**A Randomized Placebo-Controlled Clinical Trial to Evaluate the Medium-Term E**ff**ects of Oat Fibers on Human Health: The Beta-Glucan E**ff**ects on Lipid Profile, Glycemia and inTestinal Health (BELT) Study**

**Arrigo F.G. Cicero 1,\*,**†**, Federica Fogacci 1,**†**, Maddalena Veronesi 1, Enrico Strocchi 1, Elisa Grandi 1, Elisabetta Rizzoli 1, Andrea Poli 2, Franca Marangoni 2,**‡ **and Claudio Borghi 1,**‡


Received: 6 February 2020; Accepted: 28 February 2020; Published: 3 March 2020

**Abstract:** The Beta-glucan Effects on Lipid profile, glycemia and inTestinal health (BELT) Study investigated the effect of 3 g/day oat beta-glucans on plasma lipids, fasting glucose and self-perceived intestinal well-being. The Study was an 8-week, double-blind, placebo-controlled, cross-over randomized clinical trial, enrolling a sample of 83 Italian free-living subjects, adherent toMediterranean diet, with a moderate hypercholesterolemia and a low cardiovascular risk profile. Beta-glucans reduced mean LDL-Cholesterol (LDL-C) levels from baseline by 12.2% (95%CI: −15.4 to −3.8) after 4 weeks of supplementation and by 15.1% (95%CI: −17.8 to −5.9) after 8 weeks of supplementation (*p* < 0.01 for both comparison and versus placebo). Between baseline and 4 weeks Total Cholesterol (TC) levels showed an average reduction of 6.5% (95%CI: −10.9 to −1.9) in the beta-glucan sequence; while non-HDL-C plasma concentrations decreased by 11.8% (95%CI: −14.6 to −4.5). Moreover, after 8 weeks of beta-glucan supplementation TC was reduced by 8.9% (95%CI: −12.6 to −2.3) and non-HDL-C levels by 12.1% (95%CI: −15.6 to −5.3). Decreses in TC and non HDL-C were significant also versus placebo (respectively *p* < 0.05 and *p* < 0.01 to both follow-up visits). Fasting plasma glucose and self-perceived intestinal well-being were not affected by both beta-glucan and placebo supplementation.

**Keywords:** beta-glucan; fiber; lipid profile; cholesterol; intestinal function

#### **1. Introduction**

The consumption of dietary supplements to control plasma cholesterol levels has become widespread in recent years, alongside the publication of several systematic reviews and meta-analyses showing the cholesterol lowering effect of some of these products, including fibers [1–3].

A comprehensive meta-analysis of 58 clinical trials and 3974 subjects has recently showed that oat beta-glucan significantly affects the serum concentrations of low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (non-HDL-C) and apolipoprotein-B (apo-B), concluding that the inclusion of oat-containing foods in the diet may be a valid strategy to prevent the onset of cardiovascular disease [2]. In particular, another meta-analysis presented a dose-response curve

between fibre intakes and the reduction of total serum cholesterol, estimating a mean reduction for total cholesterol (TC) and LDL-C levels of 0.045 mmol/L and 0.057 mmol/L respectively, for each gram of dietary fiber [1]. These effects can theoretically play a major preventive role among the general population, since each 1% reduction in TC or LDL-C corresponds to an equivalent 1% decrease in the risk of developing a coronary heart disease event over time [3].

The mechanisms underlying the lipid-lowering properties of dietary fiber are still not fully understood [4]. The ability of soluble dietary fiber to form viscous solutions that prolong gastric emptying, and inhibit the transport of triglycerides and cholesterol across the intestine is a plausible explanation of their capacity to reduce LDL-C levels [5]. The consequences of the increased viscosity of the luminal contents manifest via the amplification of the thickness of the water layer and in the decrease of cholesterol uptake from the intestinal lumen [6].

The contribution of beta-glucans from oat and barley to the maintenance of normal blood cholesterol levels and their efficacy in the reduction of blood cholesterol levels at a dosage of 3 g per day was formally recognized by the European Food Safety Authority (EFSA) following specific applications and health claims that were authorized in the European Union (Commission Regulation (EU) 432/2012) [7]. However, the available clinical trials investigated the short-term lipid-lowering effect of beta-glucan administration on relatively small population samples and rarely involved European subjects [8,9]

In this context, we deemed interesting to evaluate the effect of a proprietary formulation of beta-glucans, at the dosage of 3 g/day on fasting plasma lipids and glucose, as well as its tolerability, in an Italian sample of subjects with mild hypercholesterolemia.

#### **2. Materials and Methods**

#### *2.1. Study Design and Participants*

The Beta-glucan Effects on Lipid profile, glycaemia and inTestinal health (BELT) Study was a medium-term, double-blind, placebo-controlled, cross-over randomized clinical trial, which enrolled a sample of Italian free-living subjects with moderate hypercholesterolemia recruited from the Lipid clinic of the S. Orsola-Malpighi University Hospital, Bologna, Italy.

Participants were required to be aged between 20 and 65 years, with moderately high levels of TC (TC ≥ 5.17 mmol/L and ≤ 6.2 mmol/L) and LDL-C (LDL-C ≥ 3.36 mmol/L and ≤ 4.91 mmol/L) and an estimated 10-year cardiovascular risk <10%, as per the country-specific risk charts from the CUORE project [10]. Exclusion criteria included having previously experienced any vascular event, suffering from type 1 or type 2 diabetes, massive hypertriglyceridemia (TG > 4.52 mmol/L), alcoholism, obesity (body mass index (BMI) > 30 kg/m2), liver failure, renal failure (estimated glomerular filtration rate (eGFR) < 0.5 mL/s), irritable bowel syndrome, inflammatory bowel diseases and food allergies, as well as having been treated in the previous 2 months with fiber based dietary supplements and/or probiotics and/or lipid-lowering drugs or any other drugs potentially able to affect the lipid metabolism.

Participants were adhering to a standardized diet for four weeks before being randomized to receive adequate supplies of either the beta-glucan supplement or placebo, in order to complete the one of two 2-month treatment sequences. The crossover to the second treatment was preceded by a 4-week wash-out period. Finally, participants were asked to return for follow-up visits two and four weeks after stopping supplementation. The study timeline is described in detail in Figure 1.

At enrollment, subjects were instructed to follow the general indications of a Mediterranean diet, avoiding an excessive intake of diary and red meat derived products.

Once every two visits, subjects were provided with a food diary to record their 3-day intake (two week-days and one week-end day), which they were requested to complete and return during their subsequent visit. In particular, the food diaries were collected and the diets analyzed at the beginning and at the end of each treatment phase.

The analysis of diet composition was performed using a dedicated software (MètaDieta®) based on a large food database that is frequently updated with values from the main official Italian databases (INRAN/IEO 2008 revision/ADI). Data were handled in compliance with the company procedure IOA87.

The study was conducted in accordance with the Good Clinical Practices and fully complied with the ethical guidelines of the Declaration of Helsinki. All subjects received an informational document describing the study and signed a consent form for study participation. The study was approved by the Bologna University Ethical Committee (Code: BELT\_2016) and registered on www.clinicaltrial.gov (ID: NCT03313713).

**Figure 1.** The flow-chart summarizes the study timeline.

#### *2.2. Treatment*

At visit at the beginning of supplementation the enrolled subjects were provided with boxes containing 28 white sachets of 15 g each; the same amount of product was provided after four weeks and at the beginning and after four weeks of the second supplementation phase. The sachets contained either 3 g oat beta-glucan (The Oatwell™ based Beta Heart®, Herbalife) or an oat-based isocaloric placebo without beta-glucan. The tested products had a similar macronutrient composition (Total energy: 50 kcal; Lipids: 1.4 g; Carbohydrates: 4 g; Proteins: 2 g) and were indistinguishable in color and taste. In particular, the Oatwell™ composition per 100 gr included 22 gr of beta-glucan soluble fiber characterized by very high molecular weight polysaccharides (>2000 kDa) and high viscosity (Table 1) [11].

Randomization was performed centrally, by computer-generated codes, and blocks were stratified by sex and age. The study staff and the investigators were blinded to the group assignment, as well as all the enrolled volunteers.

For the entire duration of the study, the subjects were instructed to take the dietary supplement regularly, dissolving the contents of one sachet in a glass of water every day in the morning.

All unused sachets were retrieved for inventory, and product compliance was assessed by counting the number of the sachets returned at the time of specific clinic visits [12]. The acceptability of the food supplements was assessed by a 10-point visual analogue score (VAS).


**Table 1.** Nutritional composition per 100 g of OatWell® 22.

#### *2.3. Assessments*

#### 2.3.1. Clinical Data and Anthropometric Measurements

Subjects' personal history was evaluated taking particular attention to cardiovascular and metabolic diseases, dietary and smoking habits assessment (both evaluated with validated semi-quantitative questionnaires) [13], physical activity, and pharmacological treatments.

Waist circumference (WC) was measured at the end of a normal expiration, in a horizontal plane at the midpoint between the inferior margin of the last rib and the superior iliac crest. Height and weight were measured to the nearest 0.1 cm and 0.1 kg, respectively, with subjects standing erect with eyes directed straight wearing light clothes and with bare feet. BMI was calculated as body weight in kilograms, divided by height squared in meters (kg/m2).

#### 2.3.2. Blood Pressure Measurements

Systolic and diastolic blood pressure measurements were performed in each subject, supine and at rest, using a validated oscillometric device, with a cuff of the appropriate size applied on the right upper arm. To improve detection accuracy, three blood pressure (BP) readings were sequentially obtained at 1-min intervals. The first one was then discarded, and the average between the second and the third was recorded [14].

#### 2.3.3. Laboratory Data

The biochemical analyses were carried out on venous blood, withdrawn after overnight fasting (at least 12 h). Plasma was obtained by addition of disodium ethylenediaminetetraacetate (Na2EDTA) (1 mg/mL) and blood centrifugation at 3000 RPM for 15 min at room temperature.

Trained personnel performed laboratory analyses according to standardized methods [15], immediately after centrifugation, to assess TC, HDL-C, TG, Apo-B, apolipoprotein A1 (Apo-A1), fasting

plasma glucose (FPG), creatinine, estimated glomerular filtration rate (eGFR) and liver transaminases. LDL-C was obtained by the Friedewald formula. Non-HDL-C resulted from the difference between TC and HDL-C.

#### 2.3.4. Safety and Tolerability

Safety and tolerability were evaluated through continuous monitoring in order to detect any adverse event, clinical safety, laboratory findings, vital sign measurements, and physical examinations. A blinded, independent expert clinical event committee was appointed by the principal investigator in order to categorize the adverse events that could possibly be experienced during the trial as not related, unlikely related, possibly related, probably related, or definitely related to the study treatment.

Even if the fiber amount supplemented was low to modify the bowel function, an *ad-hoc* semi-quantitative questionnaire (Intestinal function assessment (Supplementary Material)) was administered at each visit, in order to collect information regarding possible changes in self-perceived intestinal well-being during the different phases of the study. The questionnaire evaluated the number of daily evacuations, the stool consistency and the personal perception of discomfort, swelling, stool expulsion ease and complete expulsion [16].

#### 2.3.5. Statistical Analyses

Sample size was calculated for the change in LDL-C. Considering a type I error of 0.05, a power of 0.80 and expecting a LDL-C reduction of 7–10% with beta-glucans and 0–3% with placebo, expecting a 20% dropout rate, we calculated to enroll 80 subjects.

Data were analyzed using intention to treat by means of the Statistical Package for Social Sciences (SPSS) version 22.0 (IBM Corporation, Armonk, NY, USA) for Windows.

