Role of Phytoestrogen-Rich Bioactive Substances (Linum usitatissimum L., Glycine max L., Trifolium pratense L.) in Cardiovascular Disease Prevention in Postmenopausal Women: A Systematic Review and Meta-Analysis

The aim of this report was to determine the impact of flaxseed, soy and red clover, and their bioactive substances on the lipid profile in postmenopausal women in cardiovascular diseases prevention. We used the following databases: MEDLINE (PubMed), EMBASE and the Cochrane Library. Meta-analysis indicates that the intake of flaxseed by postmenopausal women is associated with a statistically significant reduction in total cholesterol (TC) levels (weighted-mean difference (WMD) = −0.26; 95% confidence interval (95% CI): −0.38 to −0.13; p = 0.0001), low-density lipoprotein cholesterol (LDL-C) levels (WMD = −0.19; 95% CI: −0.30 to −0.08; p = 0.0006), and high-density lipoprotein cholesterol (HDL-C) levels (WMD = −0.06; 95% CI: −0.11 to −0.01; p = 0.0150). The effect of soy protein on the lipid profile showed a significant decrease in TC levels: WMD = −0.15; 95% CI: −0.25–0.05; p = 0.0048, LDL-C levels: WMD = −0.15; 95% CI: −0.25–0.05; p = 0.0067, as well as a significant increase in HDL-C levels: WMD = 0.05; 95% CI: 0.02–0.08; p = 0.0034. Changes in the lipid profile showed a significant reduction in TC levels after the use of red clover (WMD = −0.11; 95% CI: −0.18–−0.04; p = 0.0017) and a significant increase in HDL-C levels (WMD = 0.04; 95% CI: 0.01 to 0.07; p = 0.0165). This meta-analysis provides evidence that consuming flaxseed, soy and red clover can have a beneficial effect on lipids in postmenopausal women and suggest a favorable effect in preventing cardiovascular diseases.


Introduction
Cardiovascular disease (CVD) is collection of disorders affecting the vasculature of the heart, brain and peripheral tissues, and remains the leading cause of death globally [1,2]. The most common cause of CV is atherosclerosis, which is initiated by an inflammatory reaction of the vascular endothelium [3]. The origins of these endothelial lesions are still not fully explained, but involved factors include: chronic elevations in blood pressure [4]; prolonged hyperglycemia and the resulting formation of advanced glycation end-products [5]; The search was limited to papers published in English and was conducted up to December 2018. References to selected research and review articles related to the topic of the work were also searched in order to identify additional studies.
The initial selection included the analysis of the titles and/or abstracts of all citations. After an independent and double analysis of the full texts of selected works, a decision was then made to include or exclude them. In turn, works were qualified for meta-analysis and collection of data on the clinical and methodological characteristics of the described clinical trials and for statistical evaluation.
Randomized controlled trials (RCTs) were considered eligible for inclusion if they met all of the following criteria: parallel-group design, or crossover design that contained data for the first period; a comparison with a placebo or with a no-intervention group; a followup period was at least 3 months; post-menopausal women as participants; appropriate interventions using flaxseed, soy or red clover and the presentation of sufficient information on plasma-lipid levels at baseline and after supplementation, or the net change values in both study arms. The exclusion criteria were as follows: men or premenopausal women as participants, no control group in the study, lack of sufficient information, and a study duration of less than 12 weeks. The results were reported as graphics or percent changes, and as duplicated reports.

Data Extraction
The data were extracted by the lead author and subsequently reviewed by co-authors for accuracy. Eligible studies were reviewed and the following data were abstracted: first author's name; year of publication; study location (country); follow-up period of the study; study design; number of participants in the intervention and control group; health characteristics of the population (age, menopausal status, body mass index); daily amount of flaxseed, soy protein, soy isoflavones and red clover isoflavones taken in the active arm; and data on baseline and follow-up TC, LDL-C, HDL-C and TG plasma levels.

