Combination of the effect of ginger and anti‐inflammatory diet on children with obesity with nonalcoholic fatty liver disease: A randomized clinical trial

Abstract Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in children. Following earlier reports on an increase in the prevalence of childhood obesity, NAFLD is now becoming increasingly common in children. Although no definitive cure exists, early management, early diagnosis, and treatment can reduce its complications. This study aims to determine the effectiveness of the combination of ginger and an anti‐inflammatory diet (AID) in children with obesity on fatty liver management. This randomized clinical trial was conducted on 160 children with obesity aged 8–11 years, with a mean (SD) weight of 65.01 (9.67) kg, mean (SD) height of 139.87 (7.37) cm, and mean (SD) body mass index of 33.40 (5.59) kg/m2. The study duration was 12 weeks. Children were divided into four groups: ginger (G), AID, ginger plus an AID (GPA), and control. Ginger capsules comprised 1000 mg of ginger, whereas the AID comprised fruits and vegetables, fish, turkey, and chicken (without skin) with lean meat, omega‐3 sources, nuts, legumes, probiotic products, and elimination of inflammatory food. Following the intervention, serum fasting blood sugar and high‐sensitivity C‐reactive protein levels were significantly decreased in the AID (p = .006 and .002, respectively), G (p = .04 and <.001, respectively), and GPA (p <.001 in both cases, respectively) groups. Further, in the G and GPA groups, there was a significant decrease in body mass index (p = .04 in both cases, respectively), waist circumference (p = .009 and .003, respectively), waist‐to‐height ratio (p = .02 and .005, respectively), alanine aminotransferase (p = .004 and <.001, respectively), total cholesterol (p = .0002 and .0001, respectively) and low‐density lipoprotein‐cholesterol (p < .001 and <.001, respectively). Eventually, serum aspartate aminotransferase was decreased (p < .001) and high‐density lipoprotein‐cholesterol (p = .03) was increased significantly in the GPA group. As a main finding of this study, hepatic steatosis significantly decreased in the G and GPA groups. Ginger supplementation can effectively improve NAFLD in children, and its effectiveness was further increased when combined with an AID.