A full descriptive analysis of the collected parameters was carried out as per protocol. Categorical variables were expressed as absolute number and percentage and compared with the Fisher corrected chi-square test or the Wilcoxon-Rank test, based on whether they were nominal or ordinal. Continuous variables were expressed as mean ± standard deviation (SD) or mean and standard error (SEM), and compared by analysis of variance (ANOVA) followed by post-hoc Tukey test or by Kruskal-Wallis non parametric analysis of variance followed by Dunn's pairwise test, depending on their statistical distribution (if it was normal or not). To verify the basic assumption of cross-over design, the presence of a carryover effect was excluded.

The minimum level of statistical significance was set to *p* < 0.05 two-tailed. The Dixon's Q test was always carried out to exclude the extreme values.

Efficacy analyses were performed considering the intention-to-treat (ITT) population, i.e., all subjects with at least one post-baseline control. A sensitivity analysis of the primary variable was also planned in the per-protocol population, i.e., all subjects without major protocol violations.

The primay efficacy outcome analysis was also repeated by gender, age group (20–40 vs. 40–65 years old) and weight group (body mass index <sup>&</sup>lt; 27 kg/m2 vs. <sup>≥</sup> 27 kg/m2).

Safety results were reported in all subjects who had assumed at least one dose of one study supplement.

#### **3. Results**

A total of 95 volunteers were screened, and 83 subjects underwent randomization from April through September 2017.

Subjects' characteristics at the screening visit are summarized in Table 2.

All enrolled subjects (men: 35, women: 48) successfully completed the trial according to the study design. The final distribution between men and women, when considering the treatment sequences, did not show any significant differences (*p* > 0.05). Furthermore, the study groups were well matched for all the considered variables at baseline.


**Table 2.** Pre-diet standardization parameters, expressed as mean ± SD.

HDL = High-Density Lipoprotein; LDL = Low-Density Lipoprotein; VLDL = Very-Low-Density Lipoprotein.

No statistically significant changes were observed in the dietary habits of the enrolled subjects from randomization until the end of the study, nor in total energy and macronutrient intake (Table 3).



During the placebo phase, the subjects did not experience any statistically significant change in the evaluated parameters (Table 4 and Table S1 (Supplementary Materials)).

On the contrary, the active supplementation reduced mean LDL-C levels from baseline by 12.2% (95%CI: −15.4 to −3.8) after 4 weeks and by 15.1% (95%CI: −17.8 to −5.9) after 8 weeks (*p* < 0.01 for both comparison), which corresponded to an absolute decrease of 0.59 mmol/L (95%CI: −0.8 to −0.39) at the end of the intervention period (Table S2 (Supplementary Materials)). Repeating the analysis by gender and predefined age and body mass index groups, after 8 weeks we observed a slight but significantly higher LDL-lowering effect of beta-glucans effect in women than in men (women: 16.3% (95%CI: −17.8 to −6.7) vs. men: 14.9% (95%CI: −14.1 to −5.9); *p* = 0.04), in younger subjects (16.4% (95%CI: −17.5 to −8.3) vs. older 14.7% (95%CI: −17.1 to −5.2)) while no difference has been detected as regards body mass index classes (*p* > 0.05).


**Table 4.** Anthropometric, hemodynamic, and blood chemistry parameters from the baseline/end of wash-out to the end of the placebo sequence, expressed as mean ± SD.

HDL = High-density lipoprotein; LDL = Low-density lipoprotein; VLDL = Very-low-density lipoprotein.

Considering the first-ranked secondary endpoints, the mean percentage change in TC levels between baseline and 4 weeks was a reduction of 6.5% (95%CI: −10.9 to−1.9) in the beta-glucan sequence, while non-HDL-C plasma concentrations decreased by 11.8% (95%CI: −14.6 to −4.5). Moreover, after 8 weeks of treatment, beta-glucan reduced TC by 8.9% (95%CI: −12.6 to −2.3), corresponding to an absolute decrease of 0.52 mmol/L (95%CI: −0.72 to −0.32), and non-HDL-C levels were reduced by 12.1% (95%CI: −15.6 to −5.3), which corresponded to 0.53 mmol/L (95%CI: −0.73 to −0.33) (Table S2 (Supplementary Materials)).

Descriptions and results of exploratory analyses of the other secondary endpoints, the effect on which were considered non-significant, are provided in Table 5.


**Table 5.** Changes in anthropometric, hemodynamic, and blood chemistry parameters from the baseline/end of washout to the end of the supplementation with beta-glucan, expressed as mean ± SD.

\* *p* < 0.05 versus baseline; ◦ *p* < 0.05 versus placebo; § *p* < 0.01 versus placebo. HDL = High-Density Lipoprotein; LDL = Low-Density Lipoprotein; VLDL = Very-Low-Density Lipoprotein.

After treatment discontinuation, both TC and LDL-C rapidly reversed to basal values. Lipid plasma levels assessed after 2 weeks of washout were comparable with those measured at baseline and not significantly different from those observed at the end of the wash-out period (Table 5).

The compliance with the treatment was almost complete (89%) during both treatment periods.

The tolerability profile of the beta-glucan supplement used was rated as acceptable by most subjects. During supplementation with beta-glucan, three subjects experienced moderate abdominal discomfort (reported as reversible abdominal cramps and diarrhoea) and one subjects experienced dysphagia. No one experienced adverse events regarding laboratory parameters during the trial. No volunteer discontinued the trial because of adverse events that occurred during the treatment and no effects reported in conjunction with use of the placebo.

In particular, the tested products did not exert any significant unvafourable effect on the self-perceived intestinal well-being. The results of the second-ranked secondary endpoint are summarized in Table 6.


**Table 6.** Pre-versus post-treatment effects on intestinal function of the tested products.

#### **4. Discussion**

Beta-glucans (1-3,1-4 beta-D-glucans) are polysaccharides naturally occurring in the cell wall of grains and cereals, especially barley and oats [17]. In our double-blind, placebo-controlled, cross-over randomized clinical trial, 3 g/day of oat beta-glucan were shown to safely reduce LDL-C, TC and non-HDL-C in a large sample of adults characterized by mild hypercholesterolemia and with a low cardiovascular risk profile. The trial failed to detect any significant change in FPG. On the other hand, no significant adverse effects have been assessed on the self-perceived intestinal wellbeing and of both the beta-glucan supplement and the placebo were considered accettable.

The observed effect on TC and LDL-C are larger (0.53 and 0.59 mml/L, on average, corresponding to 15.1% and 8.9% of baseline values respectively) than expected, based on the most recent meta-analysis and EFSA opinion, which estimate respectively a mean change in LDL-C of 0.3 mmol/L and 0.21 mmol/L (about 7–10% of baseline concentratios) [7,18]. The reasons of such better performance are not easily explained, but it should be considered the possibility that the tested formulation, to be dissolved in fluids before consumption, might have specific pharmaceutic properties able to enhance the efficacy of the fibers in binding cholesterol and/or its metabolites (i.e., bile salts) and/or dietary fat. However, the supply of oat beta-glucan in the form of beverages (where nutrients are in contact with free water) has been found to have, in general, a more regular effect on cholesterol reduction compared to more complex matrices [19]. Moreover, the beta-glucan used for the BELT study consists of very high molecular weight beta-glucan, which has been proved to be more effective in cholesterol lowering then that with medium and low molecular weight [11,20].

Furthermore, the observed effect might also be due to the characteristics of the considered population sample. In this regard, a reliable meta-regression analysis recently demonstrated a significant inverse association between baseline LDL-C levels and the extent of LDL-C reduction after supplementation with oat beta-glucan [2]. These results are of particular interest, also considering that the observed effect is larger than the one expected after the administration of most available lipid-lowering nutraceuticals [21,22].

Similarly, the lack of any significant effect on FPG in the trial might be due to the characteristics of the enrolled subjects, being all euglycemic. Actually, a recent meta-analysis found that oat beta-glucan intake was more effective in people with type 2 diabetes [23].

The lack of changes in metabolic parameters during supplementation with placebo may be attributed to both the enrollment in the setting of a lipid clinic (and consequently, presumably, of subject *a priori* more attentive to a healthy lifestyle) and to the run-in stabilization diet period preceding the treatment phase, during which possible dietary mistakes were corrected before the start of the trial. Thus, the results obtained with beta-glucan supplementation are more representative of what would be observed in a setting of clinical practice.

At the same time, the lack of effects of beta-glucans on intestinal function parameters in our study should be related to the Mediterranean diet pattern of the enrolled subjects and the exclusion from the enrolment of subjects affected by irritable bowel syndrome, inflammatory bowel diseases and food allergies, but also because of the low amount of supplemented fibers.

The observed results are largely supported by the mechanisms of action by wich beta-glucans can improve cholesterolemia. The LDL-C lowering effect of beta-glucans has mainly related to the their ability to act as dietary fibers, consequently entrapping bile acid micelles, impairing their ability to interact with luminal membrane transporters on the intestinal epitelium, thereby increasing the fecal cholesterol output. [24] The following decrease in bile acid level up-regulates the 7-alpha-hydroxylase expression in the liver, thus further contributing to LDL-C decrease. [11] This seems to be more evident with high molecular weight and high viscosity of the fibers [25], as the one we tested in our trial. More recent literature suggesta that a part of the LDL-C reducing effect of beta-glucans could be mediated by modulation of microbiota [26]. A part of the beta-glucans effects (i.e., the antinflammatory and immunomodulatory ones) seems to be dependent by the contact of beta-glucans with dectin-1 [27], but this could be less relevant for th metabolic activities.

The BELT Study has some relevant limitations. For instance, the observational period was not long enough to assess any adaptation phenomena possibly occurring by changes in the gut microbiota composition. For this reason, further longer-term clinical studies are needed to confirm if the dietary intake of beta-glucan enriched foods is able to maintain the observed positive metabolic effect over the time.

Secondly, we did not evaluate any marker of intestinal absorption during the study, since the aim of this study was a clinical and not a pharmacological evaluation of the effects of supplementation with beta-glucan-enriched dietary supplement in moderately hypercholesterolemic subjects.

However, the BELT Study is the first placebo-controlled clinical trial testing the effect of oat beta-glucan supplementation on a large sample of a South European population strictly adherent to the Mediterranean diet. This is of particular interest because the high fiber content of the Mediterranean diet might have theoretically reduced the effect of beta-glucan supplementation on serum lipids [28], while this actually did not occur. Moreover, the observation that both total and LDL-C concentrations tend to return to basal values after weeks of wash-out, highlights the importance of a regular and constant supplementation with beta-glucan to achieve clinically relevant results in the long term. Finally, the profile of tolerability of the product might suggest that compliance to long term treatment with beta-glucan formulation might be good.

#### **5. Conclusions**

In conclusion, the BELT study confirms the medium-term efficacy of supplementation with 3 g/day of beta-glucan in reducing LDL-C, TC and non-HDL-C in mild hypercholesterolemic subjects, even in the context of a Mediterranean setting.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/3/686/s1, Table S1: Changes in the collected parameters during supplementation with placebo in the overall study population, Table S2: Changes in the collected parameters during supplementation with beta-glucan in the overall study population.

**Author Contributions:** Conceptualization, A.P. and F.M. methodology, A.F.G.C. and F.M. formal analysis, A.F.G.C. and F.M.; investigation, A.F.G.C., F.F., M.V., E.S., E.G. and E.R. resources, F.M. and A.P. data curation, A.F.G.C., F.F. and F.M. writing-original draft preparation, A.F.G.C., F.F. and F.M. writing-review and editing, A.P. and C.B. supervision, A.P. and C.B. project administration, A.F.G.C. and C.B. funding acquisition, F.M. and A.P. All authors have read and agree to the published version of the manuscript.