Quality Assessment and Bias Risk of the Trials
The Jadad Scale is an Oxford system for assessing the quality of a clinical trial, designed to determine the minimum level of studies included in a systematic review/meta-analysis. The test may receive values from 0 (low quality) to 5 points (highest quality) [52]. This meta-analysis included studies that had a relatively high Jadad score. To explain the possible presence of bias publications, Begg's rank correlation test (Kendall Tau) and Egger's weighted regression test were applied [53,54].

Statistical Analysis and Meta-Analysis
The meta-analysis included all intervention groups from multi-arm studies. Moreover, to avoid the duplication of data from the same people in surveys covering multiple time points, only one such point was taken into account.
The data in each study were presented as numbers of subjects (N) and the mean ± standard deviations (SD). When the standard error of the mean (SEM) was employed, the conversion to SD was made according to the formula: SD = SEM × √ N. If a 95% confidence interval (95% CI) was applied, SD conversion was: SD = sqrt (N) × (upper bound-lower bound)/(2u) (equal to 3.96). When the results from the studies were presented in mg/dL, they were converted into mmol/L using standard conversion factors (the value in mg/dL was multiplied by 0.02586 for TC, LDL-C and HDL-C, and by 0.01143 for TG).
The outcome measures were the differences in the mean (MD) of components of the lipid profile between baseline and the end values for both the intervention and control groups. The missing SDs of MD were imputed using the formula: SD = sqrt ((SD "initial")2 + (SD "final")2 − (SD "initial" × SD "final") × 2R), where R is the correlation coefficient; we took an R value = 0.40 [55,56]. The outcome measures were the differences in the mean (net change in mmol/L) of elements of the lipid profile between the baseline and the end values for both the intervention and control groups.
Summary outcomes measures were presented as the mean differences between the intervention and control groups. A random-effects model was used to calculate the weightedmean difference (WMD) and 95% confidence interval (CI) for each comparison, and the combined overall effect (p < 0.05 was considered statistically significant) according to Der-Simonian and Laird [57]. Cochrane Q and I 2 statistics were used to assess the heterogeneity. The I 2 test determined whether the variance across studies was correct and not a result of a sampling error. The percentage of total variation indicated the degree of heterogeneity; I 2 values of ≤25% were considered low; >25% as moderate; and ≥75% as high heterogeneity [58]. STATISTICA Medical Software v. 11.0 StatSoft, Krakow, Poland was used for all statistical analyses.

Results
In total, a number of citations potentially related to the topic of work based on the key words-red clover = 3107; soy = 8074; and flaxseed = 4828-were identified. Building upon the title and/or abstract, exclusions were 3069 for red clover; 7991 for soy; and 4784 for flaxseed due to a lack of connection with the topic of this work. Consequently, 165 potentially relevant clinical trials qualified for further detailed qualitative analysis in the full-text assessment: red clover = 38; soy = 83; and flaxseed = 44. Among these, 130 studies were also discarded due to the failure to meet all inclusion criteria. As a result, 42 randomized controlled trials for meta-analysis. Detailed information about the literature search and study selection and identification can be found in Figure 1.

Characteristics of Included Trials
The characteristics of selected randomized controlled studies assessing the influence of flaxseed, soy protein, soy isoflavones, and red clover on lipid profile in postmenopausal women are presented in Table 1. The meta-analysis included 42 studies published in English from 1998 to 2018 .

Characteristics of Included Trials
The characteristics of selected randomized controlled studies assessing the influence of flaxseed, soy protein, soy isoflavones, and red clover on lipid profile in postmenopausal women are presented in Table 1. The meta-analysis included 42 studies published in English from 1998 to 2018 .

