Adjuvant pomegranate juice intake improves the inflammatory status of hospitalized COVID-19 patients: A randomized and placebo-controlled trial

Abstract

Background

This study aimed to evaluate the effect of pomegranate juice intake on the inflammatory status and complete blood count in hospitalized Covid-19 patients.

Methods

This randomized, double-blinded placebo-controlled trial included 48 patients with two parallel arms. In addition to the standard care provided at the hospital, the patients consumed 500 mL of whole pomegranate juice (PJ) daily or a placebo for 14 days. Inflammatory markers (C-reactive protein (CRP), interleukin-6 (IL-6), erythrocyte sedimentation rate (ESR)) and complete blood count were determined at baseline and after the 14 days of intervention.

Results

At the end of the intervention, a significant decreased was observed in primary outcomes [mean difference (95 %CI)] including IL-6 [5.24(0.87–9.61)], CRP [23.19(11.93–34.44)] and ESR [10.52(1.54–19.50)] in the PJ group vs. before the intervention. In addition, significant changes were also observed in the some of the secondary outcomes, including neutrophils, lymphocytes, platelets, platelets-to-lymphocyte(PLR) and neutrophils-to-lymphocyte (NLR) ratios (p < 0.05) in the PJ group compared to before the intervention. At the end of the intervention period, the mean change of IL-6 [− 7.09(−12.21 to − 1.96)], white blood cells [− 3.09(− 6.14 to − 0.05)], neutrophils [− 9.12(−18.08 to −0.15)], lymphocyte [7.05(0.17–13.92)], platelets [− 94.54(− 139.33 to − 49.75)], PLR [− 15.99(− 29.31 to − 2.67)], blood oxygen saturation [1.75(0.13–3.37)] and MCV [0.31(− 0.25 to 0.88)] levels were significantly different between groups while no difference was observed between the two groups in other blood indices.

Conclusion

Our results suggest that pomegranate juice intake might slightly improve the inflammatory status and CBC outcomes of COVID-19 patients and it may be beneficial.

1. Introduction

SARS-CoV-2 is the last generation of a single-stranded RNA virus family known as coronaviruses, first discovered in 2019 in Wuhan (China)¹ and only spread in mammals.² The average incubation period is 5 days and varies depending on age,³ health status, and underlying diseases, including particular cardiovascular disorders and endocrine dysfunctions.4., 5. Although this infection critically affects the respiratory tract, almost every vital organ can be damaged.⁶ The most prevalent symptoms include headache, fever, dry cough, fatigue, maldigestion, muscle pain, nausea, and vomiting.⁷ The severity of symptoms is individualized and varies from mild and moderate without any considerable symptoms to severe and critical conditions⁸ with systemic inflammation, sepsis, and organ dysfunction.⁹ Previous researches have demonstrated that many people severely affected by the virus display symptoms compatible with a cytokine storm. This disorder, often caused by the overactive immune system, is a hyperinflammatory syndrome known as sudden and fatal hypercytokinemia and multiple organ failure.10., 11. The binding of the virus to angiotensin II receptors is the main mechanism of pathogenesis, which increases the activity of T lymphocytes and enhances the production of inflammatory factors.¹² Covid-19 manifestations depend largely on the host immune system and nutritional status¹³ so that individuals who boost their immune system using drugs, supplements, and foods have been able to prevent the progression of inflammatory processes more efficiently.14., 15. Accordingly, nutritional strategies¹⁶ and immune-boosting nutrients are highly recommended.¹⁷

