Effectiveness of Eriomin® in managing hyperglycemia and reversal of prediabetes condition: A double‐blind, randomized, controlled study

This study evaluated the potential effectiveness of different doses of Eriomin® on hyperglycemia and insulin resistance associated with other metabolic biomarkers in prediabetic individuals. Prediabetes patients (n = 103, 49 ± 10 years) were randomly divided into four parallel groups: (a) Placebo; (b) Eriomin 200 mg; (c) Eriomin 400 mg; and (d) Eriomin 800 mg. Assessment of biochemical, metabolic, inflammatory, hepatic, renal, anthropometric markers, blood pressure, and dietary parameters were performed during 12 weeks of intervention. Treatment with all doses of Eriomin (200, 400, and 800 mg) had similar effects and altered significantly the following variables: blood glucose (−5%), insulin resistance (−7%), glucose intolerance (−7%), glycated hemoglobin (−2%), glucagon (−6.5%), C‐peptide (−5%), hsCRP (−12%), interleukin‐6 (−13%), TNFα (−11%), lipid peroxidation (−17%), systolic blood pressure (−8%), GLP‐1 (+15%), adiponectin (+19%), and antioxidant capacity (+6%). Eriomin or placebo did not influence the anthropometric and dietary variables. Short‐term intervention with Eriomin, at doses of 200, 400, or 800 mg/day, benefited glycemic control, reduced systemic inflammation and oxidative stress, and reversed the prediabetic condition in 24% of the evaluated patients.

The pathophysiology of prediabetes is complex and involves the combination of multiple changes in the mechanisms involved in glucose homeostasis. Similar to type 2 diabetes, prediabetes is associated with increased glucose levels, decreased insulin sensitivity, increased inflammatory cytokines, and altered incretin responses (Brannick, Wynn, & Dagogo-Jack, 2016). Prediabetes usually has no apparent signs or symptoms but may progress to type 2 diabetes with microvascular and macrovascular complications such as retinopathy, microalbuminuria, neuropathy, and cardiovascular disease. For this reason, to prevent or retard the progression to type 2 diabetes in this population is a relevant therapeutic goal. Recommendations include changes in lifestyle, such as regular physical activity combined with a balanced diet and use of antidiabetic drugs (ADA, 2018;Wasserman, Wang, & Brown, 2018). However, changes in lifestyle can be difficult to maintain in the long run, and antidiabetic drugs may be associated with side effects (Roberts, Craig, Adler, McPherson, & Greenhalgh, 2018).
Although the antioxidant and anti-inflammatory activities of citrus flavonoids have been widely recognized (Parhiz, Roohbakhsh, Soltani, Rezaee, & Iranshahi, 2015;Yi, Ma, & Ren, 2017), there is a lack of information about their actions on metabolic disorders related to prediabetes in humans, as well as information about potentially effective doses. Therefore, this study aimed to investigate the efficacy of three increasing doses of Eriomin® (200, 400, and 800 mg/day) on biochemical, metabolic, and inflammatory markers and the potential of Eriomin for reversal of the prediabetic condition.

| Individuals
Subjects were recruited through advertisement in the local media (websites, radio, and newspapers), distribution of fliers in the community, and City Health Center and University email lists. Individuals of both sexes, aged 35-60 years, and with prediabetes were considered eligible to participate in the study, because prediabetes is more prevalent in middle-aged adults, and below 60 years because the confounding factor of aging. Prediabetic subjects were selected according to the Expert Panel of the American Diabetes Association (2018), which include these following features: (a) impaired glucose metabolism, (b) glucose intolerance, and (c) glycated hemoglobin ≥5.7%.
The exclusion criteria were as follows: pregnancy; smoking; history of diabetes mellitus, cardiovascular, hepatic, renal, disease; use of dietary supplements (vitamins, minerals, bioflavonoids, probiotic, symbi-otic, or other bioactive compound); continuous use drugs; history of drug or alcohol abuse; and intense physical exercise (more than 10 hr per week).

| Sample size
The primary endpoint was serum glucose, and sample size was estimated based on a similar clinical study (Mohammadi et al., 2015).
The sample size for the parallel clinical trial was calculated statistically , with significance level of 5% and 80% power. The minimum sample size was 24 individuals per group and, in anticipation of a 15% dropout rate, 30 individuals were considered per intervention group.

| Ethics approval and consent to participate
The procedures performed in this study followed the ethical guidelines of the National Health Council (Res. 466/12) and Declaration of Helsinki (1964 The randomization scheme was performed by an independent researcher. Containers and capsules of Eriomin and placebo were identical and were prepared by a pharmacist who did not participate in the study. The container label held only the patient identification number. Thus, both the investigator and the patients were blind from the time of randomization until the analysis was complete.

