Effect of fenugreek supplementation on blood lipids and body weight: A systematic review and meta-analysis of randomized controlled trials

Journal Pre-proof Effect of fenugreek supplementation on blood lipids and body weight: A systematic review and meta-analysis of randomized controlled trials Moein Askarpour, Farkhondeh Alami, Marilyn S. Campbell, Kamesh Venkatakrishnan, Amir Hadi, Ehsan Ghaedi PII:

S0378-8741(19)33085-5

DOI:

https://doi.org/10.1016/j.jep.2019.112538

Reference:

JEP 112538

To appear in:

Journal of Ethnopharmacology

Received Date: 1 August 2019 Revised Date:

29 December 2019

Accepted Date: 30 December 2019

Please cite this article as: Askarpour, M., Alami, F., Campbell, M.S., Venkatakrishnan, K., Hadi, A., Ghaedi, E., Effect of fenugreek supplementation on blood lipids and body weight: A systematic review and meta-analysis of randomized controlled trials, Journal of Ethnopharmacology (2020), doi: https:// doi.org/10.1016/j.jep.2019.112538. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Fenugreek (Trigonella foenum-graecum L.)

Reduce cholesterol absorption

Total cholesterol (WMD = -9.37 mg/dL; 95% CI: -15.41, -3.32) Triglycerides (WMD = -13.77 mg/dL; 95% CI: -26.63, -0.91) Low density lipoprotein cholesterol (WMD = -6.59 mg/dL; 95% CI: -13.04, -0.13) High density lipoprotein cholesterol (WMD = +3.50 mg/dL; 95% CI: 1.30, 5.69)

Effect of fenugreek supplementation on blood lipids and body weight: A systematic review and meta-analysis of randomized controlled trials Moein Askarpour 1¥, Farkhondeh Alami 2¥, Marilyn S. Campbell 3, Kamesh Venkatakrishnan4, Amir Hadi 5*, Ehsan Ghaedi 6, 1** 1

Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetics,

Tehran University of Medical Sciences, Tehran, Iran 2

Student Research Committee, Department of Nutrition, Faculty of Medicine, Urmia University

of Medical Sciences, Urmia, Iran 3

Department of Kinesiology and Health Promotion, University of Kentucky, Lexington,

Kentucky, USA 4

School of Nutrition, Chung Shan Medical University, 110, Sec. 1, Jianguo North Road,

Taichung City, Taiwan, ROC 5

Halal Research Center of IRI, FDA, Tehran, Iran

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Students’ Scientific Research Center (SSRC), Tehran University of Medical Sciences (TUMS),

Tehran, Iran ¥ These two authors (M.A. and F.A.) contributed equally to this work. Running Title: Fenugreek and blood lipids Corresponding Authors: Ehsan Ghaedi Postal address: Department of Cellular and Molecular Nutrition, School of Nutrition Sciences and Dietetics, Tehran University of Medical Sciences, Poorsina Street, Enghelab Avenue, Tehran, Iran. PO Box: 14155-6446, Tel: +982188954911, Fax: +982188974462 E-mail: [email protected] Amir Hadi Halal Research Center of IRI, FDA, PO Box 81745, Tehran, Iran. E-mail:[email protected]

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Abstract Ethnopharmacological relevance Fenugreek is a traditional herbal medicine that has been used for centuries to treat hyperglycemia, muscle spasms, gastritis, constipation, edema, and other metabolic disorders. Recently, lipid-lowering effects of fenugreek have been identified. Aim of the study The aim of this systematic review and meta-analysis was to determine and clarify the impact of fenugreek supplementation on anthropometric indices and serum lipid levels. Materials and Methods We searched PubMed, Scopus, ISI Web of Science, Cochrane Library, and Google Scholar from inception to June 2019 using relevant keywords. All randomized controlled trials (RCTs) investigating the effects of fenugreek on anthropometric indices and plasma lipids in adults were included. A random-effects model was used for quantitative data synthesis. A sensitivity analysis was conducted using the leave-one-out method. Results A meta-analysis of 12 RCTs (14 arms) with 560 participants suggested a significant decrease in plasma concentrations of total cholesterol (WMD = -9.371 mg/dL; 95% CI: -15.419, -3.323, p = 0.002), triglycerides (WMD = -13.776 mg/dL; 95% CI: -26.636, -0.916, p = 0.036), and low density lipoprotein cholesterol (WMD = -6.590 mg/Dl; 95% CI: -13.042, -0.137, p = 0.045), as well as an increase in plasma high density lipoprotein cholesterol (WMD = 3.501 mg/dL; 95% CI: 1.309, 5.692, p = 0.002), while body weight (WMD = 0.223 kg; 95% CI: -0.509, 0.955, p = 2

0.551) and body mass index (WMD = 0.091 kg/m2; 95% CI: -0.244, 0.426, p = 0.596) were not altered. Conclusion Fenugreek supplementation improved lipid parameters in adults. However, to confirm these results, more studies, particularly among hyperlipidemic patients, are needed.

Key words

Fenugreek, Supplementation, Blood lipids, Meta-analysis, Systematic review

Abbreviation RCTs, randomized controlled trials; MetS, metabolic syndrome; CVD, cardiovascular disease; T2DM, type 2 diabetes mellitus; BMI, body mass index; TC, total cholesterol; TG, triglycerides; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; SD, standard deviation; CI, confidence interval; WMD, weighted mean difference;

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1. Introduction It has been estimated that 20-40% of the world’s population is directly or indirectly affected by metabolic syndrome (MetS) and its related complications, which include obesity (excess fat), dyslipidemia (elevated lipid level) and hyperglycemia (elevated sugar level). The prevalence of MetS is increasing alarmingly and shows no signs of decreasing in the near future (Ranasinghe et al., 2017). Moreover, the risk of MetS (obesity and dyslipidemic conditions) progressing into cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) is quite high (Fuller and Stephens, 2015; Han and Lean, 2016). CVD is the leading cause of death, claiming more lives than any other disease. Hence, lowering the risk factors for CVD by reducing the incidence of obesity and dyslipidemia is a major goal for many researchers. Thus, the quest for an effective drug to combat MetS and its related complications is of great interest. Many established medicines can cause unwanted side effects; therefore, selected natural agents could be an effective alternative option to combat MetS. Many natural products not only act through a wide range of biological activities, but they are also low-cost and cause fewer adverse effects (Waltenberger et al., 2016). Fenugreek (Trigonella foenum-graecum L.) is an annual herb with clover-shaped yellow seeds belonging to Fabaceae family. Fenugreek seeds are a well-known spice additive and flavoring agent in various cuisines, especially in Asian culinary dishes (Gong et al., 2016; Venkatakrishnan et al., 2019). The spice has been traditionally used in China and India for treating various illnesses or disorders including hyperglycemia, muscle spasms, gastritis, constipation, and edema (Nagulapalli Venkata et al., 2017). The major metabolites present in fenugreek are flavonoids and other polyphenols, trigonelline, 4-hydroxyisoleucine, and saponin, such as diosgenin, protodioscin; additionally, Furosap is a novel Fenugreek extract introduced 4