Pomegranate (Punica granatum) is native to the Middle East.¹⁸ This fruit is well-recognized as a rich source of anthocyanins, flavonols, proanthocyanidins, punicalagins, ellagic acid derivatives, and organic acids in the entire parts.19., 20. Pomegranate juice has been reported to exhibit antiviral activity by blocking the binding of the virus to receptors of the host cells.²¹ Anti-inflammatory properties of pomegranate polyphenolic compounds, especially punicalagins, and their gut microbiota-derived urolithins,²² are exerted through the peroxisome proliferator-activated receptor transcription factors (PPAR), inhibiting nuclear factor κB (NF-κB) activation, and mitogen-activated protein kinase (MAPK) pathway. These actions subsequently suppress the production of various pro-inflammatory mediators, including tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), and C-reactive protein (CRP), among others.23., 24. Besides, recent findings suggest that certain constituents of pomegranate peel extract exert potential benefits against SARS-CoV-2 via interaction with S-glycoprotein and angiotensin-converting enzyme 2 (ACE2).²⁵ To the best of our knowledge, there are no previous studies on the effects of pomegranate juice intake on hospitalized COVID-19 patients. Therefore, this study aims to explore the possible adjuvant effects of pomegranate juice intake on the inflammatory status and complete blood count of hospitalized COVID-19 patients.

2. Materials and methods

2.1. Study protocol

This trial was performed according to the guidelines of the Helsinki Declaration and approved by the Ethical Committee of Research, Yasuj University of Medical Sciences (IR.YUMS.REC.1399.181), and recorded at the Iranian registry of clinical trials (IRCT20150711023153N2). The study protocol was fully explained to the patients, who were asked to fill out and sign a written informed consent form prior to their participation. The protocol of this study was published in the Trial journal with https://doi.org/10.1186/s13063-021-05194-9 in April 2021.

2.2. Study design

This study was randomized, double-blinded, and placebo-controlled with a 2-arms parallel design (1:1 allocation ratio). The RCT was double blind, both for the investigators and for the enrolled participants. At baseline and after stratification for intervention, 48 subjects (hospitalized with moderate or severe infection, not critical infection)²⁶ were randomly allocated to receive either pomegranate juice (PJ) or a placebo. In addition to the standard care provided to the patients at the hospital, patients from the PJ group received 500 mL of whole natural PJ daily for 14 days (250 mL after lunch and 250 mL after dinner). Those patients from the control group received the same amount of placebo (with the same color, taste, shape, and packaging).

2.3. Sample size and randomization

Using the STATA software Version14, sample size was calculated based on the IL-6 difference after the intervention with 250 mL of pomegranate juice²⁷ and considering the α 5 %, power 80 %, and with 10 % drop, 24 people for each group, and a total of 48 people. In total, 48 patients (24 + 24) were recruited and randomized based on eight permuted blocks with block sizes of six and they were stratified according to sex and age, using random allocation software. The inclusion criteria were age > 18 years, hospitalized COVID-19 patients with a diagnosis based on RT-PCR, and signed written informed consent. Non-inclusion criteria included pregnancy or lactation, IgA < 61 mg/dL, disseminated intravascular coagulation or other types of coagulopathy, severe congestive heart failure,²⁸ or participation in any other trial within the past 30 days prior to enrollment in the present study. Possible termination of their participation included the transfer of the patient to the intensive care unit or unwillingness to continue participating in the study.

2.4. Preparation of pomegranate juice and placebo

Pomegranates from Iran (cv. Robab) were picked by hand and stored in tanks. First, the juice was obtained by pressing the whole fruit. Then the juice was pasteurized, concentrated, filtered at a temperature of 18 °C, vacuum bottled, and stored at room temperature (25 °C) until consumption. Placebo was prepared by adding 0.02 % pomegranate emulsion, prepared by a Mongolia company (Iran) containing the natural color E-122, to water.²¹ Both PJ and placebo matched in taste and color. The juice and placebo were packed in disposable bottles with codes blind to the researchers and patients. The juice and placebo were provided to each patient daily. This study protocol was approved by the Ministry of Health and followed the instructions on the product. For calculating the adherence rate, the remaining volume of pomegranate or placebo was measured in every single bottle at the end of each day, and the adherence (%) was calculated using the following formula (the lower the % obtained, the higher the adherence): mL consumed × 100/(250 mL of PJ or placebo).