| Supplements preparation
The intervention product was Eriomin®, supplement of citrus flavonoids provided by Ingredients by Nature TM, Montclair, CA. The purity was determined by HPLC and which contains 70% eriocitrin, 5% hesperidin, 4% naringin, and 1% didymin. The placebo, containing 100% microcrystalline corn starch, was formulated by an independent pharmacist, and its appearance was as similar as possible to the active supplement. Subjects were instructed to consume one capsule after dinner with a glass of water during 12 weeks. Supplement and placebo were given to participants every 2 weeks after randomization.  (Friedwald, Levy, & Friedrickson, 1972). Homeostasis Model Assessment (HOMA-IR) was calculated, and the cutoff set was at ≥2.71 (Matthews et al., 1985). Lipid peroxidation was assessed by TBARS assay (Yagi, 1998) and total antioxidant capacity by radical ABTS + assay (Re et al., 1999

| Dietary parameters
Subjects were instructed to maintain their usual diet and physical activity during the total experimental period. In the beginning of the first, fourth, eighth, and 12th weeks, registered nutritionists have analyzed usual dietary intake by a 3-day dietary record nonconsecutive, and the analysis of energy and macronutrient and micronutrient intake was performed using the DietBox®, based on the Brazilian Table of Food Composition (Unicamp, 2006).

| Compliance and adverse events
Adverse effects were previously defined by the investigators as the presence of any unfavorable and unintended signs on the health and well-being of individuals, abnormal laboratory findings, symptoms and/or diseases temporarily associated after administration of Eriomin® or placebo. During the intervention, patients were questioned biweekly by nutritionists for the eventual occurrence of nausea, vomiting, diarrhea, or any change in general well-being or illness. Conformity was assessed by counting unused capsules at each visit. Participants who consumed more than 90% of the provided capsules and completed all evaluations had good compliance and were included in the statistical analysis.
To avoid spurious association or bias between a major outcome with an independent external variable (confounding variable), such as physical activity, diet, anthropometry or lifestyle, each patient was monitored biweekly to ensure no changes on these parameters.
Physical activity was assessed individually at each consultation, asking the type and period expended on it. The diet was evaluated by a 3-day nonconsecutive dietary record, performed 1 week before the biweekly return to the nutritionist. Anthropometric measurements were taken biweekly.

| Statistical analysis
Data are presented as mean ± SD. Statistical analysis was performed using SPSS 22 (Statistical Package Social Sciences). One-way ANOVA was used to identify differences between groups in the baseline period. Two-way repeated measures ANOVA followed by Sidak post hoc test were apply to compare changes within and between groups over 12 weeks. The significance was p ≤ .05.

| Individuals
One hundred and three subjects, 49 men and 54 women, 49 ± 10 years, previously classified as prediabetic, were included in this study; 17 participants were excluded for the following reasons: low compliance to product intake (i.e., <90% compliance, n = 6), disease (n = 3), family circumstances (n = 2), moved away (n = 1), and did not attend samples withdrawal (n = 5; Figure 1). The baseline characteristics of all participants were similar among the groups: placebo and 200, 400, and 800 mg Eriomin, as shown in Table 1.  (Table 2). In addition, there was a mean reduction of 2% in HbA1c levels in the three groups supplemented with Eriomin (p ≤ .05), whereas placebo showed no change during intervention time (Table 2). All volunteers had insulin resistance at the beginning, during, and at the end of the experiment (HOMA-IR ≥ 2.71), but there was a reduction of 8% after intervention with 200 mg of Eriomin (p = .037), 7% with 400 mg (p = .043), and 6% with 800 mg (p = .042; Table 2).

| Biochemical markers
Regarding blood lipids, there was no reduction in total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides during and at the end of the experimental period in the placebo group and supplemented with Eriomin (200, 400, and 800 mg; Table 2).

| Metabolic and inflammatory markers
Levels of blood plasma GLP-1 increased 15% for all tested doses of Eriomin: 200, 400, and 800 mg (p < .001). After intervention, Eriomin supplementation also promoted an average reduction of 6.5% of glucagon (p < .001) and 5% of C-peptide levels (p < .001). No change was observed in the placebo group during treatment (Table 3).

| Liver and renal markers
Hepatic enzymes (AST, ALT, ALP, and γGT) and a marker of renal function (creatinine) remained unchanged during the experiment in all three groups supplemented with Eriomin and at placebo (Table 4).

| Anthropometry and blood pressure
Supplementation with Eriomin (200, 400, and 800 mg/day) and placebo had no effect on body weight, BMI, lean mass, fat mass, fat percentage, and hip waist ratio. However, all doses of Eriomin supplementation promoted a mean systolic blood pressure reduction FIGURE 1 Trial design of 7%. Diastolic blood pressure remained unchanged in the groups throughout the study (Table 5).