recently from fenugreek seeds (Fuller and Stephens, 2015; Srinivasan, 2019). These metabolites present in fenugreek are believed to exhibit various health benefits. Previous studies indicate that fenugreek displays an array of therapeutic properties such as anti-diabetic, anti-hyperlipidemic, anti-obesity, antioxidant, anti-inflammatory, anti-arthritic, anti-cancer and anti-microbial activities (Bahmani et al., 2016; Garg, 2016). Since fenugreek has been used in cooking and contains various biological properties, it is considered a functional food, and many researchers have considered and commercialized fenugreek products for various conditions, like T2DM (Roberts, 2011; Srinivasan, 2006). As mentioned previously, some components of fenugreek like saponins and mucilages showed hypocholesterolemic effects in animal studies. In fact, mucilage, tannins, pectin, and hemicellulose from fenugreek bind to bile acids to reduce cholesterol and fat absorption. Furthermore, they block bile salt absorption in the colon and, therefore, reduce cholesterol in the blood (Ahmad et al., 2016; Al-Sultan and El-Bahr, 2015). Several components of fenugreek seeds, like galactose and mannose, possess hypocholesterolemic effects (Wani and Kumar, 2018), which also has been shown in animal studies (Ahmad et al., 2016; Al-Asadi, 2014; AlSultan and El-Bahr, 2015; Marrelli et al., 2016; Reddy and Srinivasan, 2009; Xue et al., 2007). However, not all trials have reported the same results. As such, several human trials have demonstrated the beneficial effects of fenugreek supplementation on anthropometric indices and lipid profiles (Fedacko et al., 2016; Gholaman, 2018; Moosa et al., 2006; Rafraf et al., 2014); however, other studies have found no beneficial effects of this plant on the aforementioned parameters (Bordia et al., 1997; Chevassus et al., 2009a; Chevassus et al., 2009b; Gaddam et al., 2015; Gupta et al., 2001b; Sharma and Raghuram, 1990; Shen et al., 2013; Yousefi et al., 2017).

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To the best of our knowledge, this is the first meta-analysis conducted to explore the impact of fenugreek supplementation on various anthropometric indices, including weight and body mass index (BMI), and lipid profiles, including total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-c), and high density lipoprotein cholesterol (HDL-c), in randomized controlled clinical trials. 2. Methods This review was designed, conducted and reported based on the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (Moher et al., 2009). Since our study is a systematic review and meta-analysis of human intervention studies, it did not require approval from an ethical committee. 2.1. Search strategy The following electronic databases were searched from inception to June 2019: PubMed/ Medline, Scopus, ISI Web of Science, Cochrane Library, and Google Scholar. Medical subject heading terms and keywords used to identify studies included: ("Fenugreek" OR "Trigonella") AND ("Intervention Studies" OR "intervention" OR "controlled trial" OR "randomized" OR "randomised" OR "random" OR "randomly" OR "placebo" OR "assignment"). The search was limited to articles published in the English language. We also checked reference lists of all eligible studies to identify any additional publications (reverse snowballing). 2.2 Study selection Results from the database searches were imported into EndNote software, Version X6 (Thomson Reuters, New York), and duplicates were discarded. Then, two investigators (M.A. and A.H) 6

independently screened studies to be included in the review using predetermined inclusion criteria. Studies were first scanned on the basis of title and abstract, after which the total article was reviewed. Publications were included in the meta-analysis if they met the following inclusion criteria: 1) were an RCT with either parallel or cross-over design; 2) evaluated the effects of fenugreek on the outcomes of interest including weight, BMI, TC, TG, LDL-c, and HDL-c; and 3) presented values at baseline and at the end of follow-up in each group or presented the net change values. We excluded studies conducted in animals, children, and pregnant women. Also, trials with a treatment duration of less than two weeks and those which investigated the effects of fenugreek in combination with other components (drugs or supplements) were excluded. For duplicate publications from the same study, only the most complete reports were identified. Any possible discrepancies were resolved by discussion between the reviewers until the final decision was made. 2.3. Data extraction The standardized form was used to extract the following data from each study: 1) study characteristics (such as primary author, publication date, country origin, study design, and total sample size); 2) participant characteristics (such as gender, mean age, mean BMI, and health status); 3) intervention and comparison data (such as type and dose of fenugreek/control and intervention period); and 4) outcome measures. One reviewer (A.H) extracted aforementioned data, and all information was double-checked for consistency by a second reviewer (M.A.). Disagreements were resolved by additional reviewers. If needed, corresponding authors of the trials were contacted via email to provide any missing data. 2.4. Quality assessment

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The Cochrane Risk of Bias Tool (Higgins et al., 2011) for RCTs was used to assess the quality of the included studies. The two authors independently assessed methodological quality using seven domains: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other sources of bias. These domains were assessed as a judgment (high, low, or unclear), and discrepancies were resolved by consensus in group meetings with senior investigators. 2.5. Statistical analysis The difference in the mean values of lipid profiles was calculated for all included studies and was considered the effect size. The units of lipid parameters were collated as mg/dL. The net changes of outcomes were estimated as the difference (fenugreek minus control) between its changes (end values minus baseline) in each group. Standard deviations (SD) of the mean difference were calculated using the following formula: SD = square root [(SD pretreatment)2 + (SD post-treatment)2 – (2R × SD pre-treatment × SD post-treatment)], assuming a correlation coefficient (R) = 0.5 report (Borenstein et al., 2011). According to whether or not significant heterogeneity of outcomes was present, the continuous variable was pooled as weighted mean difference (WMD) and 95% confidence interval (CI) with a fixed-effects or random-effects model. I2 statistic was used to quantify the degree of inter-study heterogeneity (Higgins et al., 2003). If there was significant heterogeneity (I2 > 50%), subgroup analyses were conducted to identify study-level heterogeneous factors, which included participant's health condition (healthy/unhealthy), study duration (<8 or ≥8 weeks) and dose of supplementation (≤10 g/day or >10 g/day). In order to evaluate the stability of the results, a sensitivity analysis was performed by removing one study at a time and pooling the remaining ones (Tobias, 1999). The potential existence of publication bias was also examined using Begg’s and Egger’s tests 8