2.5. Blood sampling

Blood samples (10 mL) were obtained after 12 h overnight fasting to measure inflammatory indices, including IL-6, CRP, and erythrocyte sedimentation rate (ESR), and complete blood count (CBC), at the baseline and after 14 days. In the case of patients discharged earlier than scheduled, they were followed up after discharge and received PJ or placebo. The length of hospital stay, disease complications, drug and prescription dosing, and mortality rate were recorded daily. If the patient refused to continue the study for any reason, blood sample was drawn on the same day. CBC was measured with a cell counter (kh-21n, Sysmex, Japan), and CRP with a serologic kit (PAADCO, Spain) in serum. IL-6 was measured with LDN Elisa kit (KPG, Iran), and ESR was measured with an automatic ESR reader (DA-717, NOVIN GOSTAR, Iran) from whole blood.

2.6. Other determinations

A demographic questionnaire containing information about age, sex, education, occupation etc. of the patients was obtained. Besides, their medical history and medications/supplements history was also recorded. Height (wall-mounted stadiometer), weight (Seca, CA, USA), body mass index (BMI) were measured, also blood oxygen saturation was measured with pulse rate (PO 80 Pulse Oximeter, Beurer, Germany). Well-trained interviewers took a 24-hour dietary recall for 7 days (including a weekend day) to evaluate detailed nutrient intake and the food pattern. Dietary intakes were analyzed using the Nutritionist IV software (First databank Inc), and adherence rate was measured with the 7-day 24-hour dietary recall. For each patient, the amount of energy and macronutrients obtained from the 24 h recall was compared with their previous intake/intakes to ensure that the diet did not change.

2.7. Statistical analysis

All of the participants who completed the intervention period were considered for analysis. We analyzed all data using the SPSS v. 21 (SPSS, Inc., Chicago, IL, USA) and Stata v-14. The empirical distribution of data with assumption of normality was tested with the KS-SW test. The data are presented as mean [SD] for normal and median Interquartile ranges (IQR) for non-normal variables. In addition, mean difference (MD) and 95 % CI were calculated based on mean [SD] and sample size using Stata. Independent-samples t-test and Mann-Whitney U test were applied to compare the differences of selected variables between two groups for normal and non-normal variables, respectively. Paired t-test and Wilcoxon test were used for comparing differences within two groups before and after intervention for normal and non-normal variables, respectively. Furthermore, we used ANOVA-ANCOVA to assess primary outcome, despite their abnormality. In addition, the results of the primary variables were also evaluated using single imputation method. Statistical significance was set at P < 0.05.

3. Results

From 150 eligible patients, forty-eight subjects were recruited and allocated randomly into two groups, i.e., pomegranate juice (PJ) and placebo groups. Forty-three patients completed the study, 21 and 22 in the PJ and placebo groups respectively, the patient had 90 % adherence ( Fig. 1). Blinding was assessed by asking the personnel, patients, or assessors to guess the type of treatment patients received. 80 % of Patients and 60 % of assessors included were assessed. At the end of the study, more than 85 % of patients, believed they had received the pomegranate juice. Adverse events were reported by five (20 %) patients of the placebo recipients, and four (16 %) patients of the pomegranate juice recipients. The most common adverse were nausea and vomiting, reported by nine subjects (19 %).

Fig. 1.

Flow of patients through the trial.

There were no differences in age, weight, medical history, drug treatment, economic status and BMI between groups at baseline ( Table 1). The mean age of patients was 56.0[11.6] and 58.6[15.9] in the PJ and placebo groups, respectively. No differences were found regarding energy, micronutrients and macronutrients intake between two groups on the basis of the 7-d dietary records obtained throughout the intervention ( Table 2).

Table 1.

Baseline characteristics of PJ and placebo groups.

		PJ (n = 24)	Placebo (n = 24)	P-value
Age (y)		54.5 ± 14.0	57.4 ± 15.7	0.50
Weight (kg)		70 (61–75)	70 (62–72)	0.48
BMI (kg/m2)		25.9 ± 2.5	24.8 ± 1.7	0.14
Sex (male:female) (%)		50:50	50:50	1.00
Hospitalized duration (day)		6.0 ± 2.9	6.0 ± 2.9	0.96
Medical history (%)
	Diabetes	25	20.8	0.73
	Hypertension	29.2	33.3	0.75
	Dyslipidemia	25	20.8	0.73
	Others	20.8	25	0.73
Drug history (%)
	Supplements1	100	100	NA
	Antibiotic	87.5	91.7	0.63
	Antiviral	100	100	NA
	Anti-inflammatory	100	100	NA

Data are presented as number (percent) for categorical variables, mean [standard deviation] for normally distributed variables, or median (IQR) for non-normally distributed variables. Y, year; NA, not applicable; ¹Supplements included vitamin C and vitamin D.

