documented that oral intake of saffron less than 400 mg/d did not have any statistically significant side effects, compared with the control. Additionally, the maximum tolerated dosage of oral intake of saffron is 1.5 g/d. Nevertheless, the 5 and 20 g/kg are toxic and lethal dosages of oral saffron intake, respectively.23 Animal studies indicated that toxic values of oral intake or injection of saffron would have adverse effects on liver, kidney, lungs and even development of fetus by affecting the indices of blood urea nitrogen, creatinine (Cr), alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, red blood cell, white blood cell count, haemoglobin, haematocrit, platelets, alveolar size and growth retardation.24 Based on previous human investigations, 100 mg/d saffron is completely safe and appropriate.23 25

Patients under PD treatment will be randomly assigned to the intervention or control group. The patients in the treatment group will be asked to take a tablet containing 100 mg/d saffron for 12 weeks. While those in the control group will receive a placebo tablet containing 100 mg/d lactose over 12 weeks. After agreement to participate in the study, the subjects will be randomly assigned to the study groups. In the intervention group, 100 mg/d of first-class Sargol saffron powder (prepared by Mojtahedi Company) will be given, and in the placebo group, 100 mg/d of lactose powder (prepared by Pak Azma Company) will be given. To ensure the quality of tablets, the crocin content of saffron will be measured through the use of high-performance liquid chromatography method, before and after providing tablets. The preparation of tablets will be done at the Faculty of Pharmacy. The shape, colour, size and even smell of placebo and saffron tablets and their packs will be the same. These packs will be labelled A and B by a blind person. The principal investigator will instruct the participants on supplement consumption. The patients will be instructed to consume one saffron tablet per day after lunch to enhance the gastrointestinal tolerability. However, to avoid any probable interaction with other medications, they will be asked to take the tablets by maintaining at least 2–3 hours interval with other drugs.

The packs of saffron and placebo will be provided for 4 weeks. These packs will be given every 4 weeks. Therefore, the supplements will be given at the baseline, fourth and eighth weeks of intervention. Moreover, the instructions for using the supplements will be explained at the baseline, fourth week, eighth week and each phone call. Additionally, the remaining content of packs will be returned by participants every 4 weeks. Furthermore, the compliance and side effects will be evaluated by weekly phone calls and clinic visits. The calculation of the compliance rate will be conducted using this formula: compliance rate = (tablets taken/tablets prescribed) × 100. Participants with low compliance (<80%) will be excluded. The patients will be withdrawn by themselves or by the researcher for any reason, such as reluctance to continue cooperation, observing side effects, changing her/his drugs, etc. The details of subjects withdrawing will be provided in the study results. An experienced neurologist will prescribe a standard treatment for patients, which will not be changed during the study. Besides the standard treatment, they will only receive saffron or a placebo. However, they will be allowed to continue their regular care, without any restriction for concurrent care. Participants will be asked not to change their lifestyle, including PA, dietary habits and medication, during the study. If they needed to change their medicines, the neurologist would prescribe different medications and they would be excluded from the study.

Outcomes and data collection methods

The primary outcomes are the stage of PD, symptoms of PD (MS and NMS), and the serum values of CRP, TAC, MDA, GSH, zonulin and the activity of CAT. Additionally, the secondary outcomes are quality of life, mental health, sleep quality, cognitive status, anthropometric indices, BP, gastrointestinal symptoms, appetite and fatigue.

Demographic variables and socioeconomic status

An expert researcher will assess the demographic and socioeconomic status of parents through a face-to-face interview and using a general questionnaire. Such that, the following data will be assessed: age, sex, marital status, education, medical history and medications, duration of PD diagnosis, family history of PD, comorbidity, smoking and indices of socioeconomic status.

PD stage

An experienced neurologist will define the stage of PD by using the Hoehn and Yahr scale.26 In this method, the stage of PD will be stratified based on the functional disability. The modified version of this scale consisted of different stages including no signs of disease (stage 0), unilateral involvement (stage 1), unilateral plus axial involvement (stage 1.5), bilateral involvement without impairment of balance (stage 2), mild bilateral disease with recovery on pull test (mild balance impairment but recovers easily) (stage 2.5), mild to moderate bilateral disease; some postural instability; physically independent (stage 3), severe disability; still able to walk or stand unassisted (stage 4) and wheelchair-bound or bedridden unless aided (stage 5).