and by inspection of the funnel plots (Egger et al., 1997). Statistical analyses were performed using Stata, Version 11.2 (Stata Corp., College Station, TX), and results were considered statistically significant at p values < 0.05. 3. Results 3.1. Study selection A total of 1079 studies was yielded from the initial search (234 from PubMed, 259 from ISI, 101 from Cochrane and 485 from Scopus). After removing duplicates (n = 431), 648 articles remained. Then, 616 were excluded after careful evaluation of the titles and abstracts based on PICOS criteria: 1) Unrelated title (n = 381), 2) were animal studies (n = 187), or 3) other reason (review, letter, short survey and note) (n = 48). Consequently, 32 potentially relevant articles were retrieved for full-text assessment and detailed examination. Twenty full-text articles were excluded due to the following reasons: did not report data of interest (n = 10), same population (n = 3), or combination along with other herbs (n = 7). Finally, a total of 12 eligible RCTs with 14 arms met all inclusion criteria (Bordia et al., 1997; Chevassus et al., 2009a; Chevassus et al., 2009b; Fedacko et al., 2016; Gaddam et al., 2015; Gholaman, 2018; Gupta et al., 2001b; Moosa et al., 2006; Rafraf et al., 2014; Sharma and Raghuram, 1990; Shen et al., 2013; Yousefi et al., 2017) and were included in the statistical analysis. The PRISMA flow diagram is depicted in Figure 1. 3.2. Characteristics of the studies The primary characteristics of these 12 trials with 14 arms are outlined in Table 1. Overall, 560 participants were randomly assigned to and completed the studies. Of the 12 trials used in the 9

present meta-analysis, two trials were conducted in men only (Chevassus et al., 2009a; Chevassus et al., 2009b), and one trial was conducted in women only (Gholaman, 2018), whereas most of the trials (8/12) were conducted in both sexes (Fedacko et al., 2016; Gaddam et al., 2015; Gupta et al., 2001b; Moosa et al., 2006; Rafraf et al., 2014; Sharma and Raghuram, 1990; Shen et al., 2013; Yousefi et al., 2017). Moreover, one trial did not outline sex composition (Bordia et al., 1997). The included trials varied in duration from two to 162 weeks. The study design of two trials was parallel (Chevassus et al., 2009b; Sharma and Raghuram, 1990), while crossover designs were used in 10 trials (Bordia et al., 1997; Chevassus et al., 2009a; Fedacko et al., 2016; Gaddam et al., 2015; Gholaman, 2018; Gupta et al., 2001b; Moosa et al., 2006; Rafraf et al., 2014; Shen et al., 2013; Yousefi et al., 2017). Fenugreek seed power and defatted fenugreek seed power were used in eight studies with daily doses from 5.0 to 100.0 g (Bordia et al., 1997; Fedacko et al., 2016; Gaddam et al., 2015; Gholaman, 2018; Moosa et al., 2006; Rafraf et al., 2014; Sharma and Raghuram, 1990; Yousefi et al., 2017). Hydro-alcoholic extract of fenugreek seeds were chosen as the intervention in three studies with doses of 0.588 to 1.176 g (Chevassus et al., 2009a; Chevassus et al., 2009b; Gupta et al., 2001b); based on a previous study, one gram of hydro-alcoholic extract of fenugreek seed equals 9.6 gram equivalents of crude fenugreek seed (Bashtian et al., 2013). In another study, fenugreek total saponins were used at a dose of 6.3 g (Shen et al., 2013). Moreover, in some trials, participants in both groups adhered to exercise training (Gaddam et al., 2015; Gholaman, 2018) or nutritional guidance (Gaddam et al., 2015). Studies were conducted in different countries, including: India (Bordia et al., 1997; Fedacko et al., 2016; Gaddam et al., 2015; Gupta et al., 2001b; Sharma and Raghuram, 1990), France (Chevassus et al., 2009a; Chevassus et al., 2009b), Iran (Gholaman, 2018; Rafraf et al., 2014; Yousefi et al., 2017), Bangladesh (Moosa et al., 2006), and China

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(Shen et al., 2013). The BMI of the trial participants included groups with a BMI of less than 25 kg/m2 (Chevassus et al., 2009b; Fedacko et al., 2016) and groups with a BMI of greater than 25 kg/m2 (Chevassus et al., 2009a; Gaddam et al., 2015; Gholaman, 2018; Gupta et al., 2001b; Rafraf et al., 2014; Yousefi et al., 2017), although four trials did not report BMI (Bordia et al., 1997; Moosa et al., 2006; Sharma and Raghuram, 1990; Shen et al., 2013). Seven trials enrolled patients with T2DM (Bordia et al., 1997; Gholaman, 2018; Gupta et al., 2001b; Moosa et al., 2006; Rafraf et al., 2014; Sharma and Raghuram, 1990; Shen et al., 2013), and one trial enrolled individuals who were pre-diabetic (Gaddam et al., 2015). Two trials included dyslipidemic patients (Fedacko et al., 2016; Yousefi et al., 2017). Additional trials were carried out in healthy subjects (Chevassus et al., 2009b), with one including overweight but otherwise healthy subjects (Chevassus et al., 2009a). Information about the chemical composition of the preparations / extracts used in all included RCTs also depicted in Supplementary Table 1. Most of the included studies reported inadequate information and were overall taxonomically inadequate. The voucher number was not reported in any of the included studies. 3.3. Quality assessment Random allocation of participants was used in all included trials. The method of random sequence generation was described in four trials (Gaddam et al., 2015; Rafraf et al., 2014; Shen et al., 2013; Yousefi et al., 2017), whereas the remaining trials have an unclear risk of bias (Bordia et al., 1997; Chevassus et al., 2009a; Chevassus et al., 2009b; Fedacko et al., 2016; Gholaman, 2018; Gupta et al., 2001b; Moosa et al., 2006; Sharma and Raghuram, 1990). Allocation concealment of three trials has been reported (Gaddam et al., 2015; Rafraf et al.,

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2014; Yousefi et al., 2017), and the other studies indicated unclear risk of bias (Bordia et al., 1997; Chevassus et al., 2009a; Chevassus et al., 2009b; Fedacko et al., 2016; Gholaman, 2018; Gupta et al., 2001b; Moosa et al., 2006; Sharma and Raghuram, 1990; Shen et al., 2013). High risk of bias regarding the blinding of participants and personnel and outcome assessment in two trials was revealed (Bordia et al., 1997; Moosa et al., 2006). All of the studies showed low risk of bias regarding incomplete outcome data and selective reporting. Also, all trials had unclear risk of bias based on other potential threats to validity. The details of risk of bias assessment are described in Table 2. 3.4. Findings from meta-analysis 3.4.1. Effect of fenugreek supplementation on TC The overall result of our meta-analysis of 13 arms (285 intervention and 265 control subjects) indicated that there was a significant reduction in TC (WMD = -9.371 mg/dL; 95% CI: -15.419, 3.323, p = 0.002), with considerable heterogeneity between studies (p < 0.001, I2 = 87.6 %) (Figure 2). To find any source of heterogeneity, we performed subgroup analyses based on participant’s health condition (healthy/unhealthy), study duration (<8 or ≥8 weeks) and dose of supplementation (≤10 g/day or >10 g/day). Subgroup analyses for those studies which supplemented hydro-alcoholic extract the gram equivalent of crude fenugreek seed preparation that such HF extracts represent were used. Subgroup analyses revealed a significant lowering effect of fenugreek supplementation on TC in RCTs conducted in unhealthy subjects, using more than 10 grams of fenugreek, and carried out with greater than eight weeks of supplementation (Table 3). 12

