Effect of Total Anthocyanin-base Standardized Cornus Mas L. Fruit Extract on Hepatic Steatosis and Visceral Adiposity Index in Patients With Nonalcoholic Fatty Liver Disease: a Double-blind Randomized Controlled Trial

Background: Nonalcoholic Experimental evidence has proposed the benecial effects of Cornus mas L. (cornelian cherry) extract, as a rich source of anthocyanins, on steatosis and central obesity. However, very few clinical trials were conducted in this regard. We investigated the effect of total anthocyanin-base standardized cornelian cherry fruit extract on hepatic steatosis index (HSI) and NAFLD liver fat score (NAFLD-LFS), and visceral adiposity index (VAI) in patients with NAFLD. Methods: The present study was conducted as a double-blind, randomized controlled clinical trial among 50 subjects suffering from NAFLD. Subjects were randomly assigned to receive cornelian cherry extract (20 cc/d, which provides 320 mg/d total anthocyanin) or placebo for 12 weeks.

conclude, further trials with longer intervention durations are required.
Trial registration: The study protocol was registered on 30 September 2018 at Iranian Registry of Clinical Trials under code IRCT20180419039359N1 (https://www.irct.ir/trial/30707).

Background
Non-alcoholic fatty liver disease (NAFLD) refers to a range from simple steatosis to non-alcoholic steatohepatitis (NASH) (1), and it is known as the hepatic manifestation of metabolic syndrome (2). NAFLD is one of the most important causes of liver cancer and liver transplantation (1). The global prevalence of NAFLD is 25.24% (3), and its prevalence is estimated to be 27% in Asia (4), and 34% in Iran (5). Liver biopsy is the "gold standard" of steatosis and brosis diagnosis (6). However, liver biopsy is an invasive procedure and it is rarely utilized in investigations (6). Recently, the researchers have been focused on designing non-invasive, accurate and simple tools to predict and evaluate the severity of Page 4/19 NAFLD (7). Some validated simple tests such as hepatic steatosis index (HSI), and NAFLD liver fat score (NAFLD-LFS) have been developed to evaluate and estimate the severity of hepatic steatosis based on laboratory markers and anthropometric parameters (8-10). Insulin resistance, dyslipidemia and obesity are the most important causes of NAFLD (11,12). The visceral adiposity index (VAI) is an accurate and sensitive marker of cardiovascular risk factors such as insulin resistance and central obesity (13).
The recent evidence suggested that the avonoid compounds with antioxidant and anti-in ammatory properties such as anthocyanins may have bene cial effects on features of NAFLD (14)(15)(16). Fruits and vegetables are the main sources of anthocyanins (17). Cornus mas L. (cornelian cherry) fruit is one of the richest sources of anthocyanins (18). Some in vitro experiments have demonstrated that anthocyanins via phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and inhibition of lipid biosynthesis as well as triglyceride (TG) accumulation in hepatic cells and promotion of hepatic lipid clearance can attenuate the development of hepatic steatosis (19,20). In addition, cornelian cherry can ameliorate main risk factors of NAFLD such as insulin resistance, dyslipidemia and oxidative stress in mice (21,22). Clinical trials investigating the effects of cornelian cherry on features of NAFLD are scarce (23). On the other hand, animal studies have suggested that rich sources of anthocyanins can improve cardiovascular risk factors such as dyslipidemia and obesity (24,25). The clinical effects of anthocyanins on dyslipidemia and obesity are inconsistent (16, 26-30).
Therefore, due to limited number of clinical trials examining the effect of cornelian cherry on the severity of NAFLD and fat accumulation in patients with NAFLD, and lack of trials evaluating cornelian cherry/anthocyanins on above-mentioned indices, the present study was designed to investigate the effect of total anthocyanin-base standardized cornelian cherry extract on HSI, NAFLD-LFS and VAI in patients with NAFLD.