NMS and MS of PD

A neurologist will evaluate the MS and NMS symptoms of PD patients using the MDS-UPDRS scale. This scale has four parts, including (1) non-motor experiences of daily living, (2) motor experiences of daily living, (3) motor examination and (4) motor complications.27

Dietary intakes

An expert nutritionist will assess the dietary intakes during the weeks of the third, sixth, ninth and 12th weeks by using 24-hour dietary recalls. According to the protocol, the household measures will be converted to grams.28 Additionally, the Nutritionist 4 software (First Databank, Hearst Corp., San Bruno, CA, 161 USA) will be used to calculate the nutrients and calorie intakes.

Physical activity

The patients will be requested to record 1 day PAs, during the second and eighth weeks. Metabolic equivalent hours per day will be used to report the PA.

Anthropometric and blood pressure (BP)

The weight and height will be measured without shoes and with minimal clothing by a Seca scale (with an accuracy of 100 g) and a portable stadiometer (to the nearest 0.5 cm), respectively. The formula of ‘weight (kg)/height squared (m²)’ will be used for BMI calculation. A digital sphygmomanometer (OMRON, M3, HEM-7154-E, Japan; with the accuracy of 3 mm Hg) will be used for BP measurement. Such that, after 5 min of rest, BP will be measured twice, and the average of them will be reported.

Mental health

A valid and reliable Persian version of the 21-item Depression, Anxiety and Stress Scale (DASS-21) will be used for a mental health assessment, including depression, distress and anxiety.29 Each subscale will be separately investigated by seven questions with a score ranging from 0 (does not apply to me at all) up to 3 (applies to me most of the time) for each question. The score range for each subtitle is 0–21, which will be multiplied by two for interpretation. The higher scores indicate higher psychological problems. This questionnaire is a valid scale for mental health assessment in PD and previous studies used this scale for PD patients.30–32

Quality of life

The quality of life will be evaluated through the use of the validated Persian version of the 39-item PD questionnaire (PDQ-39).33 This scale consisted of eight aspects of quality of life, including cognition, mobility, emotional well-being, communication, daily activities, stigma, social support and bodily discomfort. The questions of this scale have a Likert scale of 0–4. The score of each aspect will be calculated by dividing the total number of each aspect by the maximum score of the aspect that the patient can obtain, multiplied by 100. Therefore, the score range of each aspect is 0 (best status) up to 100 (worst status). Finally, Parkinson’s Disease Summary Index (PDSI) will be calculated by summing the score of eight aspects and dividing the total score by 8.

Sleep quality

The assessment of sleep quality will be conducted through the use of the Parkinson’s Disease Sleep Scale (PDSS), which has been previously validated in Iranian PD patients.34 This questionnaire consists of 15 items with a range of scores of 0–10. The total score of each patient will be between 0 (worst status) and 150 (best status).

Cognitive status

The AMT will be used for assessment of cognitive status. This questionnaire has 10 questions. The presence of cognitive impairment will be defined as scores less than 8 out of 10. The validity and reliability of this scale were previously validated in Iran.35

Gastrointestinal symptoms

A Visual Analogue Scale will be used to assess severity and frequency of 12 gastrointestinal symptoms, including heartburn, anorexia, belching, flatulence, diarrhoea, dysphagia, vomiting, nausea, abdominal pain, bloating, constipation and faecal incontinence. The range score of this questionnaire is from 0 (best) to 100 (worst). Preziosi et al36 and Foshati et al37 38 previously used this scale for the assessment of gastrointestinal symptoms in MS patients.

Appetite

The appetite of patients will be assessed by the Simplified Nutritional Appetite Questionnaire (SNAQ).39 This questionnaire consisted of four questions. The scores of patients with the highest and lowest appetite are 20 and 4, respectively.

Fatigue

The validated Persian version of the Parkinson Fatigue Scale (PFS-16)40 41 will be used for assessment of fatigue. This questionnaire consists of 16 questions to which scores ranging from 1 to 5 were assigned. The range scores of 16–80 represent the best and worst status, respectively.

Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use

Blood collection will be conducted after 12 hours of fasting the night before and after the intervention. After obtaining a 10 cc blood sample, it will be centrifuged for 10 min at a speed of 2500 rpm at room temperature. After allocating the serum samples in microtubes, they will be frozen at −80°C. The measurement of serum zonulin and CRP will be conducted through the use of ELISA (ZellBio, Germany) and immunoturbidimetric kits assay (BIOREX, Iran), respectively. Additionally, the enzyme activity of catalase (CAT) will be measured by the commercial kits (Kiazist Life Sciences, Iran), based on the reaction of catalase with methanol and the subsequent production of formaldehyde. Furthermore, the serum values of TAC, MDA and GSH will be measured through the use of commercial kits (Kiazist Life Sciences, Iran), and using the methods of CUPric Reducing Antioxidant Capacity (CUPRAC) assay, Thiobarbituric Acid Reactive Substance (TBARS) and Thiol content assay, respectively.