3.4.2 Effect of fenugreek supplementation on LDL- c The effect of the fenugreek supplementation on LDL-c was evaluated in eight clinical trials (196 intervention and 175 control subjects), and the pooled mean difference from the inverse variance method showed a reduction in LDL-c by 6.590 mg/dL (95% CI: -13.042, -0.137, p = 0.045) with considerable between-study heterogeneity (p < 0.001, I2 = 81.1%) (Figure 3). Only high doses of supplementation showed a significant effect of fenugreek on LDL-c (Table 3). 3.4.3 Effect of fenugreek supplementation on HDL- c Overall, twelve arms (268 intervention and 249 control subjects) investigated the effects of fenugreek supplementation on HDL-c concentration. Pooled effect size showed a significant increase in HDL-c following fenugreek supplementation (WMD = 3.501 mg/dL; 95% CI: 1.309, 5.692, p = 0.002) with between-study heterogeneity (p < 0.001, I2 = 88.5%) (Figure 4). Subgroup analyses showed that HDL-c increased in RCTs with: both short and long duration, high doses of supplementation (>10g/day), and unhealthy subjects (Table 3). 3.4.4 Effect of fenugreek supplementation on TG Thirteen RCTs (285 intervention and 265 control subjects) reported the effect of fenugreek supplementation on TG concentrations, and combining effect sizes from these studies revealed a significant reduction in TG (WMD = -13.776 mg/dL; 95% CI: -26.636, -0.916, p = 0.036), with significant between-study heterogeneity (p < 0.001, I2 = 96%) (Figure 5). To find the probable source of heterogeneity, subgroup analyses were conducted. Dividing RCTs based on the aforementioned subgroups showed that TG decreased significantly following

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fenugreek supplementation in those RCTs that administered high doses of fenugreek, had a supplement duration lasting more than eight weeks, and were conducted in unhealthy subjects (Table 3). Additional analyses in patients with T2DM showed that fenugreek supplementation decreased TC (WMD = -11.010 mg/dL; 95% CI: -17.543, -4.476, p =0.001) and TG (WMD = -16.914 mg/dL; 95% CI: -31.181, -2.647, p =0.020), and increased HDL (WMD = 4.598 mg/dL; 95% CI: 0.842, 8.354, p = 0.016), indicating statistically significant results in patients with T2DM compared with healthy subjects. 3.4.5 Effect of fenugreek supplementation on anthropometric indices Nine RCTs (219 intervention and 168 control subjects) reported the effects of fenugreek supplementation on BMI, and combining effect sizes from these studies did not show any significant effects (WMD = 0.091 kg/m2; 95% CI: -0.244, 0.426, p = 0.596). Between-study heterogeneity was not significant (p = 0.578, I2 = 0%) (Figure 6). Ten RCTs (199 intervention and 167 control subjects) reported the effect of fenugreek supplementation on weight. The overall estimate of effect size did not show any significant effect (WMD = 0.223 kg; 95% CI: -0.509, 0.955, p = 0.551) and lacked between-study heterogeneity (p = 0.984, I2 = 0%) (Figure 7). Subgroup analyses revealed that fenugreek supplementation did not result in a significant effect on weight and BMI in all subgroups analyzed (Table 3). 3.5. Sensitivity analysis

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Findings did not show significance after the sensitivity analysis for weight, BMI, HDL-c and TC. However, the sensitivity analysis for TG showed that the overall estimates were influenced by the elimination of the study conducted by Fedacko et al. (-13.78 mg/dL, 95% CI: -28.76, 1.18), Gupta et al. (-11.45 mg/dL, 95% CI: -24.62, 1.7), Shen et al. (-12.56 mg/dL, 95% CI: -25.67, 0.53), Bordia et al. (-13.37 mg/dL, 95% CI: -26.93, 0.19) and Chevassus et al. (-13.18 mg/dL, 95% CI: -26.67, 0.3). The exclusion of studies carried out by Fedacko et al. (-4.05 mg/dL, 95% CI: -9.28, 1.17), Gholaman et al. (-5.35 mg/dL, 95% CI: -12.03, 1.32) and Moosa et al. (-6.21 mg/dL, 95% CI: -13.14, 0.72) changed the overall effect size for TG to a non-significant value. 3.6. Publication bias There was no evidence of publication bias for studies examining the effects of fenugreek supplementation on weight (p = 0.592, Begg`s test), LDL-c (p = 0.621, Begg`s test) and HDL-c (p = 0.731, Begg`s test). However, other parameters showed significant publication bias based on visual inspection of funnel plots or results of publication bias tests for BMI (p = 0.037, Begg`s test) and TG (p = 0.038, Begg`s test). Because of the significance indicated from publication bias tests, we performed the trim and fill sensitivity analysis, which was calculated from hypothesized negative unpublished studies. The corrected effect size of “publication bias” was unchanged for BMI; trim and fill analysis was statistically significant as well (p < 0.001). Therefore, results would not be changed if other new studies were published regarding fenugreek effects on BMI. However, trim and fill analysis from 17 unpublished studies showed that TC values changed (16.62 mg/dl 95% CI: -22.63, -10.61). Furthermore, TG values could be changed if an additional 18 unpublished studies were added; the trim and fill analysis findings changed results to (-30.84 mg/dL 95% CI: -43.34, -18.34). For both TC and TG, the trim and fill analysis indicated a greater lowering effect of fenugreek. 15

3.7. Non-linear dose-responses between dose of fenugreek supplementation and lipid profile components Dose-response analysis showed that fenugreek supplementation changed LDL-c (r = 7.7, Pnonlinearity = 0.005) significantly based on dose in a non-linear fashion. This model predicts doses and has not been investigated yet based on available studies. Significant associations were not observed for other outcomes in non-linear dose-response analyses (Figure 8). 3.8. Meta-regression analysis Meta-regression using the random-effects model was undertaken to investigate the potential association between a decrease in lipid profiles and dose of fenugreek (g/day). Meta-regression analysis indicated a linear relationship between dose and absolute changes in TC (β = -0.382; p = 0.036) (Figure 9) and LDL-c (β = -0.376; p = 0.023) (Figure 10). 4. Discussion Our meta-analysis, which reviewed 12 RCTs (14 arms) with 560 participants, supports the use of fenugreek supplementation for improving plasma lipid profiles. Compared to the control group, fenugreek supplementation demonstrated beneficial effects on plasma TC, LDL-c, HDL-c and TG levels, but not BMI or body weight. However, the considerable heterogeneity among the trials considering TC, LDL-c, HDL-c, and TG levels introduced potential doubt in the generalizability of the overall result and required additional analyses, including meta-regression and subgroup analyses. In meta-regression, the random effects model revealed an inverse correlation between dosage of fenugreek and absolute changes in TC and LDL-c, indicating a linear dose-response relationship. However, a non-linear model also indicating a dose-response relationship may better describe the correlation between fenugreek and LDL-c.

