Effect of garlic powder supplementation on blood pressure and hs‐C‐reactive protein among nonalcoholic fatty liver disease patients: A randomized, double‐blind, placebo‐controlled trial

Abstract Based on the anti‐inflammatory and antihypertensive properties of garlic, the current study was designed to evaluate the garlic powder effects on blood pressure and high‐sensitivity C‐reactive protein (hs‐CRP) among Nonalcoholic Fatty Liver Disease patients (NAFLD). This randomized, double‐blind, placebo‐controlled trial study was conducted on 110 patients with NAFLD. The patients were randomly divided into 2 groups, receiving two tablets of either 400 mg garlic or placebo daily for 15 weeks. At baseline and the end of the study, blood pressure and hs‐CRP were determined. Of 110 patients enrolled in the trial, 98 subjects were included in the final analysis. After the intervention, systolic blood pressures (SBP) (mean: −7.89; 95%CI:‒11.39 to −4.39 mm Hg), diastolic blood pressure (DBP) (mean: −5.38; 95%CI: −7.77 to −3 mm Hg), and Mean Arterial Pressure (MAP) (mean: −6:95%CI: −8.4 to −3.6 mm Hg) decreased significantly in the garlic group as compared to the placebo group. Also, the percentage of reduced hs‐CRP was significantly higher in the intervention group compared with the control group (mean: −16.1; 95%CI: −32.7 to −0.53; p = .035). Moreover, a positive correlation was observed between the percentage change in hs‐CRP and percentage changes in SBP (r = 0.221; p = .029), DBP (r = 0.166; p = .012), and MAP (r = 0.210; p = .038). Garlic supplementation can be a safe and potentially adjunct treatment to reduce blood pressure and the risk of cardiovascular disorders in patients with NAFLD.


| Study design and participants
This double-blind, randomized, controlled trial was designed to ascertain the efficacy of garlic in patients with ultrasound-diagnosed NAFLD. Participants were recruited from consecutive outpatients referring to the Metabolic Liver Disease Research Center at Isfahan University of Medical Sciences, Isfahan, Iran. Each study arm was needed 55 patients to detect a difference of 0.6 standard deviation (SD) in the Mean Arterial Pressure (MAP) response to garlic intake with an anticipated dropout rate of 20% and power 80% at a significance level of 5% (two-tailed) (Allen Jr, 2011;Ried et al., 2018) 110 patients were selected after screening based on the inclusion and exclusion criteria. The inclusion criteria were as follows: adults aged between 20 and 70 years, new case-patients with ultrasound technique-proven fatty liver, serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT) levels equal or over 40 IU/L, and stable blood pressure control by diet regimen or/and a stable dose of antihypertensive medication in the last 3 months. The exclusion criteria also were as follows: smoker patients, prior history of adverse reaction to garlic, pregnancy or lactation, other known causes of hepatic steatosis including alcohol intake, viral infections, and hepatotoxic drugs, medical history of endocrine diseases, renal disease, stroke, heart failure, hemochromatosis, Wilson's disease, and cirrhosis, or regular consumption of garlic. Patients were withdrawn from study participation when they become pregnant or need to any change their drugs.
The trial was conducted following the principles of the Helsinki Declaration. Each individual was informed of the overall objectives and procedures of this trial before obtaining the written informed consent. The study protocol was approved by a research ethics committee at Isfahan University Medical Sciences and then was registered at the Iranian Registry of Clinical Trials with the identifier number IRCT2014110819853N1.

| Randomization and trial groups
Eligible participants were stratified according to sex and degree of fatty liver diagnosed by ultrasonography and then randomly allocated (in a 1:1 ratio) to receive 400 mg garlic tablet (enteric-coated tablet containing 1.5 mg Allicin, Amin Pharmaceuticals, Isfahan, Iran) or placebo (enteric-coated tablet containing 400 mg microcrystalline cellulose; School of Pharmacy, Isfahan University of Medical Sciences) two times a day for 15 weeks. Each garlic tablet contained dried garlic powder, aerosil, magnesium stearate, and croscarmellose sodium. Placebo tablets were quite similar to garlic tablets by the means of shape, size, color, taste, and smell. This dosage was chosen based on the previous studies conducted on the blood pressure and hs-CRP (Mahdavi-Roshan et al., 2017;Ziaei et al., 2001). The trial-group assignments were concealed using sequentially numbered containers from all participants and investigators except the pharmacist who prepared randomization sequences generated with the use of a random-numbers table.