Table 2.

Dietary intake of selected nutrient during the intervention period of study in the intervention and placebo group.

Nutrients	PJ (n = 21)	Placebo (n = 22)	P-value
Carbohydrates (g)	130.6 [35.5]	145.3 ± 33.9	0.15
Proteins (g)	22.2 [9.3]	21.6 ± 6.7	0.81
Total fat (g)	21.2 (13.9–39.8)	26.2 (21.5–39.7)	0.17
Energy (kcal)	1581 (1169–1971)	1675 (1403–1855)	0.95
PUFA (g)	4.5 (2.4–18.0)	16.3 (6.1–19.9)	1.00
PUFA n-3	0.09 [21]	0.13 [25]	0.75
Magnesium (mg)	147.7 [52.7]	182.6 ± 67.9	0.06
Calcium (mg)	593.8 (324.2–902.9)	669.2 (398.6–891.5)	0.44
Phosphorus (mg)	761.1 [280.3]	822.5 ± 263.1	0.43
Potassium (mg)	2180.1 [622.5]	1999.4 ± 827.9	0.39
Cu (mcg)	1.0 [0.3]	0.9 ± 0.4	0.14
Fe (mg)	12.6 (10.4–14.2)	12.0 (10.7–15.3)	0.97
Zn (mg)	5.8 (3.0–7.8)	7.8 (5.5–8.8)	0.12
Total Fiber (g)	13.3 (11.5–13.9)	11.5 (10.2–13.5)	0.06
Vitamin C (mg)	36.2 [2.8]	36.0 ± 5.3	0.88
Vitamin C (mg) supp	290.94[2.87]	285.99[5.31]	< 0.01
Vitamin E (mg)	13.4 (11.9–15.0)	12.1 (11.0–15.0)	0.22
Vitamin D (IU) supp	3571.42	3571.42	1.00

Data are presented as mean [standard deviation] for normally distributed variables or median (IQR) for non-normally distributed variables. PUFA, polyunsaturated fatty acids, Cu, Copper, Fe, Iron, Zn, Zinc. supp, supplement, IU, international unit.

The results of Laboratory data analysis before and after intervention, between the groups, and the comparison of mean change are presented in Table 3. At the end of the trial (day 14), IL-6, CRP, neutrophils, Plts, ESR, PLR and NLR significantly decreased while Lym, and blood oxygen saturation (BOS) levels increased in the PJ group (Table 3). In contrast, RBC, Lym, Plts, and BOS significantly increased in the placebo group (Table 3). At the end of the trial, Plts (P < 0.001), MCV (P = 0.033), and PLR (P < 0.001) were significantly different between the two groups. Other values did not reach statistical significance between both groups after 14 days. In addition, the results of the primary variables were also evaluated using single imputation method and did not provide different results.

Table 3.

Values of inflammatory markers and blood variables at baseline and after the intervention^*.

