Effect of Cornus Mas L. Fruit Extract on Blood Pressure, Anthropometric and Body Composition Indices in Patients with Non-Alcoholic Fatty Liver Disease: A Randomized Controlled Trial

Faezeh Yarhosseini Shahid Sadoughi University of Medical Sciences and Health Services: Shahid Sadoughi University of Medical Sciences and Health Services Abbas Ali Sangouni Shahid Sadoughi University of Medical Sciences and Health Services: Shahid Sadoughi University of Medical Sciences and Health Services Zohreh Sadat Sangse di Shahid Sadoughi University of Medical Sciences and Health Services: Shahid Sadoughi University of Medical Sciences and Health Services Mahdieh Hosseinzadeh Shahid Sadoughi University of Medical Sciences and Health Services: Shahid Sadoughi University of Medical Sciences and Health Services Mohsen Akhondi-Meybodi Shahid Sadoughi University of Medical Sciences and Health Services: Shahid Sadoughi University of Medical Sciences and Health Services Alimohammad Ranjbar Shahid Sadoughi University of Medical Sciences and Health Services: Shahid Sadoughi University of Medical Sciences and Health Services Hossein Fallahzadeh Shahid Sadoughi University of Medical Sciences and Health Services: Shahid Sadoughi University of Medical Sciences and Health Services Hassan Mozaffari-Khosravi (  mozaffari.kh@gmail.com ) Shahid Sadoughi University of Medical Sciences and Health Services: Shahid Sadoughi University of Medical Sciences and Health Services


Introduction
Non-alcoholic fatty liver disease (NAFLD) is known as the accumulation of fat (mainly triglyceride) in the liver (more than 5% of liver weight) without evidence of alcohol consumption (1,2). The prevalence of NAFLD is high in both developed and developing countries (3)(4)(5). It is the most common chronic liver disease worldwide and the main cause of liver transplantation (6, 7). Obesity, disrupted lipid metabolism, in ammation and insulin resistance are the most important factors that contribute to the development and progression of NAFLD (8- 10). A strong association between NAFLD with both metabolic syndrome and cardiovascular disease (CVD) has been con rmed (11,12). In addition, patients suffering from NAFLD compared to the healthy subjects are at higher risk for hypertension (13,14). Modi able lifestyle factors such as weight loss and adherence to the healthy plant-based dietary patterns are the important strategies to manage NAFLD (15)(16)(17).
Cornus mas L. (cornelian cherry) fruit is a rich source of compounds with anti-in ammatory and antioxidant properties such as anthocyanins, avonoids and polyphenols (18)(19)(20). It has been reported that Cornus mas L. fruit/anthocyanins can improve obesity and hypertension through several mechanisms (21)(22)(23)(24). The effects of Cornus mas L. fruit on some factors involved in the pathogenesis of NAFLD such as insulin resistance, dyslipidemia and in ammation have been examined (25)(26)(27). A few number of studies have examined the effect of Cornus mas L. on anthropometric and body composition indices (25,26). The studies that investigated the effect of Cornus mas L. fruit in patients with NAFLD are rare (28). To the best of our knowledge, there is no study evaluating the effect of Cornus mas L. fruit on anthropometric and body composition indices in patients with NAFLD. In addition, there was no clinical trial that investigated the effect of Cornus mas L. fruit on blood pressure. Accordingly, we designed a clinical trial to examine the effect of Cornus mas L. fruit extract blood pressure variables, anthropometric and body composition indices in patients with NAFLD.

Recruitment and eligibility screening
We recruited the subjects from gastroenterology clinics a liated with Diabetes Research Center of Shahid Sadoughi University of Medical Sciences, Yazd, Iran, between May 2019 to August 2019. A total of 50 subjects met the inclusion criteria including age 25-65 years, diagnosis of NAFLD by ultrasonography, alanine aminotransferase (ALT) serum concentrations ≥ 30 U/L in men and ≥ 19 U/L in women (29)(30)(31), and resident of Yazd city. The exclusion criteria included history of alcohol abuse, viral hepatitis, liver cancer, Wilson, type 2 diabetes mellitus (T2DM), mental diseases, pregnancy, lactation, taking corticosteroids, non-steroidal anti-in ammatory drugs, hypoglycemic drugs, tamoxifen, sodium valproate, methotrexate, amiodarone, anti-retroviral agents for HIV, probiotics, antioxidant and antiin ammatory supplements such as vitamin D, vitamin E, omega-3 and resveratrol, poor compliance and unwillingness to continue the study.