| Dietary parameters
Intake of energy and macronutrients (carbohydrates, proteins, and lipids) and cholesterol, saturated fatty acid, fibers, vitamin E, and vitamin C were not significantly altered in patients supplemented with Eriomin or placebo during the 12-week intervention (Table 6).

| Adverse effects and safety of Eriomin
During the study, seven adverse events were reported: two cases in the Eriomin 800 group (one pasty stool and one headache), two cases in the 400 group (two pasty stools), one case in the 200 group (one pasty stool), and two cases in the placebo group (one pasty stool and one headache). The frequencies of these events did not differ between groups (p > .05), showing that, in general, Eriomin supplementation was well tolerated, without reports of severe or chronic adverse events. Furthermore, liver and kidney functions were unchanged in all groups during treatment. Note. Data are presented as mean ± SD. One-way ANOVA, p ≤ .05.
Abbreviations: BMI, body mass index; OGTT, 2-hr oral glucose tolerance test; HOMA-IR, homeostasis model assessment-insulin resistance; HbA1c, glycated hemoglobin; hsCRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; TNF-α, tumor necrosis factor alpha.  Under Eriomin supplementation, 5% reduction in glycemic levels and 7% reduction in the glucose tolerance test occurred. Serum HbA1c and insulin resistance, as measured by HOMA-IR, reduced by 2% and 7%, respectively. These findings were clinically relevant because after treatment with Eriomin 24% of patients reversed the prediabetic clinical condition for normal glycemia and/or lower glucose intolerance. Citrus flavonoids have been identified as antidiabetic Note. Two-way repeated measures ANOVA followed by Sidak test among groups (placebo, 200, 400, and 800 mg) over 12-week intervention period; p ≤ .05. Different letters (a, b) indicate difference within the group, and different uppercase letters (A, B) indicate difference between groups. a Difference between week 12 and 0. b Mean of the percentage differences between the groups supplemented with Eriomin. compounds because of their hypoglycemic effects, observed in vitro and in vivo studies. (Bucolo, Leggio, Drago, & Salomone, 2012;Fukuchi et al., 2008;Zhang et al., 2012). In type 2 diabetic mice, hesperidin and naringin appear to regulate the activities of hepatic enzymes involved in gluconeogenesis and glycolysis (Jung, Lee, Jeong, & Choi, 2004). These flavanones were also able to reduce the expression of glucose-6-phosphatase mRNA, phosphoenolpyruvate carboxykinase (PEPCK), and hepatic GLUT2 and increase GLUT4 expression in adipocytes (Jung, Lee, Park, Kang, & Choi, 2006). Moreover, it was shown that eriodictyol regulates the expression of peroxisome proliferator-activated receptor gamma (PPARγ) mRNA, in hepatocytes and adipocytes, which activates insulin signaling and promotes translocation of the glucose transporter GLUT4, increasing intracellular glucose uptake and consequently improving insulin sensitivity (Zhang et al., 2012).
It has been reported that prediabetic individuals have impaired serum GLP-1 secretion and incretin secreted by intestinal L cells after carbohydrate ingestion (Wang et al., 2016). GLP-1 is involved in regulating glucose metabolism, stimulating insulin secretion, and inhibiting glucagon secretion, thereby lowering plasma glucose levels (Gastaldelli, Gaggini, & DeFronzo, 2017). In our study, all doses of Eriomin promoted a 15% increase in GLP-1 levels and a 6% reduction in glucagon levels, which presumably may be associated with improved hyperglycemia in prediabetic volunteers. A recent review described new mechanisms of how citrus flavonoids act in the secretion and signaling of GLP-1 to regulate the glucose metabolism (Domínguez Avila, Rodrigo García, González Aguilar, & Rosa, 2017).
This fact was demonstrated in experiments with naringin, a citrus flavanone, which inhibited dipeptidil peptidase 4 (DPP-4) by increasing the half-life of GLP-1, improving insulin secretion and glucose uptake (Parmar et al., 2012).
Another important marker for the prediabetic condition is the low level of adiponectin, an adipokine that plays a crucial role in insulin sensitivity and regulation of glucose metabolism, and is also considered a risk factor for cardiovascular disease (Banerjee et al., 2017;Lai, Lin, Xing, Weng, & Zhang, 2015). Previous studies showed citrus    (Haidari et al., 2015;Liu et al., 2008). These data corroborate with our findings, where supplementation with Eriomin increased serum adiponectin levels by 18% and reduced the concentration of C-peptide by 5%, an important marker of beta cell function that allows differentiation between the prognosis of type 1 and type 2 diabetes .