| INTRODUC TI ON
Nonalcoholic fatty liver disease (NAFLD), known as fatty liver disease associated with metabolic dysfunction, is the buildup of extra fat in liver cells that is not caused by alcohol (Stefan, Häring, & Cusi et al., 2019). Fatty liver disease is of two types: NAFLD and nonalcoholic steatohepatitis, with the latter also including inflammation of the liver (Chalasani et al., 2018;Lazarus et al., 2021). NAFLD includes a wide range of liver disorders from hepatocellular steatosis to more severe nonalcoholic steatohepatitis, which may progress to hepatic fibrosis and cirrhosis (Marjot et al., 2020). NAFLD is present in approximately 38% of children with obesity worldwide (Draijer et al., 2019). Its prevalence in Iran is reported to vary between 42.6% and 77.1%. Fatty liver is expected to be the most common cause of chronic liver disease in children (Papandreou et al., 2012;Li et al., 2021;Chan et al., 2004). This is particularly interesting, as emerging NAFLD data associate children with an increased risk of adult morbidity and mortality, as well as cardiovascular disease and type 2 diabetes (Draijer et al., 2019).
Diet appears to be a risk factor for pediatric NAFLD (Fitzpatrick & Dhawan, 2019). However, it remains unclear which components of the diet affect the growth and development of NAFLD in children (Gibson et al., 2015). Based on epidemiological data, children with obesity with NAFLD have a higher intake of fructose, saturated fat, and less fiber than children without NAFLD (Fitzpatrick & Dhawan, 2019;Vancells Lujan, Viñas Esmel & Sacanella Meseguer, 2021). Regarding dietary models, in a population cohort study of adolescents, strict adherence to a Western diet at 14 years of age was correlated with an increased risk of NAFLD at 17 years. This association focused on body mass index (BMI; Oddy et al., 2013).
As there is no Food and Drug Administration (FDA)-approved medical treatment to treat pediatric NAFLD (Rinella et al., 2019), treatment protocols focus on lifestyle modifications to reduce disease-related risk factors (Draijer et al., 2019). Standard recommendations for children with overweight and obesity indicate weight management and physical activity (Vos et al., 2017;Chalasani et al., 2018). Previous studies in adults have shown that a diet rich in antioxidants and anti-inflammatory compounds can effectively treat NAFLD (Eslamparast et al., 2015). Furthermore, evidence has recommended the use of certain functional foods and nutraceuticals as potential therapeutic agents for NAFLD, as they may disrupt signaling pathways in the pathogenesis of NAFLD (Goodarzi et al., 2019).
Ginger (Zingiber officinale) is a flowering plant whose rhizome (ginger root or ginger) is widely used as a spice and as a popular medicine owing to its various pharmacological (as an immunoregulator and tumorigenesis inhibitor), anti-inflammatory, antiapoptotic, and antiemetic effects. Most of these medicinal effects of ginger are related to various compounds such as gingerol and shogaol. More than 40 antioxidants have been identified in ginger. The FDA has confirmed that ginger is a dietary supplement (Suman et al., 2021).
Previous studies have linked the anti-diabetes, anti-cancer, and antiinflammatory effects of ginger to its active ingredient (Thomson et al., 2002;Bhandari, Kanojia & Pillai, 2005). Ginger extract has been shown to exhibit antioxidant activity and reduce proinflammatory biomarkers (Grzanna, Lindmark & Frondoza, 2005). In addition, recent studies in patients with type II diabetes and hyperlipidemia have shown that ginger can reduce insulin resistance and serum triglyceride (TG) concentrations (Arablou et al., 2014;Shidfar et al., 2015).
Accordingly, the hypothesis of this study is that ginger supplementation and an anti-inflammatory diet (AID) can be introduced as a new therapeutic strategy for NAFLD. A randomized, doubleblind study was designed to test this hypothesis by considering the effects of anti-inflammatory dietary compounds in controlling the progression of fatty liver and the role of ginger as an antioxidant.
Therefore, this study aims to determine the combined effect of ginger and an AID on lipid profiles, blood glucose, liver enzymes, inflammatory markers, and liver fat status in children with overweight with NAFLD.

| Participants and study design
Patients were identified and recruited from Mohammad Kermanshahi Hospital, Iran. Admission criteria for selection of included patients were as follows (Ashtary-Larky et al., 2018): BMI >85th percentile, normal upper levels of alanine aminotransferase (ALT) >1.5 times the upper limit of the normal range, ultrasonography (indicated fat aggregation; grades 1-3), age 8-11 years, and not on weight loss diets. Exclusion criteria were a history of disease such as diabetes, cardiovascular, liver, and kidney disease; receiving supplements in the last 6 months; under hormone therapy; using not more than 10% of the capsules given in each followup; and unwillingness to continue the study protocol. In total, 160 children met our inclusion criteria and were selected for this study. To control for the confounding factor, a stratification approach was applied. First, patients were divided into two groups of girls and boys and then based on the amount of physical activity they were divided into three groups: low (<15 points from questionnaire), medium (15 points from questionnaire), and severe (>15 points from questionnaire). In the next step, samples in each group were divided into two categories based on calorie consumed from macronutrient intake: low (<2000 Kcal/day) and high (>2000 Kcal/ day). Finally by simple randomization, samples were divided into four groups of intervention (three groups were randomly selected for the intervention group: n = 40 for GPA, G, and AID, respectively) and a control group (n = 40). For the group receiving the AID, we recommended consumption of anti-inflammatory foods, including poultry, fish, omega-3 sources, colored fruits and vegetables, nuts and legumes, probiotic, and low-fat products, with elimination of inflammatory foods such as high-fat foods, simple sugars, fast food, processed meats (e.g. sausages, hot dogs), chips, crackers, and soft drink, for 12 weeks. Ginger powder supplement used in this study is a ready-made product. Ginger (Z. officinale) capsules contained 1000-mg ginger rhizome powder. A placebo containing wheat flour (1000 mg) was prepared in the same form and color as a ginger supplement in the Pharmacy Faculty Lab of Kermanshah University of Medical Sciences.