Trial design
We designed a double-blind randomized, placebo-controlled clinical trial (RCT) evaluating the effect of Cornus mas L. fruit extract intake for 12 weeks on blood pressure, anthropometric and body composition indices in patients with NAFLD. at the beginning, the participants signed a written informed consent that was con rmed by the ethical committee of Shahid Sadoughi University of Medical Sciences and Health Services in Yazd (IR.SSU.SPH.REC.1399.019). We registered the protocol at the Iranian clinical trials website (http://www.irct.ir) under code number IRCT20180419039359N1 (https://www.irct.ir/trial/30707).
We strati ed the participants according to their age (25-45; 45-65 years) and gender (male/female) and the participants were divided into the treatment group (n = 25) and the control group (n = 25) by a person who did not contribute to the study. To perform randomization, we used random number table that was produced by random allocation software (32). The patients and investigators were blinded until the end of the trial.

Extract preparation
We provided the Cornus mas L. fresh fruits from the forests of Ghazvin, Iran. Preparation of the Cornus mas L. fruit extract and placebo was performed in the Pharmacy Faculty of Shahid Sadoughi University of Medical Sciences. In addition, to determine total anthocyanins content of nal extract we used pH differential method (33). We reported the details of Cornus mas L. fruit extract preparation as well as total anthocyanins content determination in our previous article (28).

Intervention
Based on previous clinical trials (25,27), we considered 20 cc/d Cornus mas L. fruit extract as the optimal dosage for treatment group. This amount provided 32 mg/d total anthocyanin. 20 cc liquid extract was equivalent to 2800 mg dried extract. The safety of this dosage was con rmed by previous studies (25,26,34,35). The control group received 20 cc/d placebo without any anthocyanin. The Cornus mas L. fruit extract and the placebo that had similar appearance were packed in bottles with the same color, shape and size. The participants received the bottles every 2 weeks. We assessed the rate of compliance every 2 weeks based on extract consumption. Consumption of extracts less than 80% of the prescribed amount was considered as poor compliance. Adverse events during follow-up were checked every 2 weeks.

Dietary intake and physical activity assessment
We evaluated the dietary intake of participants at the baseline and after intervention utilizing a 3-day (1 weekend day and 2 nonconsecutive weekdays) food record. Energy and macronutrients intakes were calculated using nutritionist IV software (version 7.0; N-Squared Computing, Salem, OR, USA) (36). In addition, we used a short form of International physical activity questionnaire (IPAQ) (37, 38) to assess physical activity at the baseline and after intervention.

Blood pressure measurement
We measured diastolic blood pressure (DBP) and systolic blood pressure (SBP) under the standard protocol at the baseline and after intervention, in the nondominant arm after 10 min of rest. Measurements were performed by a trained person using a mercury sphygmomanometer device (MicrolifeBP AG1-10).

Anthropometric and body composition evaluations
Measuring height, weight, waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), body fat mass (BFM), body fat percent (BFP) and fat free mass (FFM) was performed at the baseline and after intervention by stadiometer, scale, and bioelectrical impedance analyzer (BIA) (In Body 770, Korea) based on the standard instructions.

Statistical analysis
We calculated the sample size (25 per group) based on brosis score, with 95% con dence interval, α = 0.05, power = 80% in our previous article (28). We performed the power analysis, and power = 80% was estimated. Kolmogorov-Smirnov test was utilized to evaluate the parameters distribution. We used independent t-test and chi-square test for continuous and categorical variables, respectively to compare differences between groups. Independent t-test was utilized to compare the means of variables at the end of the study, as well as the mean changes between groups. Parameters with abnormal distribution were compared between groups utilizing Mann-Whitney U test. In addition, we used paired t-test (for parameters with normal distribution), and Wilcoxon test (for parameters with abnormal distribution) to perform within group comparisons. Univariate ANCOVA was used to control confounding factors. The primary outcomes were reported based on intention-to-treat (ITT) approach. Statistical package for social science (SPSS) software (Chicago, Illinois, USA) version 24 was utilized for statistical analyses.

Characteristics and anthropometric variables
A total of 40 patients completed the study. Ten patients were excluded from the trial for reasons such as atulence (n = 1), immigration (n = 2), unwillingness to continue the study (n = 3), surgery (n = 1), and corona virus pandemic (n = 3) (Figure 1). There was no difference between the treatment group and the control groups in the baseline variables (Table 1). In addition, we found no difference between the treatment group and the control groups in dietary intakes and physical activity at the baseline and after intervention ( Table 2). The patients reported no serious adverse event related to extracts during follow-up.
One patient with history of gastrointestinal disorders, had atulence during intervention, and was excluded.  Values of total energy and carbohydrates are presented as mean ± standard deviation (SD), while for proteins, fats and physical activity are presented as median and quartile range.

Outcomes
At the baseline, there was no signi cant difference between the treatment and the control groups in terms of DBP (P = 0.46) and SBP (P = 0.98) ( Table 3). After intervention, a signi cant difference between two groups was observed in DBP (P = 0.01), but, SBP remained without signi cant difference (P = 0.07). The treatment group compared to the control group showed a signi cant reduction in DBP (-8.62 ± 11.86 vs. 0.53 ± 8.53; P = 0.009), and SBP (-8.63 ± 14.37 vs. 0.0 ± 12.67; P = 0.04) after adjusting for confounding variables (Table 3). Values are presented as mean ± standard deviation (SD).
In addition, there was no signi cant difference between the two groups in weight (P = 0.  (Table 4). In addition, we found no signi cant difference between two groups in weight (P = 0.73), WC (P = 0.20), WHR (P = 0.82), BFM (P = 0.14), BFP (P = 0.14) and FFM (P = 0.49) after intervention; but, level of HC (P = 0.03) was signi cantly higher in the control group compared to the treatment group (Table 4) (Table 4). Values are presented as mean ± standard deviation (SD).