Data collection methods

The advantages of saffron supplementation for PD and even other diseases will be explained to participants. Additionally, regular reminders will be conducted through weekly phone calls. Any reasons for non-adherence or non-retention of participants will be recorded. An intention-to-treat (ITT) approach will be used to include the patients who will not complete the intervention in the analyses.

Harms

The adherence and probable side effects will be assessed through the weekly phone calls. In case of emerging any side effects, the study will be immediately stopped and the necessary medical measures will be taken. As the presence of any side effect will be reported to the ethical committee, it will decide to exclude the subject or perform other suitable actions.

Participant timeline

The flow chart and diagram of the schedule of enrolment, interventions and assessments are provided in figure 1 and figure 2, respectively. The Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT 2025) was used to prepare the study protocol.

Figure 1

The protocol flow chart and timeline of the study.

Figure 2

Schedule of enrolment, intervention and assessment based on SPIRIT guidelines. MS, motor symptoms; NMS, non-motor symptoms; PD, Parkinson’s disease; SPIRIT, Standard Protocol Items: Recommendations for Interventional Trials.

Sample size

The sample size calculation was conducted based on the formula of (n=2× [(Z _1−α/2+Z_1−β)² × S²]/Δ²) and by considering a type I error of 5% (α=0.05), type II error of 20% (β=0.20, power=80%). We considered the most related clinical outcomes as the primary outcomes and calculated the sample size based on the primary outcomes. As the sample size based on the CRP was the biggest sample size (estimated sample size=41.9≈42), we considered CRP as the main variable for sample size estimation. However, regarding probable drop during the study, a total of 46 patients were included in each group.

The details of sample size calculation are provided below.

CRP: by considering the variances (σ²)42 and expected mean changes of CRP (σ=8.18, Δ=5), the estimated sample size was 41.9≈42.

MDA: by considering the variances (σ²)32 and expected mean changes of MDA (σ=2.39, Δ=1.5), the estimated sample size was 39.8≈40.

GSH: by considering the variances (σ²)32 and expected mean changes of GSH (σ=83.9, Δ=53), the estimated sample size was 39.29≈39.

TAC: by considering the variances (σ²)43 and expected mean changes of TAC (σ=0.7, Δ=0.45), the estimated sample size was 37.94≈38.

CAT: by considering the variances (σ²)44 and expected mean changes of CAT (σ=1.51, Δ=1), the estimated sample size was 35.75≈36.

Zonulin: by considering the variances (σ²)45 and expected mean changes of Zonulin (σ=54.2, Δ=34), the estimated sample size was 39.84≈40.

Stage based on the Hoehn-Yahr scale: by considering the variances (σ²)46 and expected mean changes of stage (σ=0.62, Δ=0.4), the estimated sample size was 37.67≈38.

MDS-UPDRS (part 2): by considering the variances (σ²)47 and expected mean changes of MDS-UPDRS (part 2) (σ=4.21, Δ=3), the estimated sample size was 30.87≈31.

Recruitment

Participant recruitment will be done in the neurology clinic of Al-Zahra and Khorshid Hospitals, affiliated with Isfahan University of Medical Sciences in Isfahan, Iran.

Such that, an expert neurologist (FK) will visit the patients and diagnose the PD based on the UKPDSBB. After informing the eligible patients about the aim of this study, written informed consent will be gathered from them.

Randomisation: sequence generation, allocation concealment mechanism and implementation

Before beginning the study, the stratified block randomisation will be conducted based on sex (men vs women) and severity of PD (Hoehn and Yahr stages of 1–2 vs 3–4).

An unaware person will do the randomisation through the use of an online tool (https://www.sealedenvelope.com/simple), with affix block size of four. Subjects will be randomly divided into the intervention (saffron, n=46) or control (placebo, n=46) groups, into a 1:1 ratio. The process of participant assignment into groups will be applied by a blind researcher. The allocation sequence will be concealed in opaque, sealed envelopes. A researcher who is not involved in recruitment or assessment will securely store the random allocation list. An independent person will do the labelling of the study packs as A and B. A second person will monitor this process. The patients, investigators and responsible persons for doing laboratory tests and statistical analyses will be blind. The randomisation codes will be locked until the trial completion

Blinding

Regarding the triple-blind design of the study, the patients, investigators and persons who will perform laboratory tests and statistical analyses will be blind. In order to have a triple-blind design, the tablets of saffron and placebo will be provided in the same shape, colour, size and smell. After that, the same packages will be used for packing the saffron and placebo. A computer labelling system will be used to label the packages as A and B. Only an independent person will be aware of the labels. The codes will be stored in opaque sealed envelopes. At the end of the trial, the codes will be unlocked.

