The effect of Spirulina sauce, as a functional food, on cardiometabolic risk factors, oxidative stress biomarkers, glycemic profile, and liver enzymes in nonalcoholic fatty liver disease patients: A randomized double‐blinded clinical trial

Abstract Objective This study sought to investigate the effect of Spirulina on cardiometabolic risk factors, oxidative stress biomarkers, glycemic profile, and liver enzymes in nonalcoholic fatty liver disease (NAFLD) patients. Methods This randomized, double‐blind clinical trial was performed on 46 NAFLD patients. Subjects were allocated to consume either Spirulina sauce or placebo, each 20 g/day for 8 weeks. Fatty liver grade, liver enzymes, anthropometric parameters, blood pressure, and serum lipids, glucose, insulin, malondialdehyde, and antioxidant capacity were assessed pre‐ and postintervention. Results Fatty liver grade was significantly different between the two groups. A significant change for ALT (alanine aminotransferase) and AST (aspartate aminotransferase) was seen between the two groups (p = .03 and .02, respectively), while ALP (alkaline phosphatase) serum levels were not significantly different within or between groups. Pertaining to glycemic profile, all variables, except HOMA‐IR, were not significantly different within or between groups. Finally, statistically significant changes were seen in both MDA (malondialdehyde) and TAC (total antioxidant capacity) among the groups (p = .04 and <.001, respectively). Conclusions Spirulina may improve fatty liver grade by modifying liver enzymes, oxidative stress, and some lipid profiles; however, there was effect of Spirulina on anthropometric characteristics and blood pressure.


| INTRODUC TI ON
Nonalcoholic fatty liver disease (NAFLD) is a global public health concern, with a high prevalence, impacting around 11%-16% of the population (Williams et al., 2011). NAFLD prevalence in Asia is reported to be 6%-25% (Fan, 2007) and ~2.9% in Iranian adults (Rogha et al., 2011). NAFLD is characterized by an increase in fat in the liver cells (Bellentani et al., 2000;Ma et al., 2020;Neuschwander-Tetri & Caldwell, 2003); although it is normal for the liver to have some fat, when it exceeds 5% of liver weight, further complications are often present (Raziel et al., 2015). Indeed, the clinical importance of this disease is attributable to the liver cell apoptosis and the destruction of liver tissue, and if not diagnosed early and properly treated, irreversible cirrhosis of the liver can occur. Concomitant to NAFLD, increased blood pressure and body fat, obesity, diabetes, and insulin resistance, components of metabolic syndrome, are routinely observed (Aller et al., 2015;Ludwig et al., 1980;Raziel et al., 2015;Wang et al., 2019).
Contemporary treatment for NAFLD generally includes lifestyle modifications, particularly diet and physical activity (Rezaei et al., 2019). In this regard, functional foods have been shown to be effective in helping control metabolic dysfunction. For instance, it has been asserted that Spirulina intake can modify cholesterol, expand the antioxidant capacity, and decrease insulin resistance and glucose uptake (Moura et al., 2011). Spirulina is a multicellular, fibrous green algae found naturally in alkaline lakes. Furthermore, it is cultivated in a controlled environment for human consumption and is considered a healthy food without side effects by the United Nations Development Program (Pham et al., 2019). Spirulina is used as a dietary compound, due to large quantities of antioxidants (Hossain et al., 2016) such as creatine, phycocyanin (containing the triple-pyrrole chromophore, identified as phycocyanobilin, which is covalently attached to apoprotein), minerals K, Na, Ca, Mg, Fe, and Zn, vitamins (tocopherols), eight crucial amino acids, unsaturated fatty acids, particularly linolenic acid, omega-3 and omega-6 fatty acids, and phenolic compounds (Ferreira-Hermosillo et al., 2010;Moura et al., 2011;Neyrinck et al., 2017;Vázquez-Velasco et al., 2017). Moreover, it purportedly possesses a good source of protein (containing 65-70% dry weight) (Moura et al., 2011;Neyrinck et al., 2017). Due to its anti-inflammatory effects, Spirulina is consumed as a dietary supplement in many countries (Neyrinck et al., 2017). Previous studies have shown positive influences of Spirulina on NAFLD, oxidative stress, hyperglycemia, hypocholesterolemia, malnutrition, anemia, allergic rhinitis, cancer, substance toxicity reduction, and arterial hypertension (Fujimoto et al., 2012;Moura et al., 2011;Neyrinck et al., 2017). Indeed, Spirulina protects against liver toxicity in murine models by enhancing cellular antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase (Premkumar et al., 2004). Moreover, it has been confirmed in animal and human studies that protection against inflammatory diseases, including colitis and arthritis, may be conferred (Pham et al., 2019). Therefore, due to the prevalence of NAFLD, and the relationship between fatty liver disease and high blood pressure, obesity, diabetes, and insulin resistance, concomitant to the limited number of human-based studies in this field, we sought to investigate the effect of Spirulina on cardiometabolic risk factors, oxidative stress biomarkers, glycemic profile, and liver enzymes in NAFLD patients. Inclusion criteria were as follows: NAFLD diagnosis by the physician (using ultrasound) and age ranges from 18 to 70 years.