**Funding:** The BELT study has been supported by a research grant from NFI to S. Orsola-Malpighi University Hospital. The NFI grant has been partially covered by an unrestricted contribution received from Herbalife Nutrition. Herbalife Nutrition had no role in the design of the study, in the collection, analyses of the data, in the preparation or approval of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **The Association between Dyslipidemia, Dietary Habits and Other Lifestyle Indicators among Non-Diabetic Attendees of Primary Health Care Centers in Jeddah, Saudi Arabia**

**Sumia Enani 1,2,\*, Suhad Bahijri 1,3, Manal Malibary 1,2, Hanan Jambi 1,2, Basmah Eldakhakhny 1,3, Jawaher Al-Ahmadi 1,4, Rajaa Al Raddadi 1,5, Ghada Ajabnoor 1,3, Anwar Boraie 1,6 and Jaakko Tuomilehto 1,7,8**


Received: 12 July 2020; Accepted: 12 August 2020; Published: 13 August 2020

**Abstract:** Diet and other lifestyle habits have been reported to contribute to the development of dyslipidemia in various populations. Therefore, this study investigated the association between dyslipidemia and dietary and other lifestyle practices among Saudi adults. Data were collected from adults (≥20 years) not previously diagnosed with diabetes in a cross-sectional design. Demographic, anthropometric, and clinical characteristics, as well as lifestyle and dietary habits were recorded using a predesigned questionnaire. Fasting blood samples were drawn to estimate the serum lipid profile. Out of 1385 people, 858 (62%) (491 men, 367 women) had dyslipidemia. After regression analysis to adjust for age, body mass index, and waist circumference, an intake of ≥5 cups/week of Turkish coffee, or carbonated drinks was associated with increased risk of dyslipidemia in men (OR (95% CI), 2.74 (1.53, 4.89) *p* = 0.001, and 1.53 (1.04, 2.26) *p* = 0.03 respectively), while the same intake of American coffee had a protective effect (0.53 (0.30, 0.92) *p* = 0.025). Sleep duration <6 h, and smoking were also associated with increased risk in men (1.573 (1.14, 2.18) *p* = 0.006, and 1.41 (1.00, 1.99) *p* = 0.043 respectively). In women, an increased intake of fresh vegetables was associated with increased risk (2.07 (1.09, 3.94) *p* = 0.026), which could be attributed to added salad dressing. Thus, there are sex differences in response to dietary and lifestyle practices.

**Keywords:** dyslipidemia; serum cholesterol; serum triglycerides; serum low density lipoprotein; serum high density lipoprotein; dietary intake; lifestyle

#### **1. Introduction**

Cardiovascular diseases (CVD) are major health problems contributing to 31% of global death in 2017 according to the World Health Organization (WHO) [1]. The prevalence of CVD in Saudi Arabia in 2004 has been reported to be 5.5% [2], accounting for almost 45.7% of deaths [3]. CVD has many modifiable and non-modifiable risk factors, including certain diseases and disorders such as diabetes, hypertension, and dyslipidemia, which commonly coexist in various populations [4–6]. Indeed, dyslipidemia, defined as any abnormalities in serum lipids is considered atherogenic, and is reported to be associated with an increased risk of ischemic heart disease [7,8]. Several studies have been conducted to investigate the prevalence of dyslipidemia in Saudi Arabia in the past reporting an overall prevalence of 20–54% [9–11]. A more recent study published in 2018 on people in the eastern region of Saudi Arabia reported that the prevalence of (diagnosed and borderline) hypercholesterolemia, hypertriglyceridemia, increased low-density lipoprotein (LDL-C)-cholesterol, and decreased high density lipoprotein (HDL-C)-cholesterol were 51%, 26.9%, 38.1%, and 90.5%, respectively [12].

Many factors contribute to the development of dyslipidemia, including genetics, sex, ethnicity, increased body mass index (BMI), dietary habits, and smoking [13]. In addition, changes in sleeping patterns, and a short duration of sleep have also been associated with dyslipidemia [14]. Studies in Saudi Arabia showed that age, sex, high BMI, and waist circumference (WC), smoking, low physical activity, as well as intake of margarine were associated with dyslipidemia [10–12]. In spite of their reported importance in controlling dyslipidemia and CVD [15,16], the association of dietary as well as other lifestyle practices, including sleeping duration, with dyslipidemia were not fully investigated in previous Saudi studies. Saudi Arabia is a very large country, with each region having its own dietary and lifestyle characteristics. Therefore, we aimed to investigate such associations in more detail in inhabitants of the city of Jeddah, the largest city in the western region, and the gateway to the two holy cities of Islam, with a population of mixed ethnicities, bringing with them their own dietary habits. We hope that our results will help in the formulation of evidence-based dietary and lifestyle guidelines for people in our region to decrease the prevalence of dyslipidemia, which can be adopted in future, more comprehensive programs for the prevention of CVD.

#### **2. Materials and Methods**

#### *2.1. Study Design and Sample Collection*

Presented data in this study were obtained between July 2016 and February 2017 from a cross-sectional survey conducted in the city of Jeddah to develop a Saudi Dysglycemia Risk Score. The Committee on the Ethics of Human Research at the Faculty of Medicine-King Abdulaziz University, Jeddah approved the study (Reference No. 338-10). A full explanation of the sampling methodology has been outlined in an earlier publication [17] and is summarized here as follows: adults (age ≥ 20 years), not previously diagnosed with diabetes, were recruited from attendees of primary health care centers (PHCC) in Jeddah, Saudi Arabia. An informed consent form was signed by consenting volunteers. Demographic, dietary, and lifestyle variables, as well as medical history, and family history of chronic diseases were collected from recruits using a predesigned questionnaire in the Arabic language which was based on validated questionnaires used in previous risk score studies [18–22] including an Arabic one [23]. The questionnaire was completed during a face to face interview by trained medical students. Anthropometric and clinical measurements (weight, height, WC, and blood pressure (BP)) were measured using standardized equipment and techniques as previously explained [17]. Weight and fat percentage were measured using a portable calibrated scale (Omron BF511; OMRON Healthcare, Kyoto, Japan). Weight and height were used to calculate BMI. A value less than 18.5 indicates underweight, while a value of 18.5–<25 kg/m<sup>2</sup> indicates healthy normal weight, 25–<30 indicates overweight, and ≥30 indicates obesity. Using WC to indicate abdominal adiposity the first cut-off value for increased risk was defined as >94 cm for men, >80 cm for women, and the second cut-off value as >102 cm for men, >88 cm for women [24,25].

The section on dietary practices consisted of food frequency questions including 17 questions covering intake of fruits, vegetables, red meat, whole grain bread/cereals, and various commonly consumed beverages including fruit juices (fresh and otherwise), carbonated beverages, energy drinks, different types of coffee such as Arabic, American, Turkish, and cappuccino, and various teas such as red, green, cinnamon, and hibiscus tea. Participants were asked to specify whether they consumed each item more than once daily, once daily, 5–6 portions per week, 1–4 portions per week, or not at all. Food models representing portion sizes of relevant food items were presented to all participants to help in making the correct decisions.

Fasting blood samples were taken, and serum was separated and stored at −80 ◦C for the estimation of the lipid profile. A flow chart outlining steps in data collection is presented in Supplementary Figure S1.

#### *2.2. Biochemical Assays*

Serum samples were sent regularly every 10 to 12 weeks to a collaborating laboratory at the National Guard Hospital in Jeddah which is accredited by the College of American Pathologist. Total cholesterol (TC), HDL-C and triglycerides (TG) levels were measured by spectrophotometric methods using Architect c8000 auto-analyzer (Abbott Park, IL, USA). LDL-C was calculated using the Friedewald equation [26].

#### *2.3. Diagnosis of Dyslipidemia*

Dyslipidemia was defined as LDL-C ≥ 3.37 mmol/L, HDL-C < 1.04 mmol/L for men and < 1.3 mmol/L for women, TC ≥ 5.18 mmol/L, TG ≥ 1.7 mmol/L or treatment with lipid-lowering drugs with all lipid levels in the normal range [27,28].

#### *2.4. Statistical Analysis*

IBM SPSS statistics version 20.0 for Windows was used to enter and analyze collected data. The baseline characteristics of the study population were calculated statistically and described as mean, standard deviations (SD), and frequencies.

Demographic, lifestyle, and clinical factors of people with high levels of TC, LDL-C, and TG, and/or low level of HDL-C, as well as dyslipidemia in general, were analyzed by comparing to those with normal lipid levels. Factors with continuous variables were analyzed using an independent t-test to compare two groups, while those with categorical variables were analyzed using the Chi-square test or Fisher's exact test, as appropriate. Unadjusted and adjusted logistic regression models were used for assessing the association between demographic, lifestyle, and dietary variables, and outcome variables: specific and general types of dyslipidemia. Stepwise regression analysis was performed to determine the dietary factors that had an influence on dyslipidemia. Independent factors included age, BMI, and WC. Only related independent variables, where *p* < 0.15 after an initial logistic regression between the dependent and independent variable was used in the corresponding stepwise regression model to avoid excluding important covariates from the final model and to include predictors that are serious or have high potential effect [29]. Values of *p* < 0.05 (two-sided test) were accepted as statistically significant.

#### **3. Results**

A total of 1477 adults were recruited by the end of February 2017 (Supplementary Figure S1). Complete data were obtained for 1385 people. Missing data were mainly due to missing, hemolysed, broken, or unlabeled blood samples. This was completely random and is not expected to affect validity. Following biochemical measurements, a total of 527 people (38%) (287 men, and 240 women) were found to be normolipidemic, and 858 (62%) (491 men, and 367 women) had dyslipidemia. Thus, there was no association between sex and dyslipidemia in general. The most prevalent type of dyslipidemia was high LDL-C found in 567 people (40.9%) (341 men and 226 men), followed by high

TC in 480 people (34.7%) (277 men and 203 women), and low HDL-C in 338 people (24.4%) (171 men and 167 women) and the least prevalent type of dyslipidemia was high TG levels in 301 people (21.7%) (218 men and 83 women). Therefore, increased LDL-C and TG were significantly more common in men (*p* < 0.05 and *p* < 0.001 respectively) whereas low HDL-C was significantly more common in women (*p* < 0.05).

#### *3.1. Association between Dyslipidemia with Anthropometric Measurements*

Demographic, clinical, and anthropometric characteristics of the study groups for men and women are presented in Supplementary Table S1. There was a significant difference in demographic and anthropometric measurements between the normolipidemic and dyslipidemic groups, with the dyslipidemia group having a significantly higher means of age, BMI, weight, body fat percentage, neck, waist and hip circumferences, and waist to hip and waist to height ratios (all *p* < 0.001, Supplementary Table S1). In addition, men and women with dyslipidemia also had significantly higher means of systolic and diastolic B*p* and significantly higher means of TC, TG, LDL-C, and lower HDL-C compared with people with normolipidemia (Supplementary Table S1). Comparing the distribution of various characteristics between the normolipidemic and the dyslipidemic groups of men and women, age, BMI, and WC were found to be significantly and directly associated with dyslipidemia (*p* < 0.001 at least, Supplementary Table S2) and all its types (*p* < 0.015 at least, Supplementary Table S2). Dyslipidemia was detected in 75% of participants over 30 years of age compared with 48.8% of those <30 years. Moreover, dyslipidemia was present in 74.8% and 72% of obese men and women compared with 53.5% and 43.7% of normal weight and 29.2% and 32% underweight men and women respectively (Supplementary Table S2a,b).

#### *3.2. Patterns of Food Intake in Studied Men and Women*

Recorded frequencies of the questionnaire were recategorized to no intake, 1–4 and 5 or more portions per week due to small numbers in some categories. The difference in the recorded pattern of food intake between men and women is shown in Table 1**.** Women reported eating more fresh and cooked vegetables than men, whereas men had a higher intake of red meat (*p* < 0.001 at least, Table 1). In addition, men reported drinking more fresh and non-fresh juice, soft drinks, energy drinks, red tea, and American coffee compared with women (*p* < 0.01 at least, Table 1) whereas women reported drinking more green tea, Arabic coffee, and cinnamon drink compared with men (*p* < 0.05 at least, Table 1). There were no sex-differences in the consumption of fruits, whole grain products, Turkish coffee, and hibiscus drink (Table 1).

