Effects of pomegranate juice (Punica Granatum) on inflammatory biomarkers and complete blood count in patients with COVID-19: a structured summary of a study protocol for a randomized clinical trial

Objectives This study is conducted to investigate efficacy of pomegranate juice on inflammatory biomarkers, C-reactive protein (CRP), interleukin 6(IL-6), erythrocyte sedimentation rate (ESR) and complete blood count (CBC) in hospitalized patients with mild to moderate coronavirus disease 2019 (COVID- 19). Trial Design This is a randomized, placebo-controlled, double-blind, parallel 2-arm (1:1 ratio) clinical trial. Participants Patients with COVID-19 admitted to hospitals in Yasuj City, Kohgiluyeh and Boyer-Ahmad Province, Iran. Inclusion Criteria Informed consent Patients 18 years of age or older Diagnosis of COVID-19 based on real-time polymerase chain reaction (RT-PCR) test Exclusion Criteria Pregnancy or lactation Immunoglobulin A (IgA) level <61 mg/dl Disseminated intravascular coagulation or any other types of coagulopathy Severe congestive heart failure Participation in any clinical trial within 30 days prior to enrollment in this RCT Other contraindications determined by the specialist. Intervention and Comparator Intervention: 500 ml pomegranate juice and standard of care hospital treatment for COVID-19 Comparator: matching placebo containing 500 ml of red water and standard of care hospital treatment for COVID-19 Both intervention and comparator to be taken twice a day, after lunch and dinner, for 14 days. Criteria for Discontinuing Transfer of patients to intensive care unit (ICU) Death Unwillingness to continue participating in the study Main Outcomes The main outcomes of this study are levels of inflammatory biomarkers, CRP, IL-6, ESR, and CBC after 14 days of treatment. Randomization Eligible patients will be randomly assigned into the intervention or control group in a 1:1 ratio. Randomization will be performed based on 8 permuted blocks with block sizes of 6 and they will be stratified according to sex and age categories. Randomization sequences will be prepared by the trial’s pharmacist using computer-generated random numbers. Blinding (Masking) This study is a double-blind clinical trial (participant, researcher). The pomegranate juice and placebo juice are packaged in identical bottles, and the researcher and all the patients will be unaware of the study assignment until the end of the study. To ensure blinding, the randomization sequences will be kept in identical, opaque, sealed, and sequentially numbered envelopes. Numbers to Be Randomized (Sample Size) The calculated total sample size is 48 patients, with 24 patients assigned into each group. Trial Status The protocol is Version 1.0, on March 3, 2021. Recruitment started on February 28, 2021, and is anticipated to be completed by May 21, 2021. Trial Registration The Name of registering trial Effects of Pomegranate Juice (Punica Granatum) on Inflammatory Biomarkers and CBC in Patients with COVID-19: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial. Iranian registry of clinical trials (IRCT) Registration Number: IRCT20150711023153N2 Date of Trial Registration February 28, 2021, retrospectively registered Full Protocol The full protocol is attached as an additional file, accessible from the Trials҆ website (Additional file 1). In the interest of expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. Supplementary Information The online version contains supplementary material available at 10.1186/s13063-021-05194-9.

people with the severe type of Covid-19 display symptoms of cytokine syndrome. This disorder which is often caused by overactive immune system, is a hyper inflammatory syndrome known as sudden and fatal hypercytokinemia and multiple organ failure (MOF) [9,10]. The main mechanism of pathogenesis is the binding of the virus to angiotensin 2 receptors, which increases the activity of T lymphocytes and thus increases the production of inflammatory factors [1].
According to the above-mentioned statement, it seems that people whom boost their immune system, with the help of drugs, supplements and foods have been able to prevent the progression of inflammatory processes and overcome the disease [1,2]. Therefore, immune boosting foods such as pomegranate could be considered as a potential therapeutic option [1][2][3]11].
Pomegranate is a fruit with numerous antiviral and antioxidant compounds, including: polyphenols, flavonoids, tannins, anthocyanins and flavonoids, vitamins E and C, magnesium, zinc, potassium, iron and quercetin, whose beneficial effects on the various diseases including cardiovascular diseases and diabetes have been studied and have an enormous capacity to neutralize free radicals mainly because of ellagic acid and pony collagen [12][13][14][15][16] [16][17][18][19]. As reported by Feng et al. these polyphenols have an inhibitory effect on NF-KB pathway which has a pivotal role in disease pathogenesis [18]. Reducing CRP and inhibiting the cytokine storm pathway through the inhibitory effects of conjugated terpenes on PPAR factors is the other protective mechanisms [15,20]. As far as our knowledge goes, this work is the first study which examines the anti-inflammatory effects of whole pomegranate juice on disease progression in covid-19 patients.