| Study design and characteristics of patients
Individuals were not included if they were diagnosed with other diseases, such as cardiovascular disease, liver disease (cirrhosis, alcoholic liver disease, viral hepatitis, autoimmune hepatitis, biliary obstruction, primary biliary cirrhosis, and hepatic injury), cancer, renal failure, or celiac. In addition, pregnant and/or lactating women, taking drugs that cause fatty liver (methotrexate, tamoxifen, valproate, etc.), taking lipid-lowering drugs, malnutrition that required nutritional support, following special diets such as vegetarian or raw vegetarian, and alcohol consumption were further exclusory factors. Finally, participants were excluded from the study if they did not follow the recommended treatment, were unwilling to continue the study, and had any acute diseases or infections during the study and if hospitalization occurred for period more than 5 days.

| Sample size
The sample size was estimated to be 19 participants in each group, with a power (1−β) of 80% and α = 0.05, in line with a previous study (Mazokopakis et al., 2014). We estimated a 20% attrition rate, and thus, 23 patients were recruited in each group.

| Randomization
We assigned 46 patients randomly in a 1:1 ratio, to the Spirulina sauce and placebo groups, respectively. To assign participants, blocked randomization with a fixed block size of 4 was used and done by a researcher who had no clinical involvement in our study.

| Blinding
To blind patients to the samples, the sauce sachets in both groups were identical in appearance, taste, and color. The sauce sachets were coded differently in each group to blind the investigator.

| Intervention
Participants in the Spirulina sauce group consumed one sachet (20 mg) of sauce containing 2 gr Spirulina per day for 8 consecutive weeks and similarly in the placebo group received one sachet (20 mg) of placebo per day. The sauce was produced in the Namakin factory in Shiraz, Iran. To produce the sauce, raw materials including oil, lemon juice, vinegar, salt, gum, spices, and water were used. 10% of Spirulina was added to the formula of sauces in the Spirulina sauce group, and to normalize the sensory properties, including the color of the sauce, natural dark green chlorophyll with international code E-141 was used in the products of the placebo. The sauces were similar in terms of fat, carbohydrate, salt, flavorings, and packaging for the Spirulina sauce and placebo groups. The chemical composition and caloric content of the sauce are shown in Table 1.