| Dietary intake and supplementation
We matched sex, physical activity level, and calorie intake in the four groups to control for confounding factors. To check AID compliance, participants complete 24-hour dietary recall weekly. A 3-day food record questionnaire (1 day off and 2 days non-off) was administered to other groups at the beginning and end of the study. The intakes of energy, macronutrients, micronutrients, and water were measured using the Modified Nutritionist IV Software (version 3.5.2, First Data-Bank; Hearst Corp., San Bruno, CA). The physical activity level of patients was evaluated using the BEACK Questionnaire (Baecke et al., 1982) at the beginning and end of the study. This questionnaire is a tool that evaluates individual's habitual physical activities over the pervious 12 months. It should be noted that the dosage of ginger used and the duration of the intervention have been determined according to previous studies (Rafie et al., 2020). For accuracy of double-blindness, capsules were coded by a person other than the study researchers, as A or B. To calculate the compliance rate of patients to the supplementation, all patients received supplementation for 4 weeks and were asked to deliver the cans of capsules at each visit, and then they were given supplements for the next 4 weeks. To control adherence to the AID, patients complete a 24-h recall (weekly consumption data). Patients were also followed up on a weekly basis by telephone to be aware of possible side effects and to ensure the use of supplements.

| Clinical and para clinical assessments
According to the WHO definition, the anthropometric measurement of the patients' height, weight, waist circumference (WC), and hip circumference was performed at the beginning and end of the intervention. BMI was calculated by dividing weight by squared height, WHR by dividing waist to hip circumference, and waistto-height ratio (WHtR) by dividing waist by height (Ebrahimzadeh Attari et al., 2017). Blood (10 cc) was taken from all participants at the beginning of the study and the end of the 12th week after a 10-12-h fasting. Samples taken were centrifuged, and their serum was separated. For further tests, the serum was stored at −80°C.
The serum TG level was measured using the enzymatic calorimetric method, while the total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) of plasma were measured by the photometric enzymatic method. The serum levels of liver enzymes, including alkaline phosphatase (ALP), ALT, and aspartate aminotransferase (AST), were measured by colorimetry (Parsazmun Co).

| Liver ultrasound
Liver ultrasound was performed by the same experienced radiologist using an Acuson Sequoia C512 scanner equipped with a 15 L8 transducer (Universal Diagnostic Solutions). Normal liver, absent steatosis (grade 0) was defined as having a normal liver echo texture; mild steatosis (grade 1) as slight and diffuse increase in fine parenchymal echoes with normal visualization of the diaphragm and portal vein borders; moderate steatosis (grade 2) was defined as moderate and diffuse increase in fine echoes with slightly impaired visualization of the diaphragm and portal vein borders; and severe steatosis (grade 3) was defined as fine echoes with poor or no visualization of the diaphragm, portal vein borders, and posterior portion of the right lobe.

| Statistical analysis
Statistical analysis was performed using STATA software version 14.2. Paired t-test and one-way analysis of variance were used to compare quantitative data and χ 2 test was used to compare qualitative data. Significance level was considered when p is <.05.

| Patient inclusion and safety
According to the flowchart in Figure 1, 40 patients in the control group and 40 patients in each intervention group completed the study. None of the patients showed any allergic reaction or side effects of supplementation.

| Participants' characteristics
A total of 160 patients were included in the study and were randomly assigned to one of the four groups (n = 40). All patients completed study and their data entered into the final analysis. Demographic characteristics are summarized in Table 1. There was no statistically significant difference between the four groups at the beginning of the study.

| Biochemical assessment
Results of the biochemical assessment are briefly presented in Table 2 and Figure 2.

Inflammatory markers
At the end of the study, serum CRP-hs in the intervention groups were significantly reduced compared with the control group (p < .001). In addition, the intragroup comparison showed that at the end of the study, compared with the beginning, the serum CRP-hs in groups AID (p = .002), G (p < .001), and GPA (p < .001) had a significant decrease.