Discussion
Based on our knowledge, our study was the rst study that examined the effect of Cornus mas L. fruit extract on blood pressure variable, anthropometric and body composition indices in patients with NAFLD. Cornus mas L. fruit extract (20 cc/d) for 12 weeks could improve blood pressure, BFM and BFP. However, weight, WC, HC, WHR and FFM remained without signi cant change.
Cornus mas L. fruit and its biological compounds through various mechanisms and pathways such as inducing endothelial nitric oxide gene expression, regulating nitric oxide synthase and increasing endogenous production of nitric oxide, modulating nuclear factor-κ B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, reducing pro-in ammatory cytokines, decreasing peroxynitrate and reactive oxygen species (ROS) levels, and attenuating vasoconstriction by regulating angiotensin-converting enzyme (ACE) and angiotensin II receptor activity can improve hypertension (22,(39)(40)(41)(42). There is no study investigating the effect of Cornus mas L. fruit on blood pressure in subjects in NAFLD. Johnson et al. (43) reported that daily consumption of blueberry, a rich source of anthocyanins, for 8 weeks in postmenopausal women with pre-and stage 1-hypertension can reduce DBP and SBP. In addition, the study of Broncel et al. (44) found that intake of 300 mg/d Aronia melanocarpa extract (another rich source of anthocyanins) for 2 months reduces DBP and SBP in subjects with metabolic syndrome. Moreover, the study conducted by Basu et al. (45) suggested that freeze-dried blueberry beverage (50 g freeze-dried blueberries) daily for 8 weeks can reduced DBP and SBP in participants with metabolic syndrome. However, a clinical study conducted by Hassellund et al. (46) demonstrated that anthocyanins supplementation (320 mg twice per day) after 4 weeks did not reduce blood pressure variables in subjects with hypertension. A meta-analysis found that anthocyanins supplementation has no effect on DBP and SBP (47). Health status of participants, duration of follow-up, type of supplement and dosage of supplement are the most important differences between these studies. It seems, difference in type of supplements is the main factor explaining discrepancies between ndings of mentioned studies. In general, anthocyanins supplementation did not show promising results in this eld, while receiving sources of anthocyanins reported the bene cial effects on blood pressure. We used Cornus mas L. fruit extract containing several biological compounds that probably have synergistic effects.
On the other hand, the evidence suggested that Cornus mas L. fruit and its main compounds by inhibiting hepatic lipogenesis, increasing hepatic lipid oxidation and clearance, regulating the expression of peroxisome proliferator-activated receptors (PPARs), increasing the activity of AMP-activated protein kinase (AMPK) pathway in the white adipose tissue, decreasing adiponectin levels and activating adiponectin signaling, decreasing levels of adipocytokines, reducing the activity of pancreatic lipase and absorption of lipids can reduce obesity (21,(48)(49)(50)(51)(52). We found no similar study investigating the effect of reported that puri ed anthocyanins supplementation (320 mg/d) derived from bilberry and black currant for 12 weeks has no effect on weight, WC, HC and WHR in patients with NAFLD. It seems, dosage and type of extract are important in this eld, and probably higher dosages of Cornus mas L. fruit extract can improve the indicators of obesity.
To comply with principals of ethics in research, we declare that our research group reported the ndings of liver function (28). We used the same data for the present article, and gure 1, sample size information, some baseline characteristics, dietary intakes and physical activity of our previous article were added to the present article.
The present study had some important advantages. This was the rst RCT that examined the effect of Cornus mas L. fruit extract on blood pressure variables and body composition indices in patients with NAFLD. In addition, the extract was standardized according to total anthocyanin content. Similar to other RCTs, this study had some limitations. Liver ultrasonography was used for diagnosis of NAFLD, while Fibroscan has higher accuracy than ultrasonography (55,56). As another important limitation, we did not measure the serum levels of anthocyanins to evaluate the bioavailability of anthocyanins.

Conclusion
Page 13/20 Overall, 12-week Cornus mas L. fruit extract intake (20 cc/d) could improve blood pressure and body fat. However, further studies utilizing higher dosages of Cornus mas L. fruit extract are required to clarify the real effects of Cornus mas L. fruit extract on obesity.

Availability of data and materials
The data and materials of the current study is available from the corresponding author on reasonable request.

Competing interests
The authors have declared no competing interests.

Funding
Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran supported this study. This was a nancial support for student thesis process including laboratory works, and providing kits.

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