The association between diet and dyslipidemia was analyzed in each sex group separately due to the sex differences in both the prevalence of some types of dyslipidemia as well as food intake patterns.

#### 3.2.1. Association between Food Intake and Dyslipidemia in Men

Comparing dietary habits in men between the two groups, a high intake of Turkish coffee was significantly associated with dyslipidemia (*p* < 0.001, Table 2). On the other hand, people with a moderate intake of 1–4 portions/week of American coffee had a lower prevalence of dyslipidemia (*p* = 0.027, Table 2). Dyslipidemia was detected in 79% of people drinking five or more portions a week Turkish coffee and in 65% of people who did not drink American coffee (Table 2). Turkish coffee consumption was associated in particular with low HDL-C and high TC (*p* = 0.036 and *p* = 0.018, respectively, Table 2). This association had a U-shape association as low HDL-C was detected in 22% in people with no intake, in 15% of those with an intake of 1–4 portions per week and in 29% in those with a weekly intake of five or more portions of Turkish coffee (Table 2). A similar U-shaped association was found between American coffee consumption and TC (Table 2).


**Table 1.** Food intake pattern by sex.


**Table 1.** *Cont*.

Data is shown as frequency and percentages of all people in the sex group. χ<sup>2</sup> is the Chi-square test value followed by its *p*-value. Significant differences between groups are shown in bold font.

After adjusting for age, BMI, and WC the weekly intake of five or more portions of Turkish coffee was associated in men with a 2.77 increased odds for having dyslipidemia compared with those with no intake (*p* < 0.001, Table 3), while a moderate and high intake of American coffee was associated with 0.55 and 0.55 decreased odds for having dyslipidemia compared with those with no intake (*p* = 0.037 and *p* = 0.031, respectively; Table 3). After performing the multivariable regression analysis for each type of dyslipidemia separately and adjusting for age, BMI, and WC, there was no longer a significant effect of Turkish coffee on any type; however, the effect of American coffee appeared only for moderate consumption (1–4 portions/week) which was only significant for high TC (OR = 0.50; 95% CI: 0.27, 0.94; *p* = 0.038) (data not shown).

There was no significant association between the consumption of carbonated drinks and dyslipidemia when analyzed by Chi-square test. However, the regression analysis showed that the weekly intake of five or more portions of carbonated drinks was associated with a 1.56 increased odds for having dyslipidemia in general (but not specific types of dyslipidemia) in men compared with those with no intake after adjusting for age, BMI, and WC (OR = 1.53 (CI: 1.04, 2.26) *p* = 0.03, data not shown).

There was a U-shaped association between some other dietary variables and dyslipidemia when analyzed by the Chi-square test. For example, fruits, fresh and cooked vegetables, and red tea consumption had a U-shaped relationship with low HDL-C in men. However, these food types were not predictors of dyslipidemia when analyzed using regression, adjusting for age and BMI (Table 3).



 and







Data are shown as frequency and percentages. χ<sup>2</sup> is the Chi-square test value followed by its *p*-value. Significant differences between groups are shown in bold font LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides.


**Table 3.** Unadjusted and adjusted Odds Ratios (OR) with its 95% Confidence Interval (CI) for dietary and lifestyle predictors of dyslipidemia in men.

Variables having a *p* > 0.15 in the initial logistic regression analysis between the dependent and independent variables were not included in the stepwise regression model. Significant differences between groups are shown in bold font.

#### 3.2.2. Association between Food Intake and Dyslipidemia in Women

Comparing dietary habits in women between the two groups of normolipidemia and dyslipidemia, a high intake of the cinnamon drink was significantly associated with dyslipidemia in general (*p* = 0.027) and high LDL-C (*p* = 0.036) and TC in specific (*p* = 0.002) (Table 4). Dyslipidemia was present in 77% of women drinking five or more portions a week cinnamon drink compared to 58% in women who do not. The weekly intake of five or more portions of the cinnamon drink was associated with 2.6 times increased odds for having dyslipidemia in women compared with those with no intake. However, after adjusting for age, BMI, and WC, these odds became insignificant (Table 5). The adjustment for age and BMI regression analysis for each type of dyslipidemia separately showed that the weekly intake of five or more portions of the cinnamon drink was associated with a 2.57 times increased odds for having high TC in women compared with those with no intake (OR = 2.57; 95% CI: 1.22, 5.42; *p* = 0.02), but this was not significant after adjustment for age, BMI, and WC.



 (83.3)

2.713 (*<sup>p</sup>* = 0.258)

 (94.3)

 (86.6)

 (84.8) 3.509 (*<sup>p</sup>* = 0.173)

 (87.9)

 (85.6)

 (86.7)

 0.311 (*<sup>p</sup>* = 0.856)

 (89.0)

 (83.5)

 (86.4)

 1.165 (*<sup>p</sup>* = 0.559)

 (87.8)

 (85.7)

 (86.5)

 0.369 (*<sup>p</sup>* = 0.831)

 18 (13.5)

 48 (14.3)

 17 (12.2)

 58 (13.6)

 15 (16.5)

 10 (11.0)

 35 (13.3)

 40 (14.4)

 8 (12.1)

 50 (15.2)

 30 (13.4)

 3 (5.7)

 36 (16.7)

 for







Data are shown as frequency and percentages. χ<sup>2</sup> is the Chi-square test value followed by its *p*-value. Significant differences between groups are shown in bold font. LDL-C, low-densitylipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides.


**Table 5.** Unadjusted and adjusted Odds Ratio (OR) with its 95% Confidence Interval (CI) for the predictors of dyslipidemia in women.

Variables having a *p* > 0.15 in the initial logistic regression analysis between the dependent and independent variables were not included in the stepwise regression model. Significant differences between groups are shown in bold font.

Moderate and high intakes of fresh vegetables were also found to be significantly associated with dyslipidemia (*p* < 0.001; Table 4) and mainly high LDL-C and TC (*p* = 0.034 and *p* = 0.008 respectively, Table 4). Dyslipidemia was diagnosed in 61% and 65% of women who ate either five or more portions of fresh vegetables/week or 1–4 times per week respectively compared to 38% of women who did not. This was associated with 2.50 and 2.07 times increased odds of having dyslipidemia, respectively, compared with those with no intake after adjusting for age, BMI, and WC (OR = 0.50 (95% CI: 1.29, 4.86) *p* = 0.007 and OR = 2.07 (95% CI: 1.09, 3.94) *p* = 0.026 respectively; Table 5).

Following the finding of this interesting results, further analysis was performed to identify the characteristics of females with high intake of fresh vegetables. Women who reported to have an intake of 1–4 and 5 or more portions per week were significantly older than those who reported no intake (*p* <0.01 at least). They also had a significantly higher mean weight and neck circumference (*p* <0.05 at least). In addition, a higher proportion of these women were previously diagnosed with dyslipidemia and taking medication, however, this was not statistically significant (Supplementary Table S3).

There was no effect of other food types on dyslipidemia in women when analyzed using regression analysis after adjusting for age, BMI, and WC.

#### *3.3. Association between Lifestyle Characteristics and Dyslipidemia in Men and Women*

After analyzing sex groups separately, only short and long sleep durations were associated with general dyslipidemia in men only (Supplementary Table S2a, *p* = 0.012). Dyslipidemia was detected in 68.5% and 68.2% of men who reported sleeping either <6 h/day, or >8 h/day respectively compared with 58.1% of people who slept 6–8 h per day. This association was particularly evident for increased TC (*p* < 0.029). There was no association between sleep duration and dyslipidemia in women.

There was no association between physical activity or daily sitting duration and dyslipidemia in general (Supplementary Table S2a,b). However, low physical activity was associated with high TC and TG in men (*p* = 0.024 and *p* = 0.048 respectively; Supplementary Table S2a). High TC and TG were detected in 39.1% and 30.9% of men reporting low physical activity compared with 31.3% and 24.5%, respectively, in men who were more physically active. In women, low physical activity was associated with low HDL-C only (*p* = 0.024; Supplementary Table S2b). Low HDL-C was detected in 41% of women reporting low physical activity compared with 32% of women who were more physically active. On the other hand, very short sitting duration (<4 h per day) was associated with decreased HDL-C in women as it was found in 36.3% of women reporting sitting for <4 h per day compared with 20–26% of those sitting for more than4h(*p* = 0.031, Supplementary Table S2b).

Smoking was not associated with dyslipidemia in general (Supplementary Table S2a,b). However, it was associated with high TG in men (*p* = 0.011; Supplementary Table S2a). High TG was detected

in 34.2% of men who smoke compared with 25.7% of non-smoking men. In women, smoking was associated with low HDL-C (*p* = 0.035; Supplementary Table S2b). Low HDL-C was detected in 50% of women who smoked compared with 36.1% of who were non-smokers.

After adjusting for age, BMI, and WC, only sleep duration and smoking were predictors for dyslipidemia in men (Table 3). Having <6 h of sleep per day was associated with increased odds of having general dyslipidemia compared with those sleeping 6–8 h (OR = 1.57 (CI: 1.14, 2.18) *p* = 0.006; Table 3). In addition, smoking was associated with increased odds of having general dyslipidemia compared with those who did not smoke (OR = 1.42 (CI: 1.00, 1.98) *p* = 0.043; Table 3).

Lifestyle characteristics were not predictors for dyslipidemia in women after adjusting for age, BMI, and WC.

#### **4. Discussion**

In this study, we investigated the association between dyslipidemia, dietary, and other lifestyle practices among Saudi adults not previously diagnosed with diabetes. Dyslipidemia was found to be associated with increased age, BMI, and WC in men and women. However, notable differences between sexes in the association of dyslipidemia with dietary and other lifestyle practices were noted. In men, an increased risk of dyslipidemia was found to be associated with high consumption of Turkish coffee, and carbonated drinks, short (<6 h) and long (>8 h) sleep duration, and smoking, while high consumption of American coffee was associated with decreased risk after adjusting for age, BMI and WC. However, only increased consumption of fresh vegetables was associated with an increased risk of dyslipidemia in women after adjusting for confounding factors.

The prevalence of dyslipidemia among Saudi adults in our study was 62% which is higher than previously reported [10,11]. The difference could be due to the variations in dietary, and lifestyle practices between regions of the Kingdom as well ethnic origin of studied populations since ethnicity among Saudi nationals can vary to some extent and influence genetic and lifestyle factors. Our study was conducted in the city of Jeddah in the Western Region of the Kingdom, while the previous study [10,11] included people from all the regions in Saudi Arabia. Another reason could be the variation in defining dyslipidemia and all its types. In this study, the used dyslipidemia definition was more comprehensive as it included laboratory-measured high TG, high fasting serum cholesterol, low fasting plasma HDL-C and/or high fasting plasma LDL-C levels, as well as treatment with hyperlipidemic drugs [27,28].

The most prevalent type of dyslipidemia in this study was high LDL-C which was detected in 40.9% of studied Saudi adults. This was followed by high TC in 34.7%, low HDL-C in 24.4%, and least of all high TG levels in 21.7% of the study population. A comparably high prevalence of high LDL-C and hypercholesterolemia in a Saudi population was reported in previous large community-based national cross-sectional studies [9–11]. However, one of these studies [9] reported a much higher prevalence for hypertriglyceridemia despite using similar definition. This could be caused by the difference in the age range of participants as the previous study [9] included people aged 30–70 years, whereas the current study included adults ≥18 years of age.

As reported in previous studies on Saudi and other populations [9–11,30,31] older age, increased BMI and WC were associated with a higher prevalence of dyslipidemia in general, and all its types. The prevalence of high TG in the current study was lower than previously reported [11] being 11.4% for young adults aged 30 years or under compared to with 32% in the older adults aged more than 30 years. This difference in prevalence is due to the variation in the cut-off point to determine hypertriglyceridemia as it was ≥ 1.7 mmol/L in our study as well as in that of Al-Amri et al. [32] whereas ≥ 1.27 mmol/L was adopted in the previous study [11]. The cut-off point in this previous study allowed a wider range of the population to be diagnosed with hypertriglyceridemia, and therefore a higher prevalence was reported.