Fasting blood sugar
There were no significant differences in serum FBS between groups at the end of the study. However, intragroup comparison showed that at the end of the study, compared with the beginning, serum FBS concentration had a significant reduction in groups AID (p = .006), G (p = .04), and GPA (p < .001).

Liver enzymes
At the end of the study, serum ALT concentration in the intervention groups significantly decreased compared with that in the control group (p < .001). Further, the intragroup comparison showed that at the end of the study, compared with the beginning, serum ALT concentration had a significant decrease in groups G (p = .004) and GPA (p < .001). In addition, serum ALP concentration F I G U R E 1 Flowchart of the study. AID, anti-inflammatory diet; G, ginger; GPA, ginger plus anti-inflammatory diet Recruited (all patient in M.K. hospital) Providing consent form, tailoring questionnaires, subject information sheet, 3-day food dairy and instructions to complete it Excluded: 12 weeks intervention Receive 1000 mg ginger powder or anti-inflammatory diet or placebo per day, end of intervention due to completion of supplementation duration according to other study and financial limitation. Assessment the food diary, anthropometric indices, blood collection and ultrasound

Analysis
Baseline visit: Delivery of the Food frequency and physical questioner, anthropometric indices and blood collection in intervention groups significantly reduced compared with that in the control group (p = .01). The intragroup comparison showed a significant reduction in groups G (p = .04) and GPA (p < .001).
Besides, there was no significant difference in serum AST concentration between the intervention groups and the control group at the end of the study. However, the intragroup comparison showed that at the end of the study, compared with the beginning, the serum AST level in the GPA group (p < .001) had a significant reduction.

Lipid profile assessment
At the end of the study, serum TC concentration in intervention groups significantly decreased compared with that in the control group (p < .001). In addition, the intergroup comparison showed that serum TC concentration in groups G (p = .0002) and GPA (p = .0001) had a significant reduction at the end of the study. Besides, serum LDL-cholesterol (LDL-c) concentration in intervention groups significantly reduced compared with the control group (p = .001), and the intragroup comparison showed that the serum LDL-c concentration in groups G and GPA significantly decreased (p < .001). Besides, there was no significant difference in serum HDL-cholesterol (HDLc) concentration between the intervention and control groups at the end of the study. However, the intragroup comparison showed that serum HDL-c concentration in the GPA group (p = .03) significantly decreased, whereas the serum TG concentration was not significantly reduced in each intervention group.

Fatty liver status assessment
The results showed a significant reduction in liver fat accumulation in the G and GPA groups at the end of the study. This reduction was higher in the GPA group (one-degree reduction: 82.5%; and 2-degree reduction: 17.5%; Figure 3).

| Secondary outcome
3.4.1 | Anthropometric assessment such as intervention and control groups. There was no significant difference in anthropometric indices before and after the intervention in the control and AID intervention groups. A significant decrease in BMI was observed in groups G (p = .04) and GPA (p = .04) at the end of the study compared with the beginning, but this difference was not significant between groups. Finally, there was a significant difference between WC and WHtR in groups G (p = .009 and p = .02, respectively) and GPA (p = .003 and p = .005, respectively).
This difference between groups was also significant (p < .001).

| Nutritional assessment
Tables 4 and 5 show the macro-and micronutrient intake within and between groups, such as intervention and control groups. At the beginning of the study (week 0), there was no significant difference in food intake within the groups. At the end of the study (week 12), protein intake in the intervention groups significantly decreased compared with that in the control group (p = .01). Besides, the intragroup comparison showed that protein intake increased significantly in groups AID (p = .02) and GPA (p = .02), compared with that in the beginning of the study.
By contrast, the intragroup comparison showed that fat intake in these two interventions groups decreased compared with that in the beginning of the study (p = .03). Besides, fat intake in the intervention groups significantly decreased compared with that in the control group (p = .01). It should be noted that there was no significant change in total energy intake within and between groups. Note: Data are expressed as mean ± standard deviation or n (%). Data are tested by a one-way analysis of variance and b chi-square test.
At the end of study, compared with the beginning, dietary intake in vitamin C, vitamin E, beta-carotene, and selenium increased significantly in groups AI (p = .02, <.001, .03, and .001, respectively) and GPA (p = .02, <.001, .01, and <.001, respectively), but there was no significant change in zinc intake in any of the groups.