Associations between Soy Isoflavones Alone (Preparation) and Lipid Profiles
A total of 13 studies were selected to analyze the effect of soy isoflavones on the lipid profile [81][82][83][84][85][86][87][88][89][90][91][92], among which the data from the Colacurici et al. [93] did not allow for the analysis of the effect of isoflavones on TC, while in the study by Dewell et al. [85], there were insufficient data on LDL-C. The results of the meta-analysis are shown in Figure 4

Associations between Red Clover and Lipid Profiles
The last analysis, presented in Figure 5, concerned the effect of red clover on the lipid profile, and included seven studies [94][95][96][97][98][99][100]. There was a significant reduction in TC levels after the use of red clover (WMD = −0.11; 95% CI: −0.18-−0.04; p = 0.0017) and a statistically significant increase in HDL-C levels (WMD = 0.04; 95% CI: 0.01 to 0.07; p = 0.0165). In the case of TC and HDL-C, no significant heterogeneity of the study effects was observed, and publication bias was not demonstrated. The p value of Begg's test was 0.4579 for TC and 0.6207 for HDL-C, while the p value of Egger's test was 0.3990 for TC and 0.5319 for HDL-C. In contrast, statistical analysis showed no significant changes in LDL-C levels after the use of red clover (WMD = −0.01; 95% CI: −0.13 to 0.10; p = 0.8230) and showed a slight decrease in TG levels, which was statistically insignificant (WMD = −0.05; 95% CI: −0.17-0.06; p = 0.3713). In the case of LDL-C and TG, the heterogeneity of the studies turned out to be high (I 2 = 49.57% and I 2 = 76.14%, respectively). The asymmetry tests showed no publication bias. The p value of Begg's test was 0.4527 for LDL-C and 0.4527 for TG, while the p value of Egger's test was 0.2560 for LDL-C and 0.6425 for TG.

Associations between Red Clover and Lipid Profiles
The last analysis, presented in Figure 5, concerned the effect of red clover on the lipid profile, and included seven studies [94][95][96][97][98][99][100]. There was a significant reduction in TC levels after the use of red clover (WMD = −0.11; 95% CI: −0.18-−0.04; p = 0.0017) and a statistically significant increase in HDL-C levels (WMD = 0.04; 95% CI: 0.01 to 0.07; p = 0.0165). In the case of TC and HDL-C, no significant heterogeneity of the study effects was observed, and publication bias was not demonstrated. The p value of Begg's test was 0.4579 for TC and 0.6207 for HDL-C, while the p value of Egger's test was 0.3990 for TC and 0.5319 for HDL-C. In contrast, statistical analysis showed no significant changes in LDL-C levels after the use of red clover (WMD = −0.01; 95% CI: −0.13 to 0.10; p = 0.8230) and showed a slight decrease in TG levels, which was statistically insignificant (WMD = −0.05; 95% CI: −0.17-0.06; p = 0.3713). In the case of LDL-C and TG, the heterogeneity of the studies turned out to be high (I 2 = 49.57% and I 2 = 76.14%, respectively). The asymmetry tests showed no publication bias. The p value of Begg's test was 0.4527 for LDL-C and 0.4527 for TG, while the p value of Egger's test was 0.2560 for LDL-C and 0.6425 for TG.

Study Limitations
Despite the results obtained in this systematic review and its meta-analysis, some limitations were found. Because of the lack of standardization in some of the study designs, such as the ingredients and doses of isoflavones and the durations and outcomes of the trials, it currently remains difficult to draw overall conclusions for all aspects of isoflavone intake. These limitations warrant further investigation with regard to the use of isoflavone in women's health. Study limitations can be also be found due to individual differences in the bioavailability of individual components of preparations as these were prepared in a variety of ways that were suitable for each study. Moreover, limitations were posed by potential publication bias, which is revealed via the asymmetry of the funnel plot and the Egger's model. Publication bias suggests that some small studies with negative findings may have been missed or unpublished. Additionally, effects on vascular function have hardly been studied and more studies are needed to better establish what the effect of flaxseed, soy, red clover are on heart and vascular function.

Conclusions
This meta-analysis provides evidence that consuming flaxseed, soy, and red clover can have a beneficial effect on lipids in postmenopausal women. Their consumption could provide an important strategy to control dyslipidemia, and therefore, natural products can be an alternative to medicaments for preventing CVD, which has some clinical relevance in anti-atherosclerotic therapy. Our data also suggest that future well-designed studies with large sample sizes and adequate durations are needed to fully investigate the effectiveness of flaxseed, soy, and red clover.