Variables		PJ (n = 21)	Placebo (n = 22)		Mean difference	95 % CI	P-valueb
IL-6 (pg/mL)
	Baseline	10.10 (6.02–19.35)	7.75 (4.02–10.12)		5.38	1.06–9.71	0.04
	Day-14	6.30 (4.15–10.10)	8.35 (4.42–12.15)		-1.70	-5.73–2.32	0.29
	Change	-3.80 (− 11.60 to − 0.57)	0.95 (− 3.77 to 3.70)		-7.09	-12.21 to − 1.96	0.00
	MD	5.24	-1.84
	95 %CI	0.87–9.61	-4.88–1.19
	P-valuea	0.00	0.46
ESR (mm/h)
	Baseline	30.00 (18.50–43.50)	23.50 (7.25–54.50)		0.16	-15.41 to 15.73	0.57
	Day-14	16.00 (9.00–30.50)	15.00 (10.50–31.25)		1.31	-9.71 to 12.35	0.86
	Change	-13.00 (− 20.50 to 0.00)	-4.50 (− 43.50 to 9.50)		1.15	-17.75 to 20.07	0.67
	MD	10.52	11.68
	95 %CI	1.54–19.50	-5.317 to 28.68
	P-valuea	0.00	0.18
CRP (mg/L)
	Baseline	33.00 (5.00–62.00)	25.00 (4.75–39.00)		10.02	-5.68 to 25.73	0.22
	Day-14	5.00 (3.00–17.00)	9.50 (5.00–30.00)		-6.22	-17.48 to 5.04	0.14
	Change	-22.00 (− 32.00 to − 1.50)	-6.00 (− 31.25 to 3.50)		-16.24	-32.06 to − 0.43	0.10
	MD	23.19	6.94
	95 %CI	11.93–34.44	-4.81 to 18.70
	P-valuea	0.00	0.13
RBC (× 1000/μL)
	Baseline	4.71 [0.73]	4.67 [0.67]		0.04	-0.39 to 0.47	0.85
	Day-14	4.81 [0.97]	4.90 [0.72]		-0.10	-0.62 to 0.42	0.70
	Change	0.09 [0.49]	0.23 [0.33]		-0.14	-0.34 to 0.12	0.29
	P-valuea	0.39	0.00
HB (g/dL)
	Baseline	13.60 [2.39]	13.40 [1.42]		0.20	-1.00 to 1.40	0.74
	Day-14	14.02 [2.83]	13.65 [1.61]		0.38	-1.03 to 1.79	0.59
	Change	0.42 [1.32]	0.24 [1.42]		0.18	-0.67 to 1.03	0.67
	P-valuea	0.16	0.43
HCT (%)
	Baseline	42.13 [6.43]	41.75 [3.99]		0.38	-2.89 to 3.66	0.81
	Day-14	43.49 [7.53]	42.43 [4.42]		1.06	-2.72 to 4.84	0.57
	Change	1.36 [4.18]	0.68 [3.43]		0.68	-1.67 to 3.03	0.56
	P-valuea	0.15	0.36
MCV (fl)
	Baseline	92.10 (86.65–94.00)	89.85 (87.27–94.55)		-0.11	-4.95 to 4.72	0.78
	Day-14	92.20 (89.30–97.80)	89.30 (86.95–92.02)		0.31	-0.26 to 0.87	0.03
	Change	1.10 (− 1.80 to 3.55)	-1.00 (− 3.42 to 1.12)		0.31	-0.25 to 0.88	0.02
	P-valuea	0.12	0.09
MCH (pg)
	Baseline	29.70 (27.85–31.40)	29.80 (28.22–30.80)		-12.03	-37.21 to 13.14	0.78
	Day-14	30.30 (27.65–31.95)	29.40 (26.80–30.00)		-10.58	-35.29 to 14.12	0.08
	Change	0.00 (− 1.05 to 1.40)	-0.35 (− 2.45 to 0.50)		1.45	-34.43 to 37.33	0.09