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High statistical heterogeneity among the studies analyzed for TC, LDL-c, HDL-c and TG levels appear to be due to differences in health conditions of participants, study duration, and supplementation dosage. Subgroup analyses revealed that the effects of fenugreek on TC and TG were most pronounced among unhealthy subjects (dyslipidemic or T2DM), with higher doses of fenugreek, and when supplementation periods were longer, whereas the effects of fenugreek on HDL-c were most pronounced with high doses and longer supplementation periods. However, the effects of fenugreek on LDL-c were only present with high doses of supplementation. Theoretical evidence has suggested promise for fenugreek’s anti-obesity properties (Marrelli et al., 2016). Our results related to BMI and weight did not support the theoretical evidence. Taken together, fenugreek significantly improved lipid profiles, with the most notable effects among TC and HDL-c. The previous Meta-analyses primarily investigated hypoglycemic effects of fenugreek. As such, one previous meta-analysis only included two studies (Suksomboon et al., 2011), showing inconsistent results among different populations. In an additional meta-analysis with 10 RCTs, fenugreek seed supplementation decreased fasting blood glucose, 2-hour postload glucose, and HbA1c, but considerable heterogeneity was found, and medium to high doses (range: 5–25 g) of fenugreek seed supplements were the most efficacious (Neelakantan et al., 2014). In the present study, we found evidence for a linear dose-response association between the fenugreek dose and the lowering effects on TC and LDL-c. While different strategies like drugs and lifestyle modifications are commonly recommended for lowering LDL-c, lower levels of LDL-c alone may not guarantee decreased risk of CVD (Han et al., 2016). However, with an increase in fasting TC of 1.0101 mmol/L, the risk of ischemic heart disease increased 2.8 fold (Varbo et al.,

17

2013). Since fenugreek shows promise in reducing TC and LDL-c, with a dose-response effect, fenugreek could be a promising agent for reducing CVD risk. The mechanisms that promote the hypolipidemic effects of fenugreek are not unequivocally elucidated; however, evidence has suggested that the sapogenin, pectin, and phytosterol components of fenugreek may contribute significantly to the overall hyperlipidemic effects (Sauvaire et al., 1991; Stark and Madar, 1993). Sapogenins, or the non-saccharide portion of saponins, enhance the excretion of biliary cholesterol in the liver, and this can further decrease serum cholesterol (Sauvaire et al., 1991). On the other hand, mucilage, tannins, pectin and hemicellulose from fenugreek bind to bile acids to reduce cholesterol and fat absorption. In fact, they block bile salt absorption in the colon (Ahmad et al., 2016; Al-Sultan and El-Bahr, 2015). Furthermore, pectin absorbs bile acids, and may therefore decrease triglyceride levels (Stark and Madar, 1993). Additionally, the estrogenic component in fenugreek may increase thyroid hormone T4 indirectly, thereby influencing lipid levels (Smith, 2003). Moreover, fenugreek also contains phytosterols (Ciftci et al., 2011), which can also positively affect blood TC and LDL-c (AbuMweis et al., 2014; Ghaedi et al., 2019a). Phytosterols act in the intestines and work as cholesterol analogs. They compete for cholesterol in absorptive micelles, resulting in reduced solubility of cholesterol (Ghaedi et al., 2019b; Ostlund Jr, 2007). Fenugreek is also particularly rich in phenolic compounds, including flavonoids, phenols and resveratrol (Benayad et al., 2014; Li et al., 2018; Rahmani et al., 2018). Primary and secondary metabolites, especially phenolic compounds(Cicerale et al., 2010), and larger amounts of fiber and complex carbohydrates (especially based on galactose and mannose) (Wani and Kumar, 2018), could explain the hypocholesterolemic effects. 4.1. Safety 18

None of the included RCTs showed renal or hepatic toxicity after supplementation with fenugreek. However, some mild gastrointestinal side effects have been reported, including dyspepsia, nausea, diarrhea, and mild abdominal distension (Gupta et al., 2001a; Neelakantan et al., 2014). All side effects were improved without withdrawal of fenugreek or any other additional treatment. While benefits of fenugreek were seen in this meta-analysis, some limitations exist with the current trials. Although random allocation was evident in all included studies, several studies did not report the method for allocation, thus adding potential bias. High bias due to inappropriate blinding of participants/personnel as well as outcome assessment was discovered within two studies in our analysis. Publication bias was indicated by funnel plots and other tests, but the trim and fill sensitivity analysis indicated more significant lowering effects for TC and TG than originally calculated. Additionally, some studies in our review had relatively small sample sizes (~5 to 10 subjects) and/or short durations (~2 weeks), requiring further investigation. Therefore, additional studies with sufficient sample sizes and study durations, especially among hyperlipidemic individuals, should be conducted to verify the results found in this meta-analysis. Based on what was found in the current study, future research should also consider whether the dosage they choose is suitable for their chosen population, and effective doses should be studied through large-scale dose-escalating trials. 5. Conclusion In conclusion, the hyperlipidemic effects of fenugreek are supported by the current body of research with potential mechanisms of action suggested. Although pooled analysis showed demonstrable improvements in TG, TC, HDL-c, and

19

LDL-c following fenugreek

supplementation, these findings must be interpreted with caution, as subgroup analyses showed different effects in subgroups, such as those with differing health status. On the other hand, differences in populations also added heterogeneity. To better understand where fenugreek is most effective, additional studies should be conducted. The results in this study indicate that fenugreek is likely to be most effective in unhealthy populations given larger doses for longer supplementation durations. Acknowledgments None. Conflict of interest The authors declare no conflict of interest. Funding source This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution A.H and E.Gh carried out the concept, design, and drafting of this study. M.A searched databases, screened articles and extracted data. A.H and E.Gh performed the acquisition, analysis, and interpretation of data. M.C and K.V. prepared the primary manuscript; A.H and M.C. critically revised the manuscript. All authors approved the final version of the manuscript. E.Gh

and

A.H

are

the

guarantors

20

of

this

study.