| Blood pressure measurements
A trained physician measured systolic blood pressures (SBP) and diastolic blood pressure (DBP) to the nearest 2 mm Hg using a calibrated mercury sphygmomanometer at the beginning and the end of the intervention. In each visit, measurements were implemented three times, at the two-minute interval, on the left arm, with the patient in a sitting comfortable position, after 10 min rest, in the morning time (8-9 a.m.). The mean of three measurements was reported as the blood pressure values. The Mean Arterial Pressure (MAP) was calculated using the following equation: MAP = (SBP + 2DBP)/3.

| Biochemical assessment
After 10-12 hr fasting condition, a venous blood sample was taken at baseline and after 15 weeks. Blood samples were immediately centrifuged at 3000 g for 10 min to separate serum and stored at −80°C until bioanalysis. Serum hs-CRP was measured by the relevant ELISA kit.

| Confounding factors measurements
Weight was measured using a digital Seca scale (Saca 831), with light clothing, to the nearest 100 grams. Height was measured using a portable stadiometer (Seca) in the standing position to the nearest 0.5 centimeters. Then, Body Mass Index (BMI) was calculated by dividing weight in kilograms by height in meters squared. Waist circumference was measured without clothing at a level midway between the lower rib margin and the iliac crest at the end of exhalation and standing position using a tape, to the nearest 0.5 centimeters.
The change in dietary intakes was measured using a three-day food record at the beginning and the end of the intervention. A trained nutritionist instructed patients on how to complete their food records. The amounts of each food item were converted to gram/day using Iranian Household Measures and then converted to the value of nutrients and calorie using Nutritionist IV software (First Data bank Inc., Hearst Corp).
Physical activity was measured using the international physical activity questionnaire (IPAQ) at the beginning and the end of the intervention. IPAQ is an instrument designed for population surveillance of physical activity among adults [15]. The Farsi-translated version of IPAQ was validated among the Iranian population. The physical activity of the participants was calculated as metabolic equivalents (MET)minutes/week. According to the guidelines for data processing and analysis of the IPAQ, participants were stratified into three categories (low, moderate, and high levels of physical activity).

| Statistical analysis
Data were analyzed using the Statistical Package for the Social Sciences (SPSS) software version 16 (SPSS Inc). The Kolmogorov-Smirnov test was used to examine the normal distribution of variables. A within-group comparison was done with the use of the Paired Sample t test and Wilcoxon test. Independent Student's t test, Mann-Whitney U test, and chi-square test were also used to ascertain differences in characteristics between two groups. We adjusted results by using the Analysis of Covariance (ANCOVA) test with baseline value, age, the grade of fatty liver, and change in energy intake, BMI, WC, and physical activity as covariates. Pearson correlation coefficients were analyzed to determine the inter-variable relationships.

| RE SULTS
A total of 205 patients were enrolled and underwent screening to participate in the trial; of whom 110 eligible patients were selected and allocated to receive either garlic tablet or a placebo tablet.
Twelve patients were excluded from data analyses as follows: in the garlic group, 3 patients were lost to follow-up, and 5 patients were withdrawn from the study; in the placebo group, 1 patient was lost to follow-up, and 3 patients were withdrawn from the study ( Figure 1). The two groups did not differ significantly in the dropout rates (14.5% vs. 7.2%; p = .22).
The participant's mean ( ± SD) age was 45.2 ± 11.6 years (range, 20-68), weight was 80.8 ± 14.6 kg (range, 53.2-117.7), and BMI was 29.5 ± 6.1 kg/m2 (range, 16.4-47.6). The majority of patients were female (59.2%); 72.5% had previous cardiovascular diseases, 52% had metabolic syndrome, and only 43.9% of patients used antihypertensive medication in which diuretics (65.1%) were more frequent drug use. Thirty-seven patients (37.8%) had mild fatty liver; 54 patients (55.1%) had moderate fatty liver, and 7 patients (7.1%) had severed fatty on ultrasound imaging. Table 1 illustrates the baseline characteristics of patients. There were no significant differences in the primary characteristics of the patients in the two groups. Dietary intake was evaluated and indicated no significant differences between groups at baseline and the end of the study (Table 2). However, the mean change of body weight was statistically significant in the garlic group compared to the placebo group (−1.98 ± 2.09 vs. −0.83 ± 2.08; p = .010).
Multivariable-adjusted mean change in blood pressure variables from baseline to 15 weeks intervention is shown in Table 3.
The mean SBP significantly reduced (mean: −6.74; 95%CI: −9.18 to −4.31 mm Hg) in the garlic group, whereas no significant change was seen in the placebo group from baseline to 15 weeks (mean: 1.15, 95%CI: −1.21 to 3.52 mm Hg). Garlic treatment significantly reduced the SBP compared with the placebo, independent of potential con-