| Outcomes and measurement
Primary outcomes of this study were changes in the severity of fatty liver (grades 1-3), and secondary outcomes included any changes in anthropometric indices, SBP (systolic blood pressure) and DBP (diastolic blood pressure), lipid profile, HOMA-IR, MDA, TAC, atherogenic index of plasma (AIP), and liver enzymes.
The demographic questionnaires, through face-to-face anamnesis interview, were completed by the main investigator. 12-hr fasting blood samples were drawn by certified clinical staff, before and after 8 weeks. Blood samples were centrifuged, and serums were kept at −7℃ in the freezer until final measurements for lipid profile, FBS, fasting plasma insulin, MDA, TAC, AIP, and liver enzymes. Height was measured using a wall-fixed tape to the nearest 0.1 cm. Body weight was measured to the nearest 0.1 kg using a scale, while participants were in light clothes. Standard methods were considered to measure waist circumference by a nonstretchable measured tape. BMI (body mass index) was computed as weight (kg)/height (m 2 ).
Blood pressure was measured twice with an interval of at least 5 min, using a mercury barometer (ALPK2), and the mean of the two measurements was considered as the subject's blood pressure. Plasma glucose levels, lipid profile, and liver enzymes were evaluated using an enzymatic colorimetric (GOD-PAP) method (Pars Azmoon Inc.).

| Liver ultrasound
Liver ultrasound was used for fatty liver measurement. A single expert radiologist was conducted ultrasound measurement. The grade of echogenicity, the embodiment of the diaphragm, borders of the liver vasculature, and incarnation of the posterior portion of the right hepatic lobe were used to assess fatty liver's grade (from grades 1 to 3).

| Statistical methods
SPSS statistical software (Version 22; IBM) was used for all data analyses. The normality of the data was evaluated by the Kolmogorov-Smirnov test. We used a paired t test for the data with normal distribution and Wilcoxon signed-rank test for nonnormally distributed data to evaluate variations within both groups.
One-way analysis of covariance (ANCOVA) was used for variations between groups, while a Kruskal-Wallis test was used for the data with non-normal distribution. Statistical significance was accepted, a priori, at p < .05. the following reasons: reluctance to continue, hospitalization, and no adherence ( Figure 1). Thus, 20 and 19 participants were analyzed in the Spirulina sauce and placebo groups, respectively.

| Baseline characteristics
The baseline characteristics of the participants are illustrated in Table 2. There was no statistically significant difference between the two groups at baseline, except for MDA.

| Fatty liver grade and liver enzymes
Changes in study outcomes are shown in Table 3. In comparison with the baseline, fatty liver grade decreased significantly in the Spirulina sauce group, concomitant to a nonsignificant increase in the placebo group. Between groups, there was a significant difference (p = .007).
Results indicated that serum concentration of ALT and AST was significantly reduced in the Spirulina sauce group, while in the placebo group, the reduction was not significant. After 8 weeks' intervention, significant changes between the two groups were seen F I G U R E 1 Flowchart of the trial for both of ALT and AST (p = .03 and .02, respectively). However, changes in ALP serum levels were not significant within or between groups.

| Lipid profiles
In the Spirulina sauce group, TG serum levels decreased and HDL-C serum levels increased significantly, in comparison with the baseline.
In addition, the changes between the two groups were significant for TG (p = .02), and marginally insignificant for HDL-C (p = .07).
Changes in LDL-C and total cholesterol serum levels did not differ within or between groups.

| Glycemic profile
In terms of glycemic profile, there were no significant differences in FBS, insulin, and QUICKI between the Spirulina sauce and placebo groups, while HOMA-IR was significantly different between groups (p = .04) ( Table 3).

| Oxidative stress biomarkers
In comparison with the baseline, MDA level decreased significantly in the Spirulina sauce group, while a significant increase was seen in TAC level. Furthermore, a significant difference was found between the two groups for MDA and TAC (p = .04 and <.001, respectively).

| Atherogenic index
The atherogenic index reduced significantly in the Spirulina sauce group when compared to the baseline; however, no significant change was found in the placebo group. In comparison with the placebo group, changes in the Spirulina sauce group were significantly greater (p = .007).