Increased production of proinflammatory cytokines, such as IL-6 and TNF-α, plays an important role in the pathogenesis of type 2 diabetes and contributes to long-term micro and macrovascular complications (Forbes & Cooper, 2013;Navarro & Mora, 2006). Prediabetic patients exhibit higher levels of these markers during disease progression (Dorcely et al., 2017). Results of our study showed that Eriomin lowered the lowgrade inflammation in prediabetic patients by reductions of serum levels of hsCRP (−12%), IL-6 (−13%), and TNF-α (−11%). Previous study in mice supplemented with eriocitrin or eriodictyol also showed decreases in the elevated levels of IL-6 and hsCRP caused by a high-fat diet (Ferreira et al., 2016). Another study showed that eriodictyol had inhibitory effects on mRNA expression of IL-6 and TNF-α (Lee, 2011). Some mechanisms have been proposed to explain the anti-inflammatory properties of citrus flavonoids, which include activation of PPARγ expression and inhibition of nuclear factor kappa B (NFκB), with consequent reduction of inflammatory cytokine secretion and increase of adiponectin (Gamo, Miyachi, Nakamura, & Matsuura, 2014;Lee, 2011).
Hyperglycemia is associated with oxidative stress, which plays an important role at the progression of diabetes, insulin resistance, and β-cell dysfunction (Rehman & Akash, 2017). In contrast, flavonoids act as antioxidants against various diseases by neutralizing the effects oxidative stress (Kawser Hossain et al., 2016). In the present study, Eriomin supplementation resulted in improved blood serum antioxidant status and reduced oxidative stress, evidenced by increased antioxidant capacity (+6%) associated with reduced lipid peroxidation marker (−17%). Our results are consistent with previous studies, which observed a reduction of lipid peroxidation on the blood serum, liver, and kidneys of diabetic rats treated with eriocitrin (Bucolo et al., 2012;Ferreira et al., 2016;Miyake, Yamamoto, Tsujihara, & Osawa, 1998). In addition, eriodictyol protected against kidney injury through activating nuclear factor-erythroid related factor 2 (Nrf2; Li et al., 2016). In diabetic individuals, the activation of Nrf2 protects pancreatic β cells against various insults, thus maintaining glucose homeostasis and also increasing insulin sensitivity (Sireesh, Dhamodharan, Ezhilarasi, Vijay, & Ramkumar, 2018). The tendency of eriodictyol to inhibit free radical-mediated events is governed by its chemical structure. Double bonds, hydroxyl groups, and carbonyl function confer antioxidant and anti-apoptotic properties of flavonoid molecules (Bucolo et al., 2012). According to Miyake et al. (1998), eriocitrin has stronger antioxidant activity than the other citrus flavonoid compounds due to its multiple hydroxyl groups.
Eriomin supplementation during 12 weeks was well tolerated by patients, because AST, ALT, ALP, and γGT levels remained at normal levels. Altered levels of these enzymes reflect damage to hepatocytes and are considered sensitive and specific clinical biomarkers for hepatotoxicity (Ozer, Ratner, Shaw, Bailey, & Schomaker, 2008). These results suggest absence or nondetectable toxicity or impairment of liver function, as previously shown of lemon flavonoid supplementation (Hiramitsu et al., 2014). In addition, a study with mice showed that eriocitrin protected against liver damage caused by consumption of the high-fat diet (Ferreira et al., 2016).
This study showed strong aspects, including (a) double-blinded, placebo-controlled design, (b) high adherence of patients attested by the low number of withdrawal, and (c) evaluation of Eriomin adverse effects and toxicity (presence of any unfavorable and unintended signs, liver enzymes and creatinine). However, some limitations were also observed, such as the relatively short duration of the study (12 weeks), and indirect observation of dietary intake and physical activity through recorded. More studies with longer intervention time and larger sample sizes are needed to better understand the effects of Eriomin in attenuating hyperglycemia in prediabetic subjects. In addition, this lack of dose response suggests that studies with doses below 200 mg should be performed. Note. Two-way repeated measures ANOVA followed by Sidak test among groups (placebo, 200, 400 ,and 800 mg) over 12-week intervention period; p ≤ .05. † Percentage difference between week 12 and 0. † † Mean of the percentage differences between the groups supplemented with Eriomin.

FIGURE 2 Percentage of individuals with prediabetic after Eriomin supplementation
In conclusion, this study showed that short-term intervention with Eriomin® benefited glycemic control, lowered the systemic inflammation and oxidative stress, and reversed the prediabetic condition in 24% of total patients evaluated for all dose tested.