	P-valuea	0.58	0.12
MCHC (g/dL)
	Baseline	32.20 (31.05–33.35)	32.45 (31.32–33.20)		0.10	-0.78 to 0.98	0.95
	Day-14	32.30 (30.55–32.95)	32.30 (31.52–33.04)		-0.26	-1.38 to 0.84	0.89
	Change	-0.10 (− 1.40 to 1.20)	0.05 (− 0.92 to 0.72)		-0.36	-1.33 to 0.60	0.78
	P-valuea	0.58	0.12
WBC (× 1000/μL)
	Baseline	9.37 [3.81]	7.72 [3.74]		1.64	-0.67 to 3.97	0.16
	Day-14	8.38 [3.52]	9.83 [3.77]		-1.45	-3.70 to 0.80	0.20
	Change	-0.99 [4.62]	2.10 [5.21]		-3.09	-6.14 to − 0.05	0.04
	P-valuea	0.33	0.07
Lym (%)
	Baseline	13.66 [8.42]	14.09 [8.29]		-0.42	-5.57 to 4.72	0.86
	Day-14	25.80 [12.62]	19.18 [11.02]		6.62	-0.66 to 13.91	0.07
	Change	12.14 [13.42]	5.09 [8.43]		7.05	0.17–13.92	0.04
	P-valuea	0.00	0.01
Mono (%)
	Baseline	4.38 [2.10]	5.68 [3.52]		-1.30	-3.10 to 0.49	0.15
	Day-14	5.66 [3.54]	5.54 [3.54]		0.12	-2.06 to 2.30	0.91
	Change	1.28 [3.70]	-0.13 [5.11]		1.42	-1.33 to 4.18	0.30
	P-valuea	0.12	0.90
Neutrophils (%)
	Baseline	80.47 [9.87]	78.45 [9.33]		2.02	-3.89 to 7.93	0.49
	Day-14	66.80 [15.00]	73.90 [14.52]		-7.09	-16.19 to 1.99	0.12
	Change	-13.66 [15.83]	-4.54 [13.21]		-9.12	-18.08 to − 0.15	0.04
	P-valuea	0.00	0.12
Plts (× 1000/μL)
	Baseline	231.23 [89.06]	239.90 [82.44]		-8.67	-61.49 to 44.15	0.74
	Day-14	168.33 [57.74]	271.54 [105.07]		-103.21	-155.78 to − 50.63	0.00
	Change	-62.90 [85.15]	31.63 [58.40]		-94.54	-139.33 to − 49.75	0.00
	P-valuea	0.00	0.01
PLR
	Baseline	25.09 (9.32–39.90)	18.10 (9.77–32.52)		-0.27	-12.49 to 11.94	0.86
	Day-14	6.90 (4.73–9.56)	16.47 (8.23–25.75)		-16.26	-30.19 to − 2.33	0.00
	Change	-21.17 (− 30.21 to − 1.96)	-1.56 (− 7.74 to 3.05)		-15.99	-29.31 to − 2.67	0.01
	P-valuea	0.00	0.30
NLR
	Baseline	6.83 (3.87–14.50)	7.40 (3.21–9.83)		0.83	-3.23 to 4.90	0.78
	Day-14	2.69 (1.45–4.72)	4.55 (2.10–8.88)		-2.55	-6.44 to 1.34	0.12
	Change	-4.72 (− 10.13 to − 0.89)	-1.57 (− .3.55 to 0.73)		-3.38	-7.84 to 1.06	0.07
	P-valuea	0.00	0.19
BOS (%)
	Baseline	89.61 [3.27]	90.77 [2.48]		-1.15	-2.94 to 0.63	0.19
	Day-14	93.14 [2.00]	92.54 [1.87]		0.59	-0.59 to 1.79	0.31
	Change	3.52 [2.99]	1.77 [2.22]		1.75	0.13–3.37	0.03
	P-valuea	0.00	0.00