In our study sex had no effect on the prevalence of dyslipidemia in general but both hypertriglyceridemia and high LDL-C were more prevalent in men than women. Similar sex-differences were reported previously [9,11]. This could be due to the higher proportion of smokers, and the higher prevalence of low sleep duration (<6 h) among men. These lifestyle factors were associated with both hypertriglyceridemia and elevated LDL-C in this study. On the other hand, low HDL-C was more prevalent among women, which is different from that previously reported [11]. Similar sex differences in the prevalence of low HDL-C was previously found in Iranian population [33], which reported a much higher prevalence in both men and women compared with that found in our study.

Emerging evidence collected from self-reported data indicated that both short and long sleep duration are associated with adverse health consequences including obesity hypertension, diabetes mellitus, and CVDs, showing a curvilinear relationship between habitual sleep duration and these conditions [34–36]. In line with this, in our study in men but not in women, both short (<6 h) and long (>8 h) sleep durations were associated with dyslipidemia, particularly with elevated LDL-C and TG levels. In contrast, a recent systemic review, meta-analysis, and meta-regression suggested that a causal relationship existed between short sleep durations and adverse health outcomes including increased risk of diabetes, hypertension, and CVD [14]. However, there was insufficient data to draw a conclusion regarding the association between short sleep duration and dyslipidemia [14,34]. On the other hand, another systematic review indicated that long sleep may have more adverse health effect than short sleep [37]. Findings from cross-sectional data for the middle-aged and older Chinese population partly supported our finding that both short and long sleep durations were associated with dyslipidemia; since their cohort data suggested that long sleep duration (>9 h) only was associated with dyslipidemia [35].

In experimental studies, sleep debt has shown to induce alterations in the endocrine functions, so that sleep debt for 4 h resulted in raising evening cortisol concentrations, suggesting that it results in the disturbance of the negative-feedback regulation of the hypothalamus-pituitary-adrenal axis [37,38]. A previous cross-sectional study reported that excess cortisol secretion was linked to increased TG concentrations in South Asians [39]. The association between sleep duration, cortisol secretion, and dyslipidemia needs to be further investigated [40]. Even though the cortisol level was not measured in our study, dysregulated cortisol secretion might be the cause for the observed association between short sleep duration and dyslipidemia.

High consumption of Turkish coffee in this study was associated with dyslipidemia in men only. This is consistent with findings from previous clinical studies [41]. In contrast, we found that high consumption of American coffee was associated with decreased risk of dyslipidemia in men also, which contradicts findings in a clinical cohort study reporting that abstention from filtered coffee for 3 weeks resulted in a decrease in TC [42]. In an attempt to explain the different findings, a previous review suggested that brewing and preparation techniques influence this association as the long contact of beans with hot water in unfiltered coffee results in the formation of cholesterol elevating compounds, such as cafestol and kahweol, and that up to 90% of these diterpenes may be carried by floating coffee bean particles [43]. The same review suggested that the predicted rise in serum cholesterol levels with consumption of five cups of Turkish coffee per day is 0.25 mmol/L whereas filtered coffee is not predicted to increase serum cholesterol levels. In addition to diterpene alcohols, other compounds are found in the complex coffee beverage [43]. Moreover, a previous meta-regression analysis revealed that coffee consumption, particularly unfiltered coffee, is related to increased LDL-C, TC, and TG in a dose dose-dependent manner [42]. Coffee also contains antioxidant and anti-inflammatory compounds such as chlorogenic acid [43]. This might explain the observed U-shaped relationship between coffee consumption and low HDL-C and high TC in the current study. The observation that Turkish coffee was related to increased dyslipidemia in men but not women in the current study cannot be explained by variation in consumption patterns between sexes since these were comparable. Similar to our findings, a previous Norwegian study reported that unfiltered coffee consumption was associated with elevated cholesterol levels in men but not women [44].

In the current study fruit and vegetable intake were associated with dyslipidemia in men in a U-shaped manner. However, following adjustment for age, BMI, and WC consumption of fruits and vegetables lost their association with dyslipidemia in men. This suggests that this association could be caused by the high intake of total food in general and thus confounded by other constituents of diet. On the other hand, the consumption of fruit and fresh vegetables was associated with an increased risk of dyslipidemia, particularly elevated TC, in women, thus contradicting the previously reported beneficial effects of fruits and vegetables on serum lipids [45]. However, following adjustment for age, BMI, and WC, only the consumption of fresh vegetables retained its association with dyslipidemia. This could be a chance finding given several statistical comparisons, or due to the residual confounding since we did not collect data on all possible factors that may be associated with dyslipidemia. Following this unexpected finding, further statistical analysis was carried out, and the mean neck circumference (NC) was found to be higher in women reporting medium and high intake of fresh vegetables. In a systemic review and meta-analysis, positive associations between NC, high TC, and LDL-C, and low HDL-C concentrations were found, and subjects with higher NC had approximately two-fold higher risk for hypertriglyceridemia compared to those with lower NC [46]. NC was not adjusted for in our study, hence its increased mean in women consuming fresh vegetables could contribute to the noted association. Another reason could be that women who knew about their dyslipidemia or were trying to lose weight had modified their diet to include more fresh vegetables, i.e., representing reverse causation. Indeed, the mean weight of women ingesting fresh vegetables was significantly higher than the mean of those reporting no intake, and a higher percentage were on lipid-lowering therapy, but the difference to those reporting no intake did not reach statistical significance. Also, the addition of salad dressings to fresh vegetable salads is a very common dietary practice. Salad dressings often contain around 40% of fat, and a considerable amount of high fructose corn syrup, which has been proven to increase lipid synthesis and is associated with dyslipidemia [47]. Furthermore, mayonnaise is a major ingredient as a salad dressing alone or as part of other popular dressings (e.g., thousand islands), and studies have found that the use of palm oil, a cheaper oil comparatively, in its preparation caused an increase in total and LDL-C compared to the use of soybean oil, which is a more expensive type [48]. Therefore, the consumption of dressing can distort potential beneficial influences of vegetable consumption on serum lipids.

Studies with more detailed dietary assessment methods such as food diaries that provide information about consumed additives to vegetables are required to get a clear picture of the influence of vegetable consumption on serum lipids in Saudis.

It was interesting to note that in men, smoking and high consumption of carbonated drinks were significantly associated with dyslipidemia only following adjustment for age, BMI, and WC. Both cigarette smoking and dyslipidemia are well-established major risk factors for cardiovascular disease. Studies on different populations reported an increased risk of dyslipidemia in smokers [1–3]. A Korean cross-sectional study [49] conducted in adults aged ≥19 years reported that there was an increased risk of low HDL-C, high TG and high LDL-C in male smokers compared with non-smokers. On the other hand, female smokers were found to have a significantly increased risk for high TC, high TG, and high LDL-C compared with non-smokers. These results emphasize the sex difference in response to the same lifestyle factors as noted in our study. A Tunisian study [50] reported an increased risk of high triglyceridemia, and low HDL-C in smokers in a dose-response manner. Our study did not investigate the amount or type of smoking, however it was noted that a higher percentage of smokers had hypertriglyceridemia. Similar findings were reported in a Chinese study of elderly adults [51] that concluded that smoking was an independent risk factor for dyslipidemia and that it had a bigger effect than other studied factors including alcohol intake, BMI, and age.

High consumption of carbonated drinks was a predictor of dyslipidemia in men. A prospective study reported that daily consumption of carbonated drinks was associated with increased risk of hypertriglyceridemia and low HDL-C. In a cross-sectional study performed in Oslo, Norway, the consumption of colas but not other carbonated drinks was associated with low serum HDL-C, as well as high TG and LDL-C [52]. The association between high carbonated drink intake and dyslipidemia could be attributed to the high sucrose content in these drinks as it was reported to be

linked to hypertriglyceridemia previously [53]. The questionnaire that was used in this study did not provide specific data about the type of carbonated drink. Future work should include more details on type of drinks, and whether it was sugar-free or not.

A number of lifestyle practices showed an association initially, but this association was lost after adjustment for confounding factors. These practices include decreased physical activity and sitting duration. Physical activity and sedentary behavior are common factors reported to be associated with serum lipid levels [54,55]. A previous review on the effect of aerobic exercise on serum lipids recommended that clinicians should rely more on physical activity and less on lipid lowering drugs to modify lipid variables, thus to reduce the risk of myocardial infractions and CVD [56]. Low physical activity was found to be associated with dyslipidemia and particularly with high TC and TG in men, but it was not a significant predictive factor. However, the questionnaire used in our study did not provide detailed information about the type, timing, and intensity of physical activity. People tend to start exercising in an attempt to control weight, and various ill-health conditions, which could explain the loss of association following adjustment for BMI and WC in our study. Specific specialized questionnaires should be used in future work to provide more detailed information about physical activity, which would aid in providing more information about the association between physical activity and dyslipidemia in the Saudi population.

Very short sitting duration (for less than 4 h) was associated with a decreased level of HDL-C in women in the current study. Short sitting duration might be associated with stress at work. Indeed, it has been observed in individuals whose career requires high labor work and low sitting hours and provides low salaries [57]. A previous cross-sectional study on women employees of a retail company in Japan reported that employees with effort-reward imbalance had a 4.4-fold higher risk of low HDL-C compared with employees who have balanced effort-reward [57]. This suggests that the stress in careers or other work-related factors could distort the relationship between physical activity and dyslipidemia and might explain the observed relationship between low sitting duration and unfavorable HDL-C profile noted in our study.

Another unexpected finding in this study was that cinnamon drink consumption was associated with increased risk of dyslipidemia, particularly elevated TC, in women before adjustment for confounding factors. This was not observed in men probably due to the higher intake of cinnamon drink in women. In contrast, a previous systematic review and meta-analysis suggested that cinnamon supplementation has an anti-lipidemic effect especially on plasma cholesterol and TG in a duration-dependent but not dose-dependent manner [58]. After adjusting for age, BMI, and WC the association ceased to exist in our study. The inverse association between cinnamon drink consumption and dyslipidemia observed might be due to the addition of sugar to the drink which is a common practice among Saudis. Another possible explanation could be due to changes in the diet by individuals who have been recently diagnosed with dyslipidemia or hyperglycemia. The current study had a cross-sectional design, which is the main limitation since it did not provide information on the duration of the recorded dietary pattern. Cinnamon drink is commonly ingested by Saudi individuals who are diagnosed with dysglycemia due to the common belief that cinnamon lowers blood sugar. Since dysglycemia occurs more in individuals with dyslipidemia particularly high LDL-C [32], changes in dietary habits such as increasing consumption of cinnamon drink or fresh vegetables in individuals diagnosed with dysglycemia or dyslipidemia can result in biased observations regarding the effect of these food types on serum lipids. In order to obtain a clear picture, future studies should include information on the duration of the recorded dietary habits, and whether dietary practices have been changed due to medical advice.

There are limitations to our study. First, its cross-sectional design did not allow for inferences about cause and effect and can suggest associations only. In addition, collected data was based on a questionnaire providing self-reported dietary and lifestyle data, hence errors of reporting are expected. However, conducting face to face interviews by trained data collectors is believed to minimize such errors. In addition, our study had a relatively small sample size, but it was enough to detect several associations.

In spite of the limitation, our study has many points of strength. The first being that bias was avoided by a random selection of PHC and included volunteers. Secondly, standardized methods for data collections by well-trained data collectors were used. Moreover, a comprehensive definition was used to diagnose dyslipidemia, and all collected samples were analyzed in one accredited lab to avoid variations leading to misclassification.