| D ISCUSS I ON
The current study showed that the administration of garlic supplement (Allium sativum) to patients with NAFLD can reduce the blood pressure and the risk of CVDs independent of potential risk factors.
We also found a positive relationship between change in hs-CRP and blood pressure. To the best of our knowledge, no attempts were made to determine the effect of garlic on blood pressure and hs-CRP in patients with NAFLD.
Our results showed that garlic intake can reduce SBP, DBP, and MAP in patients with NAFLD independent of potential risk factors.
These findings are consistent with recent meta-analysis reports. Xiong et al. reported that the administration of garlic powder can reduce SBP by 6.7 mm Hg and DBP by 4.8 mm Hg in hypertensive patients (Xiong et al., 2015a). Another meta-analysis study in subjects with and without hypertension revealed that garlic intake can reduce the SBP by 16.3 mm Hg and DBP by 9.3 mm Hg in patients with hypertension, while it did not alter the SBP and DBP in patients without hypertension (Reinhart et al., 2008). Furthermore, regression analysis indicated a positive relationship between baseline values of blood pressure and reduction in blood pressure after the administration of garlic (Ried et al., 2008)). Two main mechanisms of action for the BP-lowering properties of compounds in garlic have been offered, including the blockage of the Angiotensin-II pathway and regulation of endothelialdependent vasodilation through intracellular hydrogen sulfide (H2S) and nitric oxide (NO) production (Ried and Fakler, 2014). Likewise, we found that reduction in blood pressure was parallel with the reduction in hs-CRP, as a systemic inflammation marker produced in the liver. This finding is consistent with the previous study that showed a positive association between blood pressure and serum concentration of hs-CRP in hypertensive patients (Dar et al., 2010). Thus, the BPlowering of garlic might be partially mediated by its well-documented anti-inflammatory properties.
It is well documented that chronic inflammation has central roles in the pathogenesis of CVDs. In clinical practice, serum concentration of hs-CRP has been used to reliably predict the CVDs F I G U R E 1 Flow diagram of the study based on CONSORT statement risk (Danesh et al., 2004;Ridker et al., 2000). Therefore, the current trial showed that the garlic treatment can contribute to reducing the risk of CVDs in patients with NAFLD independent of potential risk factors. Our findings are consistent with a recent meta-analysis study in which the administration of garlic significantly decreased serum concentrations of hs-CRP by 0.82 mg/Dl (Taghizadeh et al., 2019). These findings are worth noting because cardiovascular events are the leading cause of premature death in patients with NAFLD. Likewise, emerging evidence suggests that NAFLD is an independent risk factor for CVDs and most likely has a crucial role in the cardiovascular sequelae of metabolic syndrome.
Several pathological conditions are involved in the development of CVD in patients with NAFLD, including systemic inflammation, oxidative stress, endothelial dysfunction (NO), and structural alterations in the arterial wall (Francque et al., 2016). Garlic mainly contains organosulfur compounds with antioxidant, anti-inflammatory, and BP-lowering properties (Iciek et al., 2009). Garlic and its constituents have been shown to inhibit the activation of NF-kB as the main transcription factor for many inflammatory mediators, such as TNFα, IL-1, IL-6, CXCL1, CXCL2, ICAM-1, and VCAM-1, in response to various.
The main limitation of the current trial is a small number of patients having a severe grade of fatty liver, which can affect its generalizability to this group. However, this trial is strengthened by stratified randomization, well-matched baseline values, low dropout rate, high compliance rate to treatment, and adjusting the results for potential confounding factors. Further research is needed to compare antihypertensive drugs with garlic powder among NAFLD subjects for choosing the best intervention to control hypertension. It would also be important to assess the effect of garlic supplement on other cardiovascular diseases in patients with NAFLD and to compare it with another drug therapy strategy.

| CON CLUS ION
In conclusion, our present trial indicates that garlic supplementation can be useful to reduce blood pressure and the risk of cardiovascular disorders in patients with NAFLD. Note: The mean change was adjusted for baseline value, age, grade of fatty liver, energy intake, BMI, WC, and physical activity.

TA B L E 3
Changes in blood pressure from baseline to 15 weeks of follow-up F I G U R E 2 Association between percentage change in hs-CRP and percentage changes in SBP (a), DBP (b), and MAP (c)