| Anthropometrics indexes and blood pressure
In comparison with the baseline, weight and BMI increased in the placebo group and decreased in the Spirulina sauce group. However, none of the changes were significant within and between groups.
Similarly, the reduction in waist circumference within and between groups was not significant. Systolic and diastolic blood pressure reduced in both groups; however, this was nonsignificant within and between groups (Table 4).

| Dietary intakes and Physical activity
Energy and macronutrients' dietary intakes were not significantly different between groups during the intervention (Table 5). Similarly, physical activity did not show significant change between two groups.
There were no significant correlations between MDA and fatty liver grade, ALT, AST, Insulin, and HOMA-IR, where all correlation coefficients and accompanying p-values are shown in Table 6. b Paired t test was used for determination of the difference between baseline and endpoint values in each group.

| D ISCUSS I ON
c ANOVA was used for examining the significance of the difference in changes of variables between the two groups. BMI, body mass index; SBP, systolic blood pressure; and DBP, diastolic blood pressure. d ANCOVA was used for examining the significance of the difference between the two groups with baseline values as a confounder.

Variables
Week 0 Week 8 Changes p-value b p-value c p-value e
b Paired t test was used for determination of the difference between baseline and endpoint values in each group.
c ANOVA was used for examining the significance of the difference in changes of variables between the two groups.
d Values are the mean and SD of fatty liver grade when it was coded as 0 (no fatty liver), 1 (mild fatty liver), 2 (moderate fatty liver), and 3 (severe fatty liver). e ANCOVA was used for examining the significance of the difference between the two groups with baseline values as a confounder.
b Paired t test was used for determination of the difference between baseline and endpoint values in each group.
c ANOVA was used for examining the significance of the difference in changes of variables between the two groups.
d ANCOVA was used for examining the significance of the difference between the two groups with baseline values as a confounder. (Torres-Durán et al., 1998). The antioxidant and anti-inflammatory property of Spirulina, and its constituents, might contribute to a reduction in inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factorα (TNFα) and an increase in anti-inflammatory cytokines such as adiponectin, indicating an improvement in oxidative stress, in NAFLD patients (Blé-Castillo et al., 2002;Browning et al., 2006;Samuels et al., 2002). Additionally, Spirulina is reportedly able to modulate the gut microbiota and to activate the immune system, a mechanism that may be involved in the improvement of hepatic inflammation (Moura et al., 2011).
Although our study revealed a significant improvement in MDA, there was no significant correlation between MDA with fatty liver grade and liver enzymes. Comparing the differences in both MDA and TAC between the groups, significant changes were evident.
Concordant with our study, evaluating the effect of Spirulina, for 45 days, on liver-injured mice, demonstrated a reduction of MDA and ALT and an increase of superoxide dismutase (SOD) activity (Ding et al., 2004). Moreover, these outcomes are similar to the find-  (Han et al., 2006). Although the exact mechanism for the apparent hypolipidemic effects of Spirulina is not fully understood (Nagaoka et al., 2005), it has been claimed that the C-phycocyanin protein, a main ingredient of Spirulina, has the potential to improve lipid profile by enhancing glutathione peroxidase and superoxide dismutase activity, free radical scavenging, downregulating NADPH (nicotinamide adenine dinucleotide phosphate), oxidase expression, and lipid peroxidation. Furthermore, reductions in cholesterol and bile acid reabsorption have been suggested (Sharma et al., 2011;Upasani & Balaraman, 2003). Glycolipid H-b2 and phycocyanin, other ingredients of Spirulina, can, reportedly, prevent pancreatic lipase activity, while its gamma-linolenic acid (GLA), αlinolenic acid, linolenic acid, and niacin contents have a role in the regulation of cholesterol synthesis and hypolipidemic effects of Spirulina (Han et al., 2006;Mazokopakis et al., 2014). Furthermore, the activity of lipoprotein lipase and hepatic triglyceride lipase is shown to be increased following Spirulina supplementation (Serban et al., 2016). Another protective effect of Spirulina arises from its specific low-calorie, low-fat, cholesterol-free, and high-protein nature (Browning et al., 2006;Westerbacka et al., 2005), which might Note: The correlation between data was examined using partial correlation by controlling MDA in baseline.