#### **5. Conclusions**

In this studied population, there was a high prevalence of dyslipidemia among men and women which was found to be associated with increased age, BMI, and WC. Dyslipidemia was associated with several lifestyle and dietary factors, which was sex-specific. After adjusting for age, BMI, and WC, short (<6 h), and long (>8 h) sleep duration, and high consumption of Turkish coffee and carbonated drinks were associated with increased risk of dyslipidemia in men, while high consumption of American coffee was associated with a decreased risk. However, in women, only increased consumption of fresh vegetables was associated with an increased risk of dyslipidemia after adjusting for confounding factors.

This highlights the necessity of the adjustment of these modifiable risk factors since individuals with dyslipidemia are at increased risk of developing CVD. More detailed cohort studies are needed to reach firmer conclusions and lead to prevention recommendations. Nevertheless, results from this study provide useful information for the planning of future preventive actions against CVD.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/8/2441/s1, Figure S1: Recruitment flow diagram, Table S1: Demographic, anthropometric, clinical and biochemical characteristics of studied groups, Table S2: Comparison of demographic, anthropometric, and lifestyle characteristics of normolipidemia vs. dyslipidemia groups presented as number of people (%) for overall dyslipidemia, and abnormalities in different lipid parameters in men (a) and women (b), Table S3: Anthropometric and clinical characteristics of vegetable intake groups in women.

**Author Contributions:** Conceptualization, all authors; methodology, S.B., H.J., R.A.R., G.A., J.A.-A., A.B., and J.T.; software, H.J., R.A.R., G.A., J.A.-A., and S.E.; validation, S.B., H.J., R.A.R., G.A., J.A.-A., and J.T.; formal analysis, S.E., S.B., and H.J.; investigation, S.B., H.J., R.A.R., G.A., J.A.-A., A.B., and J.T.; resources, S.B., H.J., R.A.R., G.A., J.A.-A., and A.B.; data curation, S.B., H.J., R.A.R., G.A., and J.A.-A.; writing—original draft preparation, S.B., S.E., B.E., and M.M.; writing—review and editing, all authors; visualization, S.B., S.E., and J.T.; supervision, S.B., H.J., R.A.R., G.A., and J.A.-A.; project administration, S.B. and G.A.; funding acquisition, S.B., G.A., and J.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by King Abdulaziz University, grant number (2-140-1434-HiCi).

**Acknowledgments:** We thank the Deanship of Research in King AbdulAziz University in the highly cited program for supporting this work. We also thank Lubna Al-Shaikh for cleaning the data.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

BMI, body mass index; BP, blood pressure; CVD, cardiovascular disease; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; PHCC, primary health care centers; SD, standard deviation; TC, total cholesterol; TG, triglycerides; WC, waist circumference; WHO, World Health Organization.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

**A Randomized, Double-Blinded, Placebo-Controlled, Clinical Study of the E**ff**ects of a Nutraceutical Combination (LEVELIP DUO**®**) on LDL Cholesterol Levels and Lipid Pattern in Subjects with Sub-Optimal Blood Cholesterol Levels (NATCOL Study)**

#### **Arrigo F.G. Cicero \*, Sergio D'Addato and Claudio Borghi**

Medical an Surgery Sciences Department, Dyslipidemia and Atherosclerosis Research Unit, Alma Mater Studiorum University of Bologna, 40138 Bologna, Italy; sergio.daddato@unibo.it (S.D.); claudio.borghi@unibo.it (C.B.)

**\*** Correspondence: arrigo.cicero@unibo.it; Tel.: +39-512142224

Received: 8 September 2020; Accepted: 11 October 2020; Published: 14 October 2020

**Abstract:** Phytosterols and red yeast rice are largely studied cholesterol-lowering nutraceuticals, respectively inhibiting the bowel absorption and liver synthesis of cholesterol. Our aim was to test the effect of combined nutraceutical-containing phytosterols and red yeast rice vs. a placebo on the lipid profile. We performed a parallel arms, double-blind, placebo-controlled clinical trial, randomizing 88 moderately hypercholesterolemic subjects to treatment with a combined nutraceutical containing phytosterols (800 mg) and red yeast rice, standardized to contain 5 mg of monacolins from *Monascus purpureus*, with added niacin (27 mg) and policosanols (10 mg) (LEVELIP DUO®)*,* or placebo. The mean LDL-Cholesterol (LDL-C) change at Week 8 was −32.5 ± 30.2 mg/dL (−19.8%) in the combined nutraceutical group and 2.5 ± 19.4 mg/dL (2.3%) in the placebo group. The estimated between-group difference of −39.2 mg/dL (95% CI: −48.6; −29.8) indicates a statistically significant difference between treatments in favor of the combined nutraceutical (*p* < 0.0001). Total Cholesterol (TC), non-HDL cholesterol (non-HDL-C), Apolipoprotein B, TC/HDL-C and LDL-C/HDL-C improved in a similar way in the combined nutraceutical group only. No significant changes in other clinical and laboratory parameters were observed. In conclusion, the tested combined nutraceutical was well tolerated, while significantly reducing the plasma levels of LDL-C, TC, non-HDL-C, ApoB, TC/HDL-C and LDL-C/HDL-C ratios in mildly hypercholesterolemic patients. Trial registration (ClinicalTrials.gov): NCT03739242.

**Keywords:** monacolins; LDL-cholesterol; phytosterols; red yeast rice; nutraceuticals; clinical trial; endothelial function

#### **1. Introduction**

Hypercholesterolemia is a largely prevalent cardiovascular disease risk factor in the general population, and its early reduction seems to be an effective preventive strategy [1]. However, pharmacologically treating moderately hypercholesterolemic subjects without other cardiovascular risk factors in the primary prevention for cardiovascular disease is still debated [2]. In fact, the guidelines suggest managing these subjects with a more conservative approach, stressing the need for a therapeutic lifestyle promotion, eventually added with lipid-lowering nutraceuticals and/or functional foods [1,3].

In the last few decades, a relatively large number of nutraceuticals and functional foods has been studied for their ability to decrease cholesterolemia in humans [4]. The most clinically studied include soluble fibers, phytosterols, soy proteins, monacolins from red yeast rice, berberine, and garlic and artichoke extracts [5]. In particular, the most recent guidelines for dyslipidaemia management from the European Atherosclerosis Society and from the European Cardiology Society suggest increasing the amounts of fiber and omega 3 polyunsaturated fatty acids in the diet, while adding phytosterols and monacolins as dietary supplements, when a lifestyle intervention is needed to reduce cholesterolemia [6].

Plant sterols and stanols (phytosterols) are natural constituents of the plant cell membrane [7]. From a chemical point of view, they are very similar to cholesterol, with minor differences in the relative positions of ethyl and methyl groups. Based on this similarity, phytosterols may compete with dietary and biliary cholesterol for micellar solubilization in the intestinal lumen, impairing intestinal cholesterol absorption [8]. Moreover, several clinical trials have consistently shown that an intake of 2–3 g/day of plant sterols is associated with a significant lowering (between 4% and 15%) of low-density lipoprotein-cholesterol (LDL-C) [9,10]. The variability in the observed LDL-reduction is mainly related to genetic factors [11]. Based on the available data, the European Food Safety Agency (EFSA) accepted a health claim for the phytosterols' LDL-C-lowering effect [12].

Red yeast rice is a nutraceutical obtained by the fermentation of rice (*Oryza sativa*) as result of a yeast (in general *Monascus purpureus*), whose typical red coloration is due to the presence of some specific pigments, by-products of the fermentative metabolism process [13]. Monascus yeast produces a family of substances called monacolins, including monacolin K. Monacolins act as reversible inhibitors of the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the key enzyme in cholesterol biosynthesis [14].

A recent meta-analysis of 20 randomized clinical trials, including 6663 subjects, showed that, after 2–24 months of treatment, red yeast rice (RYR) reduced LDL-C on average by 39.4 mg/dL compared to placebo, which was comparable to the reduction achieved with regular-dosed statins (pravastatin 40 mg, simvastatin 10 mg, lovastatin 20 mg) [15]. Based on these data, the EFSA has expressed a scientific opinion supporting the health claims for the relationship between the administration of RYR and the control of plasma LDL-C levels [16]. Even if recently the same EFSA raised some concerns about RYR's safety [17], a recent large metanalysis of 53 randomized clinical trials, including 8535 subjects, has shown that monacolin K administration is not associated with an increased risk of Statin Associated Muscle Symptoms (SAMS) (odds ratio (OR) = 0.94, 95% confidence interval (CI) 0.53, 1.65) for daily doses of monacolin K of between 3 and 10 mg [18].

In this context, the primary objective of the study was to evaluate the effects of LEVELIP DUO® on LDL-C blood levels in subjects with sub-optimal blood cholesterol levels over an 8-week period. The secondary objective was the evaluation of effects of the tested combined nutraceuticals on other lipoproteins and on the estimated cardiovascular risk.

#### **2. Materials and Methods**

This parallel-armed, double-blind, randomized clinical trial was carried out in 90 moderately hypercholesterolemic subjects, non-smokers, pharmacologically untreated, in primary prevention for cardiovascular diseases, consecutively enrolled in the ambulatory service of cardiovascular disease prevention in the Medical and Surgical Sciences Department of the University of Bologna.

The inclusion criteria were age between 30 and 75, and LDL-C level between 115 and 190 mg/dL, confirmed in at least two sequential checks prior to signing the consent form.

The exclusion criteria were as follows:


The study was fully conducted in accordance with the Declaration of Helsinki, its protocol was approved by the Ethical Committee of the University of Bologna, and informed consent was obtained from all patients before inclusion in the study (Clinical trial.gov ID NCT03739242).

The study design has been detailed in Figure 1.

**Figure 1.** Study design.

At the enrollment visit (T-1, Day-14), patients were given standard behavioral and qualitative (not quantitative) dietary suggestions to correct unhealthy habits. Standard diet advice was given by a dietitian and/or specialist doctor. A dietitian and/or specialist doctor periodically provided instructions on dietary intake recording procedures as part of a behavior modification program, and then later used the subject's food diaries for counseling. In particular, the subjects were instructed to follow the general structure of a Mediterranean diet, avoid excessive intakes of dairy products and red meat-derived products during the study, and maintain overall constant dietary habits [19]. Individuals were also generically encouraged to increase their physical activity by walking briskly for 20 to 30 min, 3 to 5 times per week, or by cycling [20].

After 2 weeks of diet and physical activity (Randomization visit, T0), if the LDL-C and TG values were confirmed, the patients were randomly allocated to a treatment with LEVELIP DUO® or placebo, one tablet after dinner. LEVELIP DUO® is a registered combination of phytosterols (800 mg) and other registered ingredients including red yeast rice standardized to contain 5 mg monacolins from *Monascus purpureusi,* with niacin (27 mg) and policosanols (10 mg) (Dif1/Stat®) [21,22]. The red yeast rice extract used was certified to be highly purified in monacolins, without chromatographically detectable levels of dehydromonacolins, decalin derivatives and contaminants. The phytosterol dose was chosen based on the minimal efficacious dose identified by the meta-analysis of randomized clinical trials, performed by Demonty et al. [23].

The active products and placebo were administered as indistinguishable tablets (kindly provided by Menarini IFR, Firenze, Italy). The treatment then continued for 8 weeks. Clinical and laboratory data have been obtained at the baseline (T0), after 4 weeks (T1) and at the end of the trial (8 weeks, T2).

A computer-generated randomization list produced by the trial statistician randomized patients to one of the two treatment groups (LEVELIP DUO® or placebo) in a 1:1 ratio. A paper-based randomization procedure assigned a randomization number to the patient, which has been used to link the patient to a treatment arm and specified a unique kit number for the package of treatment to be dispensed to the patient. The randomization numbers were generated using a procedure to ensure that treatment assignment is unbiased.