Material and Methods: Participants & recruitment:
Out of the total number of hospitalized patients, 48 patients with definitive diagnosis based on PCR test, who also have other inclusion criteria, are included in the study by availability sampling.

Study Design & randomization:
The present study will be performed as a Randomized, Placebo-Controlled, Double-Blind, Parallel-Group clinical trial on patients with Covid-19 admitted to Shahid Jalil Hospital in Yasuj (Kohgiluyeh & Boyr Ahmad province, south-west of Iran). The study protocol was approved by the Ethics Committee of the School of Nutrition and Public Health () in Yasuj University of Medical Sciences and is in agreement with the declaration of Helsinki of the world medical association. This study was also registered in the Iranian Center for Clinical Trials (). Patients will be randomly divided into two groups of intervention (n=24), and placebo (n=24), by block random sampling method, which randomizes participants into 4 different blocks in each group, based on gender and age . Intervention group receive 500 ml of natural whole pomegranate juice twice a day, after lunch and dinner and control group receive the same amount of placebo with the same color and taste as natural juice. The pomegranate juice and placebo will be administered to patients by two nurses in opposite shifts. Patients will be followed for 14 days and the objectives of the study will be fully explained to the patient and their companions. An informed written consent form will be also obtained.

Preparation of pomegranate juice:
Pomegranates of Iranian species (robab) will be picked by hand and stored in tanks. All fruits will be crushed with skin. The juice will be pasteurized, concentrated and filtered at a temperature of 18 ° C. The product will be packaged in a disposable bottle and given to the patient daily. According to the manufacturer's recommendation, pomegranate juice will be stored at room temperature (25 ° C) until consumption [17].

Preparation of placebo drink:
Placebo is prepared by the addition of 0.02 % pomegranate emulsion of Mongolia Company to water. This protocol is approved by the Ministry of Health and is in accordance with the instructions on the product. The color is also matched to the natural pomegranate juice by edible color with the international code of E-122 [21].

Inclusion criteria:
 written informed consent form

Clinical, Para clinical and dietary intake assessments:
A demographic questionnaire containing information about age, sex, habitat, education, occupation etc., will be taken from patients. Medical history and medications/supplements history will also be asked of the patient or his companion.
Height, weight and waist to hip ratio (WHR) will be measured by standard methods [1] as well as the body mass index (division of weight in kilograms to the height in meters squared).
For the purpose of evaluating detailed information about nutrient intake and the food pattern, a 24-hour dietary recall for 3 days (including a weekend day) will be taken by a well-trained interviewer. Dietary intakes will be entered into Nutritionist IV software for further analysis.
10 cc blood samples will be obtained after 12 hour overnight fasting to measure complete blood count (CBC) and inflammatory indices including CRP, ESR & IL-6, at the baseline and by the end of week-2. In the case of patient discharge earlier than scheduled, they will be followed up after discharge and receive pomegranate juice or placebo. The length of hospital stay, disease complications, drug and prescription dosing and mortality rate will be recorded on a daily basis.
If the patient refuses to continue the study for any reason, blood sample will be drawn on the same day.

Follow up:
Subsequent follow-up is to detect and minimize the effects of the confounding factors, as well as to accurately carry out the intervention protocol and will be done by well-trained, committed researchers.

Primary objectives
To assess of the effect of Punica granatum juice on inflammatory biomarkers and CBC in patients with  Secondary objectives  To assess and compare the mean or median serum concentrations of interleukin 6 before and after the intervention and between the intervention group with placebo  To assess and compare the mean or median serum concentrations of ESR before and after the intervention and between the intervention group with placebo  To assess and compare the mean or median serum concentrations of CRP before and after the intervention and between the intervention group with placebo  To assess and compare the mean or median of CBC parameters before and after the intervention and between the intervention group with placebo

Data analysis
The data will be analyzed by SPSS software version 25. After examining the normality distribution of variables using Kolmogorov-Smirnov test, quantitative normal variables will be reported as mean±standard deviation, quantitative non-normal variables will be reported as median IQR, and qualitative variables as number (percentage). Independent T Test and Mann Whitney test will be used to compare the mean of variables between two groups. Paired T test or Wilcoxon test will be used to analyses within groups means. P-Value <0.05 will be considered as statistically significant. If the patient refuses to continue the study for any reason, statistical methods (Intention-to-treat analysis) will be performed.