p-values of <.05 were considered to indicate significance.
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; HOMA-IR, homeostasis model assessment of insulin resistance; MDA, malondialdehyde TA B L E 6 Correlation analysis between MDA with fatty liver grade, ALT, AST, insulin, and HOMA-IR in patients with nonalcoholic fatty liver disease contribute to weight loss and thus support a reduction in TG levels and decelerate the HDL apoA-I catabolism, with a simultaneous decline in the secretion of HDL apoA-I (Han et al., 2006;Nagaoka et al., 2005;Ng et al., 2007;Nseir et al., 2012).
In terms of glycemic profile, HOMA-IR decreased significantly, and while FBS and insulin decreased, these changes were not statistically significant. In Parikh et al., 2 g/day Spirulina supplementation for 8 weeks revealed a statistically significant effect of Spirulina on reducing fasting blood glucose and postprandial blood glucose levels as well as HbA 1c level (Parikh et al., 2001). Some other studies have also shown that Spirulina consumption could reduce blood glucose levels (Aissaoui et al., 2017;Huang et al., 2018). Spirulina can putatively reduce blood glucose levels by stimulating β cell activity and increasing glucose transport to peripheral tissues (Layam & Reddy, 2006); moreover, it has been posited that the hypoglycemic  (Mazokopakis et al., 2014). However, discordant with our results, another study found spirulina could elicit positive effects on body weight, which might be owing to the relatively higher dose and a longer period of intervention (6 g/day for 6 months in comparison with 1 g/day for 3 months in our study). The potential effect of Spirulina on weight loss might be attributable to its low-fat and low-carbohydrate content (Mazokopakis et al., 2014).
Additionally, Spirulina is a source of phenylalanine, a potent releaser of cholecystokinin, which might help to regulate the brain's appetite center (Ballinger & Clark, 1994;Gibbs et al., 1976;Silverstone & Goodall, 1984).
Although the effect of Spirulina maxima in the inhibition of synthesis and releasing of arachidonic acid, and its vasoconstricting metabolites, and increasing nitric oxide synthesis (a well-known vasodilatation metabolite) is shown in some studies (Mascher et al., 2006), another potential mechanism to ameliorate blood pressure increases might be attributed to the inhibition of platelet aggregation, anti-inflammatory effects, and the high potassium and low sodium contents of Spirulina (Guan et al., 2007;Hsiao et al., 2005).
In our study, we did not find any significant ameliorating effects of Spirulina on blood pressure; however, this might be due to the normal baseline blood pressure levels and weight of our participants.

| Strengths and limitations
This study represents one of the first randomized double-blinded clinical trial human studies to have evaluated the effect of Spirulina in NAFLD patients. In addition, potential errors in grading fatty liver were decreased due to the radiologist being blinded to the group allocation of the patients. However, despite the strength and novelty of our study, several limitations must be considered. For instance, a relatively short period of intervention and a low sample size conceivably hindered the identification of effects of Spirulina in some parameters; while a lack of measurement of inflammatory factors could have further impacted the overall results. Therefore, in light of the aforementioned strengths and limitations, further studies with longer intervention duration, larger sample size, and additional evaluation of inflammatory factors are suggested.

| CON CLUS ION
We found that 8 weeks' supplementation of Spirulina improved fatty liver grade, oxidative stress markers, TG, and HOMA-IR; however, no significant change was found in blood pressure and anthropometric indices.

ACK N OWLED G M ENTS
The authors thank the cooperation of the participating patients.
The present manuscript was financially supported by Baqiyatallah University of Medical Sciences, Tehran, Iran, with grant number: 98000190. The author proclaimed that they have no other related financial interest.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interest.