Throughout the study, we instructed patients to take the first dose on the day after they were given the study product in a blinded box. At the end of the study, all unused products were retrieved for inventory. Product compliance was assessed by counting the number of product doses returned at the times of specified clinic visits.

At each visit, enrolled patients were interviewed about eventual changes in lifestyle and possible adverse events raised in the previous weeks. Then, anthropometric measurement and vital signs were recorded, and a plasma sample was obtained after a 12-h overnight fast. Venous blood samples were drawn by a nurse from all patients between 8:00 a.m. and 9:00 a.m. The serum used was centrifuged at 3000 g for 15 min at ambient temperature. Immediately after centrifugation, the samples were frozen and stored at −80 ◦C for no more than 3 months. The following parameters were evaluated via standardized methods [24,25]: total cholesterol (TC), HDL-C, TG, apolipoprotein B100 (apoB), glucose, creatinine, serum uric acid, liver transaminases, gamma-glutamyl transferase, and creatinine phosphokinase (CPK). All measurements were performed by trained personnel in the Lipid Clinic laboratory of the Medicine and Surgery Sciences Department, by the S. Orsola-Malpighi University Hospital. Since hypertriglyceridemia was an exclusion criterion, the LDL-C level was estimated by the application of the Friedewald's formula (LDL-C = TC − HDL-C − TG/5).

As post-hoc analysis, the cardiovascular disease risk was estimated with a validated nation-specific algorithm (Progetto CUORE) [26].

Considering the primary endpoint of the study as the reduction from baseline to week 8 in LDL cholesterol level, and the data available from literature [27], a reduction in LDL level of approximately 10% is expected after the intake of the nutraceutical. Therefore, assuming a baseline LDL level of 145 ± 19 mg/dL, a power of 90% and a 5% two-sided alpha level to detect a difference in mean change in LDL from baseline to week 8 equal to 15 mg/dL between the nutraceutical and the placebo group, the total number of patients to be evaluated should be 35 per treatment arm in a 1:1 ratio (NQuery Advisor, 7.0). Allowing for an approximate 20% dropout rate, at least 88 patients should be randomized—44 patients in each treatment group.

Statistical tables, figures, listings and analyses were produced using SAS® for Windows release 9.4 (64-bit) or later (SAS Institute Inc., Cary, NC, USA). For each secondary efficacy variable, an ANCOVA model was used to estimate the treatment's effect on the changes from baseline at week 8, considering the planned treatment group as the factor and the baseline value of the parameter as the continuous covariate. The results are reported as Least Squares Means together with associated two-tailed 95% CI. The difference in Least Squares Means between the nutraceutical combination group and placebo group was estimated with two-tailed 95% CI and *p* value. If the assumption of the normality of the residuals was violated, the ANCOVA model was fitted to rank transformed data. A *p* value less than 0.05 was considered significant for all tests.

#### **3. Results**

Enrolled patients were age- and sex-matched. We enrolled 38 men (19 randomized to the combined nutraceutical, 19 to placebo) and 47 women (24 randomized to the combined nutraceutical, 23 to placebo). The baseline characteristics of patients assigned to the different treatments (active and placebo) were similar, and no significant differences were observed regarding the studied parameters (Table 1). Patient disposition is outlined in Figure 2.


**Table 1.** Baseline characteristics of the enrolled subjects: non-statistically significant difference has been detected between the treatment groups (values reported as mean ± standard deviation; No significant difference between groups has been observed).

**Figure 2.** Patient disposition.

Overal, 85 subjects (94.4%), 43 in the active treatment group and 42 in the placebo group, were compliant (compliance levels between 80% and 120%). One patient (2.33%) in the active treatment group had compliance lower than 80%, whereas 1 patient (2.33%) in the placebo group had compliance greater than 120%.

Dietary habits remained overall unchanged during the study.

The primary variable of the study was the change from baseline at week 8 in LDL cholesterol (Figure 3).

The LDL-C levels decreased from visit to visit in the combined nutraceutical group, and, on the contrary, increased in the placebo group. The mean change at week 8 was −32.5 ± 30.2 mg/dL (−19.8%) in the combined nutraceutical group and 2.5 ± 19.4 mg/dL (2.3%) in the placebo group. The ANCOVA model results for the estimated change from baseline at week 8 in LDL-C levels, adjusting for the baseline value of LDL-C, provided Least Squares Means equal to −34.5 mg/dL (95% CI: −41.1; −27.9) for the combined nutraceutical group and 4.6 mg/dL (95% CI: −2.0; 11.3) for the placebo group. The estimated between-group difference of −39.2 mg/dL (95% CI: −48.6; −29.8) indicates a statistically significant difference between treatments in favor of the combined nutraceutical (*p* < 0.0001).

**Figure 3.** Plasma LDL-Cholesterol level percentage change from the baseline to 4 and 8 weeks in subjects treated with the tested combined nutraceutical or placebo.

The Least Squares Means of TC changes from baseline at week 8, adjusting for baseline value, were −33.0 mg/dL (95% CI: −40.4; −25.7) for the combined nutraceutical group and 8.2 mg/dL (95% CI: 0.8; 15.7) for the placebo group. The estimated between-group difference of −41.2 mg/dL (95% CI: −51.7; −30.7) and a *p* < 0.0001 suggest a statistically significant difference between treatments in favor of the combined nutraceutical.

The Least Squares Means of non-HDL cholesterol change from baseline at week 8, estimated adjusting for baseline value, were equal to −34.2 mg/dL (95% CI: −41.1; −27.3) in the combined nutraceutical group and 7.5 mg/dL (95% CI: 0.5; 14.5) in the placebo group. The between-group difference of −41.7 mg/dL (95% CI: −51.6; −31.8) was significantly in favor of treatment with the combined nutraceutical (*p* < 0.0001).

The Least Squares Means of Apo B change at week 8, adjusting for baseline value, were −15.2 mg/dL (95% CI: −19.6; −10.8) for the combined nutraceutical group and 2.0 mg/dL (95% CI: −2.4; 6.5) for placebo. The between-group difference of −17.3 mg/dL (95% CI: −23.5; −11.0) indicates a statistically significant difference between treatments in favor of the combined nutraceutical (*p* < 0.0001).

The Least Squares Means of the TC/HDL-C ratio change from baseline at week 8, adjusting for baseline value, were −0.8 (95% CI: −1.0; −0.6) for the combined nutraceutical group and 0.1 (95% CI: −0.1; 0.3) for placebo. The estimated between-group difference was −0.9 (95% CI: −1.2; −0.6), and a *p* < 0.0001 indicates a significantly greater change in the TC/HDL ratio in subjects treated with the combined nutraceutical.

The Least Squares Means of the LDL-C/HDL-C ratio change from baseline at week 8, adjusting for baseline value, were −0.8 (95% CI: −0.9; −0.6) for the combined nutraceutical group and 0.02 (95% CI: −0.1; 0.2) for the placebo group. The between-group difference of −0.8 (95% CI: −1.1; −0.6) was statistically significant (*p* < 0.0001).

Body weight, BMI, waist circumference, blood pressure, FPG, HDL-C, TG, GOT, GPT, gGT, SUA, eGFR and CPK did not significantly change in both groups during the study (Table 2).

During the study, no treatment-emergent adverse event was reported. The trends of hematology and clinical chemistry values did not indicate any safety concerns.


**Table 2.** Laboratory parameter changes during the study (values reported as mean ± standard deviation).

\* *p* < 0.01 vs. baseline; ◦ *p* < 0.01 vs. control. TC = Total Cholesterol, ApoB = Apolipoprotein B, FPG = Fasting Plasma Glucose, ALT = Alanine Aminotranferase, AST = Aspartate Aminotranferase, gGT = γ-glutamyl transferase, SUA = Serum Uric Acid, eGFR = estimated glomerular filtration rate, CPK = creatine phosphokinase.

**Figure 4.** Change in estimated 10-year cardiovascular disease risk in the two treatment groups (post-hoc analysis; \* *p* < 0.05).

#### **4. Discussion**

The Mediterranean diet remains a milestone in cardiovascular disease prevention, [28] even if its impact on LDL-cholesterolemia is limited. For this reason, the ESC/EAS guidelines [6] and the International Lipid Expert Panel (ILEP) [5] consider the use of some dietary supplements (namely, red yeast rice and phytosterols) as a support to a balanced diet in order to improve cholesterolemia control. Many trials evaluating the effect of nutraceuticals on lipid pattern are not adequately designed, being often not double-blinded and underpowered.

In our double-blind, placebo-controlled, randomized clinical trial, we observed that the LEVELIP DUO® was able in the short-term to significantly reduce the plasma levels of LDL-C, TC, non-HDL-C, and apoB, and the TC/HDL-C and LDL-C/HDL-C ratios. In particular, the estimated between-group difference in LDL-C was −39.2 mg/dL (95% CI: −48.6; −29.8). This effect was observed after 4 weeks of treatment and confirmed after 8 weeks, excluding short-term adaptation phenomena. This is compatible with the mechanism of action of the nutraceutical components of the tested product. The LDL-C reduction that was achieved is near to that 39.8 mg/dL, which is estimated to be associated in long-term

trials with a corresponding 22% reduction in cardiovascular mortality and morbidity [29]. This is also in line with our observation that the estimated cardiovascular risk was significantly modified by the tested treatment, but not by the placebo. This impressive result was partly expected. In fact, it confirms what we already observed in a previous smaller pilot study, where the association of phytosterols and red yeast rice was able to increase red yeast rice's LDL-C-lowering efficacy [30]. On the other side, because the mechanisms of action of red yeast rice and phytosterols should be additive or synergistic, they represent the natural alternative to the synergistic association of statins with ezetimibe [27].

In this context, the tested combined nutraceutical was shown to be effective and well-tolerated. In 2017, the ILEP [5] classified the association of phytosterols and red yeast rice as recommendation IIa (should be considered), based on a level of evidence classified as B (Data derived from single randomized clinical trial or large non-randomized studies). Considering the results of our current trial and of our previous one [31], combined with the ESC/EAS suggestion, the class of recommendation could probably improve.

We can argue that the greater part of the observed effect of LEVELIP DUO® is be related to the contents of phytosterols and red yeast rice. However, the minor components of the products could have also minimally contributed to the final effect. In particular, EFSA approves the health claim of niacin supporting energy metabolism and macronutrient metabolism [32]. Policosanols seem to have a small impact on hypercholesterolemia, however their efficacy when consumed with red yeast rice has been observed in a large number of trials [5].

Our study has some relevant limitations. The first one is the relatively low number of subjects investigated per treatment group, while, however, the study was sufficiently powered to detect differences between treatment groups. Of course, we could have used a cross-over design, but we feared losing the volunteers' compliance to the treatment if the study duration was excessive. The second one is the lack of the measurement of markers of cholesterol absorption and synthesis, as cholesterol hyperabsorbers could have manifested a more significant LDL-C reduction than standard cholesterol absorbers [31]. Finally, the study was relatively short, so that we do not know if the observed effect could be confirmed in the long term. However, since the monacolins' and phytosterols' mechanisms of action are the same as those of statins and ezetimibe, drugs that have largely proven to maintain their efficacy over decades, we could reasonably assume that this evidence could be translated to the tested combined nutraceutical. Moreover, the study was adequately powered, so that we can be confident in the reported results.

#### **5. Conclusions**

LEVELIP DUO® was well-tolerated while significantly reducing the plasma levels of LDL-C, TC, non-HDL-C, ApoB, TC/HDL-C and LDL/HDL-C ratios in mildly hypercholesterolemic patients. Further long-term studies are needed to confirm the maintenance of this impressive effect over time.