| DISCUSS ION
To   Our findings are consistent with an observational study on 430 young Americans aged 21-35 years (Wirth et al., 2016) and the study by Aslani et al. (2019), which indicated no significant association between DII and BMI.
Our study showed a significant reduction of FBS in all three intervention groups compared with the control group. Similar to our study, Rafie et al. (2020)  Note: Data are tested by a t-test (intragroup) and b one-way analysis of variance (intergroups).
F I G U R E 4 (a) Intra-and intergroup comparisons of the changes from baseline to the end of the intervention for anthropometric variables in the four groups. (b) Intra-and intergroup comparisons of the changes from baseline to the end of the intervention for anthropometric variables in the four groups. AID, anti-inflammatory diet; BMI, body mass index; G, ginger; GPA, ginger plus anti-inflammatory diet; HC, hip circumference; WC, waist circumference; WHR, waist-to-hip ratio; WHtR, waist-to-height ratio.
glucose 6-phosphatase activity, ginger supplementation can reduce the degradation of glucose 6-phosphate into glucose, thereby reducing blood glucose (Nammi, Sreemantula & Roufogalis, 2009;Shanmugam et al., 2011). Ginger also appears to reduce glucose uptake by inhibiting enzymes in glucose metabolism, such as α- Another finding of our study was the antilipidemic effect of ginger. Our results indicated that daily consumption of ginger compared with placebo significantly reduced LDL-c and TC levels and increased HDL-c level when consumed with an AID. However, serum TG levels did not change in any of the intervention groups.
The results of previous clinical trials that investigated the effect of ginger on lipid profiles are, however, contradictory. In the study by Rahimlou et al. (2016) an intervention using ginger reduced serum Note: Data are tested by a t-test (intragroup) and b one-way analysis of variance (intergroups). animal studies (Nammi, Sreemantula & Roufogalis, 2009;ElRokh et al., 2010). Cholesterol reduction by ginger can be explained as follows: ginger can increase the conversion of cholesterol in to bile acids by increasing the activity of cholesterol-7α-hydroxylase, leading to a decrease in serum cholesterol levels (Alizadeh-Navaei et al., 2008). Biocompounds in ginger have also been reported to suppress cholesterol synthesis in rat liver cells (Bordia, Verma & Srivastava., 1997). The findings of this study regarding the lack of effect of ginger supplement on serum HDL-c are consistent with those of some previous studies (Rafie et al., 2020;Mozaffari-Khosravi et al., 2014;Alizadeh-Navaei et al., 2008& Bordia, Verma & Srivastava., 1997. This is probably because changes in HDL-c levels are less affected by dietary components.