RBC, red blood cell; HB, hemoglobin; WBC, white blood cell; HCT, hematocrit; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; MCH, mean corpuscular hemoglobin; Lym, lymphocytes; Eos, eosinophils; Plt, platelets; ESR, erythrocyte sedimentation rate; PLR, platelets-to-lymphocytes ratio; NLR, neutrophils-to-lymphocytes ratio; BOS, blood oxygen saturation; IL-6, interleukin 6; CRP, C-reactive protein.

^*Data are presented as mean [SD] for normally distributed variables, or median (IQR) for non-normally distributed variables, Mean Difference and 95 %CI. Normal values of IL6 < 6 pg/mL, ESR < 15 mm/h for males and < 20 mm/h for females, and CRP< 10 mg/l.

^aP-value based on paired t-test or Wilcoxon test for within groups differences.

^bP-value based on Independent-samples t-test and Mann-Whitney U test for the difference between the two groups at the end of the intervention.

4. Discussion

In the present trial, there was a significant decrease in CRP, IL-6, ESR, neutrophils, platelets, PLR, and NLR, whose increased values have been associated with COVID-19 infection.²⁹ Besides, RR and BOS levels were improved after PJ intake. Since severe disease cases can result in alveolar damage and progressive respiratory failure,³⁰ BOS is objective signs of respiratory compromise, which are associated with markedly elevated mortality.³¹ Therefore, our results suggest that, in addition to the therapeutic treatments for these patients, the intake of PJ improved their inflammatory status and CBC outcomes, which might be helpful as an adjuvant strategy.

In this study, the investigated blood indicators showed changes in the recovering stage of Covid-19. These changes included an increase in hemoglobin, a decrease in WBC, an increase in RBC, a decrease in neutrophils, and an increase in lymphocytes. Although some of these changes in the intervention group were significant compared to before the intervention, there was no difference between the two groups. These changes were consistent with other studies.32., 29., 33. One of these changes is the lymphopenia in patients with Covid, which could be due to apoptosis caused by viral infection and is in line with the increase of inflammatory cytokines,³⁴ and these small positive changes can be attributed to the anti-inflammatory effect of pomegranate juice. In some studies, it has been shown that these changes occur due to the tight or irreversible binding of bioactive substances, especially punicalagin, to the CD4 site on the envelope glycoprotein GP120.35., 36., 37.

Covid-19 is described as a chronic inflammatory disorder, which is marked by an increment of inflammatory cytokines such as IL-6 and CRP.³⁸ Our study indicated that, a significant decrease in the level of CRP and IL-6 in the PJ group after intervention period. Again, the associations between changes in plasma IL-6 and CRP has contributed to the reduction of inflammation in the PJ group. Our observations are consistent with other studies,39., 27., 40. which reported a decrease in plasma levels of IL-6 and CRP following consumption of pomegranate juice in chronic patients.41., 42. However, our study showed no significant effect on the serum levels of CRP in PJ group compared with placebo group following intervention period, which may be due to the dose of PJ, small sample size or/and short duration of intervention. The beneficial effects of pomegranate are attributed to polyphenols⁴³ that are mostly from the anthocyanin subcategory⁴⁴ which have anti-inflammatory properties.

We acknowledge some limitations in the present study, although our trial could also open new research scenarios. The sample size was relatively small considering the high inter-individual variability associated with polyphenol-related effects, which might have prevented more differences between the PJ and placebo groups. In addition, the intervention period was relatively short as it was adjusted to the hospitalization of the patients, which could have limited the effects. Another limitation of this study was the lack of measurement of blood vitamin D levels. Another limitation of this study was the lack of detailed investigation of the precise content and amount of polyphenols in pomegranate juice. It is also necessary to mention that the results of this study cannot be generalized to all brands of pomegranate juice. However, the threshold polyphenolic content to exert effects is unknown. In this line, a dose-response trial could have yielded relevant information on the necessary dosage and cause-effects relationship after PJ consumption. Besides, finding a possible association between urine and (or) blood pomegranate-derived metabolites and the effects observed could have provided additional information on the main drivers of the effects.

CRediT authorship contribution statement

SBP is the Chief Investigators; he conceived the study. SBP and MY led the proposal and study development. MY, SM, MRSI, BS, SA, AP and ZS contribute to data collection, SBP, MY, OS study design, and development of study. SBP, JCE and MY are revising the manuscript. MH, NR, MY and SBP contributed to statistical interpretations. All authors read and approved the final manuscript.

Funding

This study was funded and supported by Yasuj University of Medical Sciences (Grant no.: 990913), Yasuj, Kohgiluyeh and Boyer-Ahmad Province, Iran. The funders did not have a role in the design of study, the intervention, collection, analysis, interpretation of data, and writing the manuscript.

Declaration of Competing Interest

The authors declare no conflict of interest.

Appendix A. Supplementary material

Supplementary material

Availability of Data and Materials

The final dataset of trial will be available upon request from the primary investigator via e-mail at panahande.b@gmail.com, after obtaining permission from Regional Ethics Committee.