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Ranasinghe, P., Mathangasinghe, Y., Jayawardena, R., Hills, A., Misra, A., 2017. Prevalence and trends of metabolic syndrome among adults in the asia-pacific region: a systematic review. BMC public health 17(1), 101. Reddy, R., Srinivasan, K., 2009. Fenugreek seeds reduce atherogenic diet-induced cholesterol gallstone formation in experimental mice. Canadian journal of physiology and pharmacology 87(11), 933-943. Roberts, K.T., 2011. The potential of fenugreek (Trigonella foenum-graecum) as a functional food and nutraceutical and its effects on glycemia and lipidemia. Journal of medicinal food 14(12), 1485-1489. Sauvaire, Y., Ribes, G., Baccou, J.-C., Loubatieres-Mariani, M.-M., 1991. Implication of steroid saponins and sapogenins in the hypocholesterolemic effect of fenugreek. Lipids 26(3), 191-197. Sharma, R., Raghuram, T., 1990. Hypoglycaemic effect of fenugreek seeds in non-insulin dependent diabetic subjects. Nutrition research 10(7), 731-739. Shen, L., Li, J., Lu, F., Yu, H., Zheng, N., 2013. Clinical study on diabetic foot treated with fenugreek extract. Chin. J. Trad. Med. Traumatol. Orthop. 21(2), 8-9. Smith, M., 2003. Therapeutic applications of fenugreek. Alternative Medicine Review 8(1), 20-27. Srinivasan, K., 2006. Fenugreek (Trigonella foenum-graecum): A review of health beneficial physiological effects. Food reviews international 22(2), 203-224. Srinivasan, K., 2019. Fenugreek (Trigonella foenum-graecum L.) Seeds Used as Functional Food Supplements to Derive Diverse Health Benefits, Nonvitamin and Nonmineral Nutritional Supplements. Elsevier, pp. 217-221. Stark, A., Madar, Z., 1993. The effect of an ethanol extract derived from fenugreek (Trigonella foenumgraecum) on bile acid absorption and cholesterol levels in rats. British Journal of Nutrition 69(1), 277287. Suksomboon, N., Poolsup, N., Boonkaew, S., Suthisisang, C.C., 2011. Meta-analysis of the effect of herbal supplement on glycemic control in type 2 diabetes. Journal of Ethnopharmacology 137(3), 1328-1333. Tobias, A., 1999. Assessing the influence of a single study in the meta-anyalysis estimate. Stata Technical Bulletin 8(47). Varbo, A., Benn, M., Tybjærg-Hansen, A., Jørgensen, A.B., Frikke-Schmidt, R., Nordestgaard, B.G., 2013. Remnant cholesterol as a causal risk factor for ischemic heart disease. Journal of the American College of Cardiology 61(4), 427-436. Venkatakrishnan, K., Chiu, H.-F., Wang, C.-K., 2019. Popular functional foods and herbs for the management of type-2-diabetes mellitus: A comprehensive review with special reference to clinical trials and its proposed mechanism. Journal of Functional Foods 57, 425-438. Waltenberger, B., Mocan, A., Šmejkal, K., Heiss, E., Atanasov, A., 2016. Natural products to counteract the epidemic of cardiovascular and metabolic disorders. Molecules 21(6), 807. Wani, S.A., Kumar, P., 2018. Fenugreek: A review on its nutraceutical properties and utilization in various food products. Journal of the Saudi Society of Agricultural Sciences 17(2), 97-106. Xue, W.-L., Li, X.-S., Zhang, J., Liu, Y.-H., Wang, Z.-L., Zhang, R.-J., 2007. Effect of Trigonella foenumgraecum (fenugreek) extract on blood glucose, blood lipid and hemorheological properties in streptozotocin-induced diabetic rats. Asia Pac J Clin Nutr 16(Suppl 1), 422-426. Yousefi, E., Zareiy, S., Zavoshy, R., Noroozi, M., Jahanihashemi, H., Ardalani, H., 2017. Fenugreek: A therapeutic complement for patients with borderline hyperlipidemia: A randomised, double-blind, placebo-controlled, clinical trial. Advances in integrative medicine 4(1), 31-35.

23

Legends of figures: Figure 1. PRISMA flow diagram of study selection process Figure 2. Forest plot of the effect of fenugreek supplementation on TC Figure 3. Forest plot of the effect fenugreek supplementation on LDL- c Figure 4. Forest plot of the effect of fenugreek supplementation on HDL- c Figure 5. Forest plot of the effect of fenugreek supplementation on TG Figure 6. Forest plot of the effect of fenugreek supplementation on BMI Figure 7. Forest plot of the effect fenugreek supplementation on weight Figure 8. Non-linear dose-response relations between fenugreek supplementation and absolute (unstandardized) mean differences of LDL-c Figure 9. Random-effects meta-regression plots of the association between dose of fenugreek and weighted mean difference of TC. Meta-regression analysis indicated a linear relationship between dose of fenugreek (g/day) and absolute changes in TC (β = -0.382; p = 0.036). There was significant decreasing trend in TC along with higher fenugreek doses. Figure 10. Random-effects meta-regression plots of the association between dose of fenugreek and weighted mean difference of LDL-c. Meta-regression analysis indicated a linear relationship between dose of fenugreek (g/day) and absolute changes in LDL-c (β = -0.376; p = 0.023). There was significant decreasing trend in LDL along with higher fenugreek doses.

24

Table 1. Characteristics of the 12 included studies. Author, Publication Year

Location

Sample size Mean age (intervention) (range)

Bordia et al., 1997 Chevassus et al., 2009 Chevassus et al., 2009-A Chevassus et al., 2009-B Fedacko et al., 2016

India France France France India

30 18 12 12 29

NR 38 22 22 45.8

Mean BMI Gender Intervention/Control (Kg/m2) NR 27.3 22.5 22.5 24.3

NR M M M M/F

FSP/ PB H/ PB H/ PB H/ PB DFSP/ PB

Daily dose (g) 5 1.176 0.588 1.176 60

Duration (week)

Patient features

13.5 6 2 2 12

T2DM Overweight Healthy Healthy Hypercholesterolemi a Pre T2DM

Design Outcomes

RP RP RC RC RP

TC, HDL-c, TG TC, LDL-c, HDL-c TC, HDL-c, TG TC, HDL-c, TG TC, LDL-c, HDL-c, TG Gaddam et al., 2015 India 52 (30-70) 26.6 M/F NG+EX+ DFSP/ NG+EX 10 162 RP TC, HDL-c, LDL-c, TG Gholaman et al., 2018-A Iran 10 NR 33.1 F FSP/ PB 15 8 T2DM RP TC, LDL-c, HDL-c, TG Gholaman et al., 2018-B Iran 10 NR 32.5 F EX + FSP/ EX 15 8 T2DM RP TC, LDL-c, HDL-c, TG Gupta et al., 2001 India 12 49.1 26.5 M/F H/ PB 1 9 T2DM RP TC, LDL-c, HDL-c, TG Bangladesh 15 (30-65) NR M/F FSP/ control 50 6 T2DM RP TC, LDL-c, HDL-c, Moosa et al., 2006 TG Rafraf et al., 2014 Iran 44 40.8 28 M/F FSP/ PB 10 8 T2DM RP TC, LDL-c, HDL-c, TG Sharma et al., 1990 India 5 42 NR NR FSP/ control 100 20 T2DM RC TC, TG Shen et al., 2013 China 17 (41-67) NR M/F TFGs/ PB 6.3 12 T2DM RP TC, TG Yousefi et al., 2016 Iran 24 37.2 27.4 M/F FSP/ PB 8 8 Hyperlipidemia RP TC, LDL-c, HDL-c, TG BMI, body mass index; DFSP, defatted fenugreek seed power; EX, exercise training; F, female; FSP, fenugreek seed power; H, Hydro-alcoholic extract of fenugreek seeds; HD, hypo-caloric diet; HDL-c, high density lipoprotein cholesterol; LDL-c, low density lipoprotein cholesterol; M, male; NG, nutritional guidance; NR, not reported; PB, placebo; TC, total cholesterol; TFGs, fenugreek total saponins; TG, triglyceride; T2DM, type2 diabetes mellitus.