Abbreviations
This study showed that ginger supplementation plus an AID can reduce serum hs-CRP levels. The hs-CRP is mainly produced in the liver, and its serum level is associated with the rate of liver inflammation. In the study by Arablou et al. (2014), a daily intake of ginger (1.6 g) significantly reduced serum hs-CRP level in patients with diabetes. In addition, a study by Atashak et al. (2012) showed that ginger powder could reduce serum hs-CRP concentration in men with obesity, which is similar to the results of our study. It seems that the efficacy of ginger on inflammation is due to the positive effect of some active ingredients (gingerol and zerumbone) in inhibiting NF-κB (nuclear factor kappa B) and TNFα (tumor necrosis factor alpha).
Ginger inhibits NF-κB activity by inhibiting the TNFα gene, and thus inhibits the production of acute-phase positive proteins such as CRP (Kim et al., 2004). By contrast, the present study contradicts the findings by Hart et al. (2021), who examined the effect of an AID on serum CRP. In a cross-sectional study of adolescents aged 10-17, Yilmaz et al. (2012) reported decreased CRP adherence to an AID.
Our findings showed that consumption of ginger compared with control resulted in a reduction in serum ALT and ALP concentrations. However, there was no significant change in AST levels.
These findings were consistent with the findings of a previous study. Rahimlou et al. (2016) showed that ginger supplementation significantly reduced serum ALT and gamma-glutamyl transferase (GGT) levels compared with placebo. Further, these results were also observed by Rafie et al. (2020). The effect of ginger on liver fibrosis in rats showed that treatment with a ginger extract induced a significant reduction in AST and ALT levels as well as a protective effect against liver fibrosis. Although the exact mechanism of this protective effect of ginger is not yet fully understood, insulin resistance is one of the reasons for elevated liver enzymes. Insulin resistance activates lipolysis in adipose tissue and increases the free flow of fatty acids to the liver, leading to steatosis and liver damage.
In this regard, Pagano et al., reported that ginger supplementation reduces the indicators related to insulin resistance and improves liver enzymes (Pagano et al., 2021).
In the AID group, there was a significant decrease in ALP levels. Darbandi et al. (2020) have shown that adherence to an AID is TA B L E 5 Intra-and intergroup comparisons of the changes from baseline to the end of the intervention for nutritional variables in the four groups Note: Data are tested by a t-test (intragroup) and b one-way analysis of variance (intergroups).
Abbreviations: AID, anti-inflammatory diet; G, ginger; GPA, ginger plus anti-inflammatory diet. associated with lower liver enzymes, including AST, ALT, and GGT. It seems that weight loss inhibits the accumulation of lipid in hepatocytes, resulting in a reduction of inflammatory response via NF-κB activation and cytokine production, which leads to a decrease in liver damage. Lack of a significant decrease in liver enzymes in the present study may be due to the low sample size.
Finally, the main finding of this study was a significant reduction in liver steatosis in groups G and GPA. It was also observed that this decrease in hepatic steatosis in the groups receiving ginger was increasingly more significant than the AID group. Results of this study were, however, inconsistent with the studies by Rafie et al. (2020) and Rahimlou et al. (2016). Animal studies have also shown that the active compounds in ginger can reduce fatty liver disease (Liu et al., 2013). Thus, when following an AID in combination with ginger supplementation, this positive regulation likely becomes more tangible (Nayebi Far & Ghasemi, 2021).
Finally, based on the between-intervention groups comparison in this study, it was concluded that ginger (G group) is more effective than the AID (AID group) in the treatment of fatty liver in children.
By contrast, taking an AID and ginger together (GPA group) is even more effective than taking ginger alone. Therefore, it can be concluded that ginger as a nonmedicinal supplement can improve fatty liver in children with obesity, while adherence to an AID will increase its effectiveness.
The strength of this study was that it is the first to evaluate the effect of two interventions alone and together on pediatric fatty liver. However, there are some limitations, such as the small sample size and lack of complete adherence to diet by some participants.
Nevertheless, it should be noted that these are not weaknesses just for our study. Still, it is one of the limitations of lifestyle-modification studies, because according to the results of published studies, only 30% of patients participating in lifestyle-modification programs for the treatment of NAFLD were able to achieve the desired weight loss (>5%) at the end of 1 year (Vilar-Gomez et al., 2015).

ACK N OWLED G M ENTS
This work was extracted from the MSc thesis (Nutrition) of Miss Negin Kamari, Faculty of Nutritional Sciences and Food Industry, Kermanshah University of Medical Sciences, Kermanshah, Iran.

FU N D I N G I N FO R M ATI O N
This study was approved and supported by a grant from Kermanshah University of Medical Sciences, Kermanshah, Iran (grant number 990165).

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analyzed during this study are available from the corresponding author on reasonable request and permission for use was received by the Ethics Committee of Kermanshah University of Medical Sciences.

The Medical Ethics Committee of Kermanshah University of Medical
Sciences has approved the implementation of this research (IR.

KUMS.REC.1399.085). Moreover, it has been recorded in the Iranian
Clinical Trial Registry (IRCT20181111041611N3) and is available through www.irct.ir. All participants signed a written informed consent form.