According to the criteria of the Medical Ethics Committee, the packs of saffron and placebo will be blind, unless observing any side effect. In this case, the pack will be revealed to realise the kind of intervention (placebo or saffron). After that, any unblinding will be recorded in the study and the Iranian Registry of Clinical Trials, and the Ethics Committee of Isfahan University of Medical Sciences.

Data management

The process of coding, security and storage of data will be overseen by one researcher. Immediately, the data from forms will be entered electronically at the participating site. The data entry and values will be double-checked. A researcher will securely store the forms. The normality of variable distribution will be evaluated through the use of Q–Q plot. In the case of non-normal distribution, log transformation will be applied. The χ² test and independent sample t-test will be used to compare the values of qualitative and quantitative variables between groups, respectively. To assess the changes in each group, a paired-sample t-test will be conducted. To adjust the potential confounders, a one-way analysis of covariance will be performed. SPSS software version 26 will be used for doing statistical analyses. The statistically significant results will be considered as p<0.05.

The current study will not have any interim analysis. The study will be stopped for patients with adverse side effects that have a significant effect on health status, including death, severe allergic reactions, disability, etc. However, by doing ITT analysis, these subjects will not be excluded from analyses. Assuming that data are missing at random, the missing values will be handled through the use of expectation-maximisation (EM) algorithm. In this method, the missing data will be estimated according to the maximum likelihood method by incorporating baseline characteristics, treatment allocation and all available outcome measures into the estimation model.

The details of withdrawing each subject, especially observing any side effects, will be reported. No subgroup analyses are planned.

Data monitoring committee

The academic committee of Isfahan University of Medical Sciences will impartially and continuously monitor the data. To ensure that the study is run based on the ethical principles to protect the patient’s health and dignity, the Ethics Committee and the Vice-Chancellor of Isfahan University of Medical Sciences will supervise all aspects of the study. In the case of ethical violations, the study will be modified or terminated. The study coordination and regular communication among the study team will be conducted by the principal researcher and through the supervision by a steering committee.

Trial monitoring

The current study will adhere to the guidelines of the Ethics Committee of Isfahan University of Medical Sciences. This committee will oversee the quality, validity and adherence to the ethical standards during monitoring sessions. Additionally, a report will be submitted to the auditor every month.

Ethics and dissemination

This study was confirmed by the Ethics Committee of Isfahan University of Medical Sciences with the code of IR.MUI.PHANUT.REC.1402.072. Written informed consent to participate will be obtained from all participants. Final findings will be presented in future publications and scientific congresses.

Protocol amendments

Any changes in the study protocol including study sample size, objectives, participants, or study procedures will be reported to the ethical committee of Isfahan University of Medical Sciences and approved by this committee. Moreover, the details of changes will be reported at the Iranian registry of clinical trial website (https://www.irct.ir/).

Consent or assent

The principal researcher will inform the patients and their caregivers about the purpose of the study, the gains and the probable risks. Before the initiation of the study, the written informed consent will be signed by participants. The end stage patients (Hoehn and Yahr score=5) who might have severe cognitive impairment will not be included in the study. However, the caregiver or adult children of the patients with cognitive impairment (AMT score less than 8), who are not able to make a decision, will also sign the informed consent. The consent form provided to participants includes their agreement to use the data in future studies related to this one. However, they will not be obligated to consent to other studies beyond this.

Confidentiality

A unique code will be assigned to each subject to store research data. The personal participants will be confidential in publications.

Ancillary and post-trial care

At the end of the study, the supplementation with saffron and placebo will be stopped and patients will continue their standard treatment. In case of emerging any side effect, the participant will be excluded from the study and appropriate free treatment will be provided for them.

Planned study dates

According to the previously recorded registration in the Iranian Registry of Clinical Trials (IRCT), the expected recruitment start date was 21 June 2024, and the expected end date was 21 April 2025. However, as the manuscript review process for the protocol article took 1 year, the publication of this article was delayed.

Protocol and statistical analysis plan

The protocol of this study was registered in the Iranian Registry of Clinical Trials.

The full statistical analysis plan will be accessible by a logical request from the corresponding author.

Data sharing

The statistical code and data sets will be provided by the corresponding author (G.A), through a logical request.