Recruitment and eligibility screening
A total of 50 Patients with NAFLD were recruited from gastroenterology clinics affiliated with Diabetes Research Center and Shahid Sadoughi University of Medical Sciences, Yazd, Iran. The inclusion criteria were as follows: age 25–65 years, alanine aminotransferase (ALT) levels of higher than 30 U/L in men and higher than 19 U/L in women, NAFLD diagnosed by a gastroenterologist, resident of Yazd city, and written consent signed by the patient. The exclusion criteria were as follows: history of alcohol abuse (an average daily alcohol consumption of ≥ 10 g for women and ≥ 20 g for men), viral hepatitis, liver cancer, psychological disorders, pregnancy, lactation, taking corticosteroids, non-steroidal anti-inflammatory drugs, hypoglycemic drugs, tamoxifen, sodium valproate, methotrexate, amiodarone, anti-retroviral agents for HIV, probiotics, antioxidant and anti-inflammatory supplements (such as vitamin D, vitamin E, omega-3, and resveratrol) and unwillingness to continue the study.
Study design
This study was conducted as a double-blind placebo-controlled clinical trial for 12 weeks. We investigated the effect of cornelian cherry fruit extract on HSI, NAFLD-LFS and VAI in patients with NAFLD. Before signing the informed written consent, the participant was notified about the risks and benefits of the study. The protocol was approved by the ethical committee of Shahid Sadoughi University of Medical Sciences and Health Services in Yazd (IR.SSU.SPH.REC.1400.020). The registration of the study protocol was performed on 30/09/2018 at the Iranian clinical trials website (http://www.irct.ir), under code: IRCT20180419039359N1 (https://www.irct.ir/trial/30707).
Intervention
The intervention group received 20 cc/d cornelian cherry extract, which provides 320 mg/d total anthocyanin. Preparing placebo in the same appearance, color, and texture as the cornelian cherry extract was performed by the Pharmacy Faculty of Shahid Sadoughi University of Medical Sciences. The cornelian cherry extract and the placebo were packed in containers with the same color, shape, and size. The bottles containing the extract or placebo as were labeled as A or B by a person who was unaware about the trial details.
Preparation of extract
From the forests of Ghazvin, Iran, fresh cornelian cherry fruits were provided in November 2020 and frozen at -18°C. Assessment of the fruits’ authenticity was performed in the Department of Pharmacognosy, School of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, based on their voucher number (SSU0029). Preparation of the cornelian cherry extract was performed based on the standard protocols (31). Determining the total anthocyanin content of the obtained extract was performed based on pH differential method (32). The microbiological assessments for the final extract were done. The placebo was provided using purified water and red color such as carmoisine color similar to the extract in a similar container.
Randomization and blinding
Participants were divided and stratified based on age, gender, body mass index (BMI) and grade of fatty liver into two groups including intervention (cornelian cherry fruit extract, n = 25) and control (placebo, n = 25) by a person who was not involved in the study, using the computer-generated random numbers (33). The participants, investigators and laboratory staff were blinded to the treatment allocation. Randomization codes were unlocked only after all individuals completed the study.
Compliance rate
Cornelian cherry extract and placebo bottles were given to the participants every 2 weeks. Participants were asked to return the bottles with their remaining contents at the end of each 2 weeks. To evaluate the compliance rate, the consumption of cornelian cherry extract and placebo was monitored at the end of each 2 weeks of intervention. At the end of the intervention, the remaining contents of bottles were recorded for each participant. Consuming less than 80% of the administered extract or placebo was defined as poor compliance. Participants with poor compliance were excluded from the study and their data was not analyzed at the end of the study.
Dietary intake, physical activity and anthropometric evaluations
To evaluate dietary intake of subjects, a 3-day (1 weekend day and 2 nonconsecutive weekdays) food record was used at weeks 0 and 12. In addition, the short form of International Physical Activity Questionnaire (IPAQ) was utilized for assessment of physical activity at weeks 0 and 12. Height was measured in the standing position at weeks 0 and 12 using a Seca stadiometer with an accuracy of 0.5 cm. Measuring weight, and waist circumference (WC) as the important confounding factors was performed based on standard protocols with light clothes and without shoes by a Seca scale with an accuracy of 100 g. Using the following formula, BMI was calculated: weight (kg)/height (m)2.
Steatosis and visceral adiposity indices assessment
HSI, NAFLD-LFS and VAI were calculated for each participant at weeks 0 and 12, based on the following formulas:
HSI = 8 × (ALT/AST ratio) + BMI + 2 (if female) + 2 (if diabetes mellitus) (10).
NAFLD-LFS = -2.89 + 1.18 × metabolic syndrome (yes = 1; no = 0) + 0.45 × type 2 diabetes (yes = 2; no = 0) + 0.15 × fasting insulin (mU/L) + 0.04 × AST (U/L) − 0.94 × AST/ALT (8).
VAI men = [WC/39.68 + (1.88 × BMI)] × (TG/1.03) × (1.52/HDL-c) (13).
VAI women = [WC/36.58 + (1.89 × BMI)] × (TG/0.81) × (1.31/HDL-c) (13).
Laboratory assessments
Laboratory assessments was performed at weeks 0 and 12. 10 cc blood was drawn after 12 hours fasting and centrifuged for 10 minutes at a speed of 3600 rpm. Serum samples poured into the microtubes were immediately frozen at -75° C. ALT, aspartate aminotransferase (AST), total cholesterol (TC), TG, low density lipoprotein-cholesterol (LDL-c) and high density lipoprotein-cholesterol (HDL-c) were measured by routine enzymatic assays with Pars Azmoon, Iran, kits using an autoanalyzer. Measuring insulin was performed by ELISA reader (Epoch, Bio Teck, USA) utilizing Monobind, USA, kit. Measurements were performed based on standard methods in laboratory of Nutrition Department, Yazd, iran.
Statistical analysis
Based on the study of Sangsefidi et al. (23), with α = 0.05, power = 80%, and considering 10% drop-out rate, the optimal sample size was estimated to be 25 per group. SPSS version 24 (SPSS, Inc.) was used for data analysis. Using Kolmogorov–Smirnov test, the normal distribution of variables was assessed. Comparing the qualitative variables between two groups was performed using Chi-Squared test. To compare the means of normal variables at baseline, at the end of study, and compare the mean changes of normal data between two groups, an independent t-test was utilized. Mann-Whitney U test was used to compare abnormal data between the two groups at baseline and after the intervention as well as comparing the mean changes of abnormal data between two groups. Paired t-test was utilized to compare the normal variables in each group, and Wilcoxon test was utilized to compare the abnormal data in each group. P value < 0.05 was considered significant.