Recruitment and eligibility screening
A total of 50 Patients with NAFLD were recruited from gastroenterology clinics a liated 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-in ammatory drugs, hypoglycemic drugs, tamoxifen, sodium valproate, methotrexate, amiodarone, anti-retroviral agents for HIV, probiotics, antioxidant and anti-in ammatory 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 noti ed about the risks and bene ts 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 nal extract were done. The placebo was provided using puri ed water and red color such as carmoisine color similar to the extract in a similar container.

Randomization and blinding
Participants were divided and strati ed 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 de ned 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:

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 Sangse di 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 signi cant.

Results
Characteristics of the participants Fifty subjects were assigned into two groups. During the follow-up, ten participants were excluded due to gastrointestinal symptoms such as atulence (n = 1), immigration (n = 2), participant's personal decision to discontinue the study (n = 3), performance of surgery (n = 1), and incidence of corona virus (Covid 19) pandemic (n = 3). Finally, 40 subjects completed the study (cornelian cherry group: n = 22, placebo group: n = 18). No serious adverse events were reported by the participants. However, only one participant in the cornelian cherry group reported atulence; this subjects had a history of gastrointestinal disorder, namely atulence (Fig. 1). There was no signi cant difference between the two groups in the baseline variables (Table 1). In addition, at baseline as well as the end of the study, there was no signi cant difference between the two groups in confounding factors such as dietary intakes and physical activity ( Table 2).

Outcomes
At the baseline, there was no signi cant difference between the cornelian cherry extract and placebo groups in the terms of HSI, NAFLD-LFS and VAI. In addition, no signi cant difference was observed between the two groups in the terms of HSI (P = 0.56), NAFLD-LFS (P = 0.31) and VAI (P = 0.45) ( Table 3). P † : resulted from comparisons between two groups by independent t-test.