Table 2. Cochrane risk of bias among included studies Author, year

Sequence generation

Allocation concealment

Blinding of participants and personnel Bordia et al., 1997 U U H Chevassus et al., 2009 U U L Chevassus et al., 2009 U U L Fedacko et al., 2016 U U L Gaddam et al., 2015 L L L Gholaman et al., 2018 U U L Gupta et al., 2001 U U L Moosa et al., 2006 U U H Rafraf et al., 2014 L L L Sharma et al., 1990 U U U Shen et al., 2013 L U U Yousefi et al., 2016 L L L L, low risk of bias; H, high risk of bias; U, unknown risk of bias

Blinding of outcome assessment H L L L L L L H L U U L

Incomplete outcome data L L L L L L L L L L L L

Selective outcome reporting L L L L L L L L L L L L

Other potential threats to validity U U U U U U U U U U U U

Table 3. Subgroup analysis to assess the effect of fenugreek supplementation on body mass index, body weight and lipid profile. Sub grouped by

BMI Total Dose of Intervention ≤10 g/day >10 g/day Body Weight Total Intervention Duration (Weeks) < 8 weeks ≥ 8 weeks Health Status Healthy Unhealthy Dose of Intervention ≤10 g/day >10 g/day TC Total Intervention Duration < 8 weeks ≥ 8 weeks Health Status Healthy Unhealthy Dose of Intervention ≤10 g/day >10 g/day Diabetes Status DM

No. of trials

WMD (95% CI)

9

0.091

-0.244

0.426

P Value

P for heterogeneity

I2 (%)

0.596

0.578

0

P for between subgroup heterogeneity

0.820 5 4

0.056 0.134

-0.392 -0.370

10

0.223

0.806 0.602

0.181 0.955

36.0 0

0.551

0.984

0

4 6

0.143 0.274

-1.029 -0.664

1.315 1.211

0.811 0.567

0.987 0.817

0 0

3 6

0.100 0.159

-2.133 -0.629

2.333 0.947

0.930 0.693

1 0.941

0 0

6 4

0.196 0.232

-1.292 -0.610

1.683 1.073

0.797 0.589

0.919 0.815

0 0

13

-9.371

0.002

0.000

87.6

-0.509

0.504 0.639 0.955

0.865

0.562

0.967

-15.419

-3.323

0.000 4 9

-1.551 -12.182 9.079 -13.022 -20.810 -5.233

0.775 0.001

0.008 0.000

74.6 86

3 10

3.457 -4.543 11.458 -12.910 -19.126 -6.693

0.397 0.000

0.777 0.000

0.0 87.2

9 4

-5.101 -15.994 5.791 -16.581 -25.855 -7.307

0.359 0.000

0.000 0.000

88 87.4

8

-11.010 -17.543

0.001

0.000

86.6

0.000

0.010

0.000 -4.476

Healthy

3

3.457

8

-6.590

-4.543

11.458

0.397

0.777

0

0.045

0.000

81.1

LDL-c Dose of Intervention ≤10 g/day >10 g/day HDL-c Total Intervention Duration < 8 weeks ≥ 8 weeks Health Status Healthy Unhealthy Dose of Intervention ≤10 g/day >10 g/day Diabetes Status DM Healthy TG Total Intervention Duration < 8 weeks ≥ 8 weeks Health Status Healthy Unhealthy Dose of Intervention ≤10 g/day >10 g/day Diabetes Status DM

-13.042

-0.137

0.000 4 4

-0.290 -1.539 0.960 -14.820 -20.819 -8.822

0.650 0.000

0.801 0.230

0 30.3

12

3.501

0.002

0.000

88.5

5 7

2.817 4.111

1.666 0.472

3.968 7.749

0.000 0.027

0.908 0.000

0 93.7

3 9

2.723 3.692

-0.809 1.215

6.254 6.170

0.131 0.003

0.794 0.000

0 91.6

8 4

2.396 4.898

-1.932 2.260

6.725 7.536

0.278 0.000

0.000 0.000

88.2 91.2

7 3

4.598 2.723

0.842 -0.809

8.354 6.254

0.016 0.131

0.000 0.794

92.7 0.0

13

-13.776

0.036

0.000

96.0

1.309

5.692

0.703

0.955

0.116

0.004

-26.636

-0.916

0.000 5 8

-8.168 -22.284 5.948 -17.523 -34.551 -0.495

0.257 0.044

0.005 0.000

73.2 97.1 0.000

3 10

-2.974 -28.304 22.357 -16.914 -31.181 -2.647

0.818 0.020

0.011 0.000

77.9 96.5 0.000

9 4

-17.619 -37.334 2.097 -8.600 -16.386 -0.814

0.080 0.030

0.000 0.001

90.5 81.5

8

-16.914 -31.181

0.020

0.000

96.5

0.000 -2.647

Healthy

3

-2.974 -28.304

22.357

0.818

0.011

77.9

BMI, body mass index; HDL-c, high density lipoprotein cholesterol; LDL-c, low density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride, DM: Diabetes Mellitus

Records screened (n = 648)

Full-text articles assessed for eligibility (n = 32)

Identification

Screening

Exclusion based on duplicate records (n = 431)

Eligibility

Searching

Records identified (n = 1079) through PubMed (n = 234), ISI (n = 259), Scopus (n = 485) and Cochrane Library (n = 101) searching

Figure 1

Records excluded based on title and abstract searching (n = 616)

• • •

Unrelated title (n = 381) Animal studies (n = 187) Other reason (review, letter, short survey and note) (n = 52)

Full text (n = 20) was excluded due to • Inadequate information (n = 10) • Intervention contained other components (n = 7) • Same population (n = 3)

Studies included in quantitative synthesis (n = 12)

%

Study ID

WMD (95% CI)

Weight

Bordia et al. (1997)

-13.50 (-28.08, 1.08)

6.79

Gupta et al. (2001)

-0.60 (-19.44, 18.24)

5.37

Moosa et al. (2006)

-11.65 (-15.32, -7.98)

10.78

Chevassus et al. (2009)