Discussion
The worldwide prevalence of NAFLD is increasing, and has become a main problem in the health care (3,4). Currently, there is no approved pharmacological treatment in this regard (34), and the main strategy to manage NAFLD is lifestyle modi cation including adherence to the healthy dietary patterns, weight loss, and body exercise (34,35). Recent evidence suggested that rich sources of anthocyanins due to their antioxidant and anti-in ammatory properties can have a therapeutic effect in the management of NAFLD (14,15,21,25). We investigated the effect of cornelian cherry extract as the one of the richest sources of anthocyanins on steatosis indices (HSI and NAFLD-LFS) and VAI in patients with NAFLD. The present study demonstrated that intake of cornelian cherry extract (20 cc/d, providing 320 mg anthocyanins/d) for 12 weeks has no bene cial effect on HSI, NAFLD-LFS and VAI indices in patients with NAFLD.
As mentioned, liver biopsy as the gold standard method to detect the severity of NAFLD is an invasive test (6). On the other hand, non-invasive methods like magnetic resonance imaging and ultrasonography are expensive (7,36,37). Therefore, investigators designed and developed valid, non-invasive and inexpensive tools like HSI and NAFLD-LFS indices to estimate and evaluate the severity of NAFLD (7,8,10). There are limited number of clinical trials examining the effect of cornelian cherry on hepatic steatosis. Consistent with our ndings, the study of Sangse di et al. (23) reported that intake of cornelian cherry extract for 3 months has no therapeutic effect on hepatic steatosis, and levels of liver enzymes such as ALT and AST; however, compared to the cornelian cherry group the brosis score signi cantly increased in the placebo group. Another study that conducted among hyperlipidemic adult patients demonstrated that intake of Vaccinium arctostaphylos L. fruit, as a rich source of anthocyanins, had no signi cant effect on ALT and AST levels (38). In addition, clinical trials evaluating the effects of other sources of anthocyanins such as Hibiscus sabdariffa extract (27) and barberry juice (39) on liver enzymes in patients with NAFLD showed ndings similar to our results. Furthermore, Qin et al. (28), and Yang et al. (40) found that pure anthocyanins has no signi cant effect on liver enzymes in dyslipidemic and prediabetic subjects, respectively. On the other hand, the trial of Zhang et al. (41) that conducted among patients with NAFLD demonstrated that brosis score was higher in the placebo group compared with who consumed pure anthocyanins for 12 weeks. In addition, the study of Zhang et al. (41) showed that pure anthocyanins supplementation can decrease levels of ALT; however, AST remained without signi cant change. Moreover, based on the study of Stote et al. (26) that conducted among patients with NAFLD, intake of freeze-dried blueberries, as a rich source of anthocyanins can improve levels of liver enzymes. Some discrepancies in the results can be due to the differences in the health status of participants, duration of the study, and sources as well as dosages of anthocyanins. The exact mechanisms of cornelian cherry/anthocyanins in this eld are still unclear. The early evidence suggested that anthocyanins by inhibiting nuclear factor κ-B (NF-κB) signaling pathways, modulating gene expression of in ammatory markers such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) can reduce cellular in ammation as an important factor involved in the pathogenesis of NAFLD (15,39,42,43). On the other hand, anthocyanins by reducing reactive oxygen species and hepatic lipid peroxidation, as well as increasing the activity of antioxidant enzymes can improve oxidative stress (15,38,44).
Generally, the therapeutic effects of cornelian cherry are attributed to their anti-an ammatory and antioxidant properties (15,45). In ammation and oxidative stress are the main contributors in the progression of steatosis to brosis (12,46,47). Based on the mentioned mechanisms as well as the clinical evidence, anthocyanins may have a bene cial effect on brosis, but not steatosis. However, further clinical trials in this eld are required to make a correct conclusion.
Our study demonstrated that intake of cornelian cherry extract has no impact on VAI. VAI is an accurate and sensitive indicator of cardiovascular risk factors such as insulin resistance and visceral obesity (13).
There is no clinical trial evaluating the effect of cornelian cherry/anthocyanins on VAI. This index is based on anthropometric variables and lipid pro le (13). Results of the studies that investigated the effect of cornelian cherry/anthocyanins on anthropometric variables and lipid pro le are not integrated.
The study of Asgary et al. (30) demonstrated that intake of cornelian cherry for 6 weeks has no signi cant effect on lipid pro le in subjects with dyslipidemia. In addition, the study of Lee et al. (48) reported that 12-week consumption of cranberry can't reduce WC in patients with T2DM. However; two studies found that supplementation with anthocyanins for 24 weeks showed some lipid-modifying effects in subjects with dyslipidemia (44,49). It seems, the main reason of this inconsistency between ndings is difference in the duration of follow-up. Studies with longer-term interventions showed bene cial effect of anthocyanins on dyslipidemia. Anthocyanins may improve dyslipidemia by mechanisms such as modulation of β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase activity, inhibition of cholesteryl ester transfer protein (CEPT), and reduction of the apolipoprotein B and apolipoprotein C-III-lipoprotein, which are TG transporters (15,16,28,42). On the other hand, Asgari et al. (30) demonstrated that 6-week intake of cornelian cherry has no effect on body composition. In addition,  (14). In addition, anthocyanins by inhibiting the activity of the pancreatic lipase, and decreasing intestinal fat absorption can reduce visceral fat accumulation (14,19).
The present study had some strengths. For the rst time we used accurate, simple and inexpensive indices such as HSI and NAFLD-LFS to evaluate the effect of cornelian cherry extract on hepatic steatosis in patients with NAFLD. In addition, the cornelian cherry extract was standardized based on its total anthocyanin content. Moreover, diagnose of NAFLD was performed by broscan, which has higher accuracy than ultrasonography. Furthermore, several confounding variables such as anthropometric indices, dietary intakes, physical activity, and baseline values of variables were controlled. However, the present trial had some limitations such as small sample size, and short duration of follow-up.

Conclusion
In conclusion, we found that intake of 20 cc/d cornelian cherry extract for 12 weeks has no effect on steatosis and cardiovascular risk. To perform a correct as well as comprehensive conclusion, longer-term interventions investigating the effect of cornelian cherry on features of NAFLD are needed.

Consent to publish
Not applicable.

Availability of data and materials
Not applicable.

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 is a nancial support for student thesis process including laboratory works, and provide kits as well as fresh cornelian cherry fruits.
Authors  Figure 1 ow chart of eligibility, screening, and follow-up.