7.33 (-7.88, 22.54)

6.57

Chevassus et al. (2009)

3.90 (-9.40, 17.20)

7.28

Chevassus et al. (2009)

0.05 (-13.25, 13.35)

7.28

Shen et al. (2013)

-54.14 (-96.93, -11.35)

1.70

Rafraf et al. (2014)

-20.10 (-21.75, -18.45)

11.14

Gaddam et al. (2015)

6.40 (-3.90, 16.70)

8.47

Fedacko et al. (2016)

-33.14 (-41.39, -24.89)

9.29

Youse? et al. (2016)

-2.44 (-16.96, 12.08)

6.82

Gholaman et al. (2018)

-10.10 (-16.41, -3.79)

10.01

Gholaman et al. (2018)

-12.50 (-22.69, -2.31)

8.51

Overall (I-squared = 87.6%, p = 0.000)

-9.37 (-15.42, -3.32)

100.00

NOTE: Weights are from random effects analysis -96.9

Figure 2

0

96.9

Study

%

ID

WMD (95% CI)

Weight

Gupta et al. (2001)

-0.60 (-16.18, 14.98)

8.74

Moosa et al. (2006)

-10.10 (-23.99, 3.79)

9.77

Rafraf et al. (2014)

-0.38 (-1.66, 0.90)

17.71

Gaddam et al. (2015)

0.90 (-6.85, 8.65)

14.18

Fedacko et al. (2016)

-23.59 (-33.61, -13.57)

12.47

Youse? et al. (2016)

5.97 (-7.04, 18.98)

10.33

Gholaman et al. (2018)

-14.10 (-22.29, -5.91)

13.85

Gholaman et al. (2018)

-10.30 (-19.67, -0.93)

12.95

Overall (I-squared = 81.1%, p = 0.000)

-6.59 (-13.04, -0.14)

100.00

NOTE: Weights are from random effects analysis -33.6

Figure 3

0

33.6

%

Study ID

WMD (95% CI)

Weight

Bordia et al. (1997)

1.60 (-1.87, 5.07)

8.91

Gupta et al. (2001)

0.85 (-6.81, 8.51)

4.78

Moosa et al. (2006)

3.00 (1.70, 4.30)

11.04

Chevassus et al. (2009)

3.10 (-1.19, 7.39)

7.98

Chevassus et al. (2009)

0.00 (-8.80, 8.80)

4.03

Chevassus et al. (2009)

3.86 (-4.94, 12.66)

4.03

Rafraf et al. (2014)

12.95 (9.52, 16.38)

8.97

Gaddam et al. (2015)

-3.10 (-5.50, -0.70)

10.09

Fedacko et al. (2016)

1.93 (1.14, 2.72)

11.32

Youse? et al. (2016)

-0.47 (-3.61, 2.67)

9.29

Gholaman et al. (2018)

6.10 (3.75, 8.45)

10.14

Gholaman et al. (2018)

10.00 (6.97, 13.03)

9.42

Overall (I-squared = 88.5%, p = 0.000)

3.50 (1.31, 5.69)

100.00

NOTE: Weights are from random effects analysis -16.4

Figure 4

0

16.4

Study

%

ID

WMD (95% CI)

Weight

Bordia et al. (1997)

-18.70 (-36.90, -0.50)

8.16

Gupta et al. (2001)

-62.40 (-106.40, -18.40)

4.55

Moosa et al. (2006)

-12.80 (-20.96, -4.64)

9.38

Chevassus et al. (2009)

-21.26 (-43.33, 0.81)

7.57

Chevassus et al. (2009)

17.71 (2.47, 32.95)

8.58

Chevassus et al. (2009)

-8.86 (-34.13, 16.41)

7.08

Shen et al. (2013)

-48.72 (-105.64, 8.20)

3.35

Rafraf et al. (2014)

-39.86 (-42.11, -37.61)

9.72

Gaddam et al. (2015)

5.40 (-16.59, 27.39)

7.59

Fedacko et al. (2016)

-16.82 (-22.31, -11.33)

9.58

Youse? et al. (2016)

-6.48 (-43.00, 30.04)

5.46

Gholaman et al. (2018)

-3.06 (-9.35, 3.23)

9.53

Gholaman et al. (2018)

-1.45 (-8.66, 5.76)

9.46

Overall (I-squared = 96.0%, p = 0.000)

-13.78 (-26.64, -0.92)

100.00

NOTE: Weights are from random effects analysis -106

Figure 5

0

106

Study

%

ID

WMD (95% CI)

Weight

Ghattas et al. (2008)

0.10 (-1.12, 1.32)

7.55

Ghattas et al. (2008)

0.30 (-0.84, 1.44)

8.62

Lu et al. (2008)

0.15 (-0.99, 1.29)

8.59

Hassanzadeh et al. (2013)

1.85 (0.32, 3.38)

4.81

Gaddam et al. (2015)

-0.15 (-1.07, 0.77)

13.15

Kaur et al. (2016)

-0.28 (-1.05, 0.49)

18.87

Youse? et al. (2016)

0.02 (-1.02, 1.06)

10.47

Gholaman et al. (2018)

-0.04 (-0.88, 0.80)

16.01

Gholaman et al. (2018)

0.27 (-0.70, 1.24)

11.93

Overall (I-squared = 0.0%, p = 0.578)

0.09 (-0.24, 0.43)

100.00

NOTE: Weights are from random effects analysis -3.38

Figure 6

0

3.38

Study

%

ID

WMD (95% CI)

Ghattas et al. (2008)

-0.10 (-2.01, 1.81) 14.67

Ghattas et al. (2008)

0.40 (-1.47, 2.27)

15.40

Chevassus et al. (2009)

0.10 (-3.36, 3.56)

4.47

Chevassus et al. (2009)

0.10 (-3.36, 3.56)

4.47

Poole et al. (2010)

0.10 (-5.34, 5.54)

1.81

Hassanzadeh et al. (2013)

2.50 (-1.73, 6.73)

3.00

Gaddam et al. (2015)

-0.15 (-2.94, 2.64) 6.89

Kaur et al. (2016)

-0.78 (-4.64, 3.08) 3.59

Gholaman et al. (2018)

-0.21 (-1.74, 1.32) 22.79

Gholaman et al. (2018)

0.77 (-0.76, 2.30)

22.91

Overall (I-squared = 0.0%, p = 0.984)

0.22 (-0.51, 0.96)

100.00

NOTE: Weights are from random effects analysis -6.73

Figure 7

0

6.73

Weight

20 10 0 -10 -20 -30 0

20

40

60

Dose (g/day) 95% CI Mean Difference (mg/dl)

Figure 8

predicted Mean Difference (mg/dl)

20 0 WMD -20 -40 -60 0

20

40 Dose (g/day)

Figure 9

60

10 0 WMD -10 -20 -30 0

20

40 Dose (g/day)

Figure 10

60