**Author Contributions:** Conceptualization, A.F.G.C. and C.B.; methodology, A.F.G.C. and S.D.; investigation, A.F.G.C. and S.D.; writing—original draft preparation, A.F.G.C.; writing—review and editing, S.D. and C.B.; supervision, C.B.; project administration, C.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Menarini IFR, Firenze, Italy.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Efficacy and Safety of Armolipid Plus®: An Updated PRISMA Compliant Systematic Review and Meta-Analysis of Randomized Controlled Clinical Trials**

**Arrigo F. G. Cicero 1,2,3,\*, Cormac Kennedy 4, Tamara Kneževi´c 5, Marilisa Bove 1,2, Coralie M. G. Georges 6, Agne Šatrauskien ˙ e˙ 7,8, Peter P. Toth 9,10 and Federica Fogacci 1,2,3**


**Abstract:** Armolipid Plus® is a multi-constituent nutraceutical that claims to improve lipid profiles. The aim of this PRISMA compliant systematic review and meta-analysis was to globally evaluate the efficacy and safety of Armolipid Plus® on the basis of the available randomized, blinded, controlled clinical trials (RCTs). A systematic literature search in several databases was conducted in order to identify RCTs assessing the efficacy and safety of dietary supplementation with Armolipid Plus®. Two review authors independently identified 12 eligible studies (1050 included subjects overall) and extracted data on study characteristics, methods, and outcomes. Meta-analysis of the data suggested that dietary supplementation with Armolipid Plus® exerted a significant effect on body mass index (mean difference (MD) = <sup>−</sup>0.25 kg/m2, *<sup>p</sup>* = 0.008) and serum levels of total cholesterol (MD = −25.07 mg/dL, *p* < 0.001), triglycerides (MD = −11.47 mg/dL, *p* < 0.001), highdensity lipoprotein cholesterol (MD = 1.84 mg/dL, *p* < 0.001), low-density lipoprotein cholesterol (MD = −26.67 mg/dL, *p* < 0.001), high sensitivity C reactive protein (hs-CRP, MD = −0.61 mg/L, *<sup>p</sup>* = 0.022), and fasting glucose (MD = <sup>−</sup>3.52 mg/dL, *<sup>p</sup>* < 0.001). Armolipid Plus® was well tolerated. This meta-analysis demonstrates that dietary supplementation with Armolipid Plus® is associated with clinically meaningful improvements in serum lipids, glucose, and hs-CRP. These changes are consistent with improved cardiometabolic health.

**Keywords:** Armolipid Plus®; red yeast rice; berberine; nutraceutical; supplementation; lipids; blood pressure; fasting plasma glucose

#### **1. Introduction**

Atherosclerosis cardiovascular diseases (ASCVD) are the leading cause of mortality worldwide, and the main cause of death in persons under 75 years old in Western countries, with a huge social and economic impact [1]. Pooling data from 204 countries, the Global Burden of Disease (GBD) Study recently showed that prevalent cases of total CVD nearly

**Citation:** Cicero, A.F.G.; Kennedy, C.; Kneževi´c, T.; Bove, M.; Georges, C.M.G.; Šatrauskiene, A.; Toth, P.P.; ˙ Fogacci, F. Efficacy and Safety of Armolipid Plus®: An Updated PRISMA Compliant Systematic Review and Meta-Analysis of Randomized Controlled Clinical Trials. *Nutrients* **2021**, *13*, 638. https://doi.org/10.3390/nu13020638

Academic Editor: Xavier Pinto Received: 18 January 2021 Accepted: 12 February 2021 Published: 16 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

doubled from 271 million in 1990 to 523 million in 2019, and the number of CVD deaths steadily increased from 12.1 million in 1990, reaching 18.6 million in 2019 [2].

High serum levels of low-density lipoprotein cholesterol (LDL-C) are the most important risk factor for the development of ASCVD [3]. The American Heart Association (AHA) 2016 update on heart disease and stroke statistics verified that only 75.7% of US children and 46.6% of US adults have total cholesterol (TC) within the advised ranges (< 170 mg/dL for untreated children and < 200 mg/dL for untreated adults), with comparable rates for other Western countries [4,5].

To reach the LDL-C target, the international guidelines recommend lifestyle changes and lipid-lowering therapy depending on the severity of dyslipidemia and global CV risk [6,7]. Specific lifestyle interventions for hypercholesterolemia include a diet low in saturated fat, moderate to high-intensity physical activity, smoking cessation, as well as weight loss for overweight and obese patients [8,9]. If maintained over the long term, these lifestyle modifications can reduce LDL-C by 5% to 15% and improve ASCVD risk [10]. However, patients unable to reach their target LDL-C goals through lifestyle interventions can consider using lipid-lowering nutraceuticals [11], as also suggested by the International Lipid Expert Panel [12].

Nutraceuticals with a detectable lipid-lowering effect can be divided into natural inhibitors of hepatic cholesterol synthesis, inhibitors of intestinal cholesterol absorption, and enhancers of the excretion of LDL-C on the basis of their mechanisms of action [12]. However, the lipid-lowering effect of most nutraceuticals occurs through multiple mechanisms. The possibility that they act synergistically on multiple stages of lipid-induced vascular damage makes them potential candidates for improving the lipid-lowering effects when used in combination with diet, medications, or other nutraceuticals [13].

Armolipid Plus® is a widely tested and used proprietary formulation of six naturally occurring substances containing red yeast extract (200 mg, corresponding to 3 mg of monacolin K), policosanols (10 mg), and berberine (500 mg), in addition to folic acid (0.2 mg), astaxanthin (0.5 mg), and coenzyme Q10 (2 mg), with a detectable effect on serum lipids, blood pressure (BP), fasting plasma glucose (FPG), and several markers of insulin resistance with a good safety profile [14].

Given the increasing number of good quality studies on this nutraceutical combination, the aim of our systematic review and meta-analysis was to evaluate the efficacy and safety of Armolipid Plus® on the basis of the available randomized, blinded, controlled clinical trials.

#### **2. Materials and Methods**

The study was designed according to guidelines inthe 2009 preferred reporting items for systematic reviews and meta-analysis (PRISMA) statement [15], and was registered in the PROSPERO database (Registration number CRD42020212600). Due to the study design (meta-analysis), neither institutional review board (IRB) approval nor patient informed consent were required.

#### *2.1. Search Strategy*

PubMed, EMBASE, SCOPUS, Google Scholar, Web of Science by Clarivate, and ClinicaTrial.gov (accessed on 1 February 2021) databases were searched, with no language restriction, using the following search terms: "Armolipid Plus®" AND ("Cholesterol" OR "LDL" OR "Triglycerides" OR "Body mass index" OR "BMI" OR "Plasma Glucose" OR "Glycemia" OR "Insulin"). The wild-card term "\*" was used to increase the sensitivity of the search strategy, which was limited to studies in humans. The reference list of identified papers was manually checked for additional relevant articles. In particular, additional searches for potential trials included the references of review articles on the topic of the meta-analysis and relevant abstracts from selected congresses. The literature was searched from inception to 3 February 2021.

All paper abstracts were screened by two reviewers (F.F. and A.F.G.C.) in an initial process to remove ineligible articles. The remaining articles were obtained in full text and assessed again by the same two researchers, who evaluated each article independently and carried out data extraction and quality assessment. Disagreements were resolved by discussion with a third party.

#### *2.2. Study Selection Criteria*

Original studies were included if they met the following criteria: (i) being a clinical trial with either a multicenter or single-center design, (ii) having an appropriate controlled design for Armolipid Plus®, (iii) investigating the effect of Armolipid Plus® on plasma lipids, (iv) testing the safety of Armolipid Plus®, and (v) reporting all the adverse events that occurred during the supplementation.

Exclusion criteria included the following: (i) lack of a control group for Armolipid Plus® administration, (ii) lack of blinding, (iii) lack of sufficient information about plasma lipids at baseline or follow-up, and (iv) lack of sufficient information about the prevalence and specification of adverse events. Studies were also excluded if they contained overlapping subjects with other studies.

#### *2.3. Data Extraction*

Data abstracted from the eligible studies were: (i) first author's name; (ii) year of publication; (iii) study design; (iv) main inclusion criteria and underlying disease; (v) treatment duration; (vi) study groups; (vii) number of participants in the active and control group; (viii) background lipid-lowering treatment; (ix) age and sex of study participants; (x) weight, body mass index (BMI), waist circumference, systolic BP (SBP), diastolic BP (DBP), TC, triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), LDL-C, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase (CPK), FPG, fasting plasma insulin (FPI), homeostatic model assessment for insulin resistance (HOMA-IR) and high sensitivity C reactive protein (hs-CRP) at baseline; and (xi) discontinuation of treatment and adverse events occurred during the trials. All data extraction and database typing were reviewed by the principal investigator (A.F.G.C.) before the final analysis, and doubts were resolved by mutual agreement among the authors.

#### *2.4. Quality Assessment*

A systematic assessment of risk of bias in the included studies was performed using the Cochrane criteria [16]. The following items were used: adequacy of sequence generation, allocation concealment, blind addressing of dropouts (incomplete outcome data), selective outcome reporting, and other probable sources of bias [17]. Risk-of-bias assessment was performed independently by 2 reviewers (F.F. and A.F.G.C.); disagreements were resolved by a consensus-based discussion.

#### *2.5. Data Synthesis*

Meta-analysis was entirely conducted using Comprehensive Meta-Analysis (CMA) V3 software (Biostat, NJ) [18].

Net changes in the investigated parameters (change scores) were calculated by subtracting the value at baseline from the one after intervention, in the active-treated group and in the control one. All values were collated as mean change from baseline. Standard deviations (SDs) of the mean difference were obtained as follows, as reported by Follman et al.: SD = square root [(SDpre-treatment) <sup>2</sup> + (SDpost-treatment) <sup>2</sup> − (2R × SDpre-treatment × SDpost-treatment)], assuming a correlation coefficient (R) = 0.5 [19]. If the outcome measures were reported in median and range (or 95% confidence interval (CI)), mean and SD values were estimated using the method described by Wan et al. [20]. The findings of the included studies were combined using a fixed-effect model or a random-effect model (using the DerSimonian–Laird method) and the generic inverse variance method based on the level of inter-study heterogeneity, which was quantitatively assessed using the Higgins index (I2) [21]. For continuous parameters, effect sizes were expressed as absolute mean differences (MD) and 95%CI, standardized by the change score in SD. For treatment

emergent adverse events, odd ratios (OR) and 95%CI intervals were calculated using the Mantel–Haenszel method [22]. A safety analysis was performed by excluding studies with zero events in both arms. If one or more outcomes could not be extracted from a study, the study was removed only from the analysis involving those outcomes. Adverse events were considered for the analysis only if they occurred in at least two of the included clinical trials.

In order to evaluate the influence of each study on the overall effect size, a sensitivity analysis was conducted using the leave-one-out method (i.e., removing one study at a time and repeating the analysis) [23]. Two-sided *p*-values ≤ 0.05 were considered statistically significant for all tests.

#### *2.6. Publication Biases*

Potential publication biases were explored using a visual inspection of Begg's funnel plot asymmetry, Begg's rank correlation test, and Egger's weighted regression test [24]. The Duval and Tweedie "trim and fill" method was used to adjust the analysis for the effects of publication biases [25]. Two-sided *p*-values < 0.05 were considered statistically significant.

#### **3. Results**

#### *3.1. Flow and Characteristics of the Included Studies*

After database searches were performed according to inclusion and exclusion criteria, 445 published articles were identified, and the abstracts were reviewed. Of these, 112 were excluded because they were not original articles. Another 314 were eliminated because they did not meet the inclusion criteria. Thus, 19 articles were carefully assessed and reviewed. An additional 7 studies were excluded because of a lack of a controlled design for Armolipid Plus® administration (*n* = 3) or lack of blinding (*n* = 4). Finally, 12 studies were eligible and included in the meta-analysis [26–37]. The study selection process is shown in Figure 1.

**Figure 1.** Flow chart of the number of studies identified and included in the systematic review.

Data were pooled from 12 clinical trials comprising 24 treatment arms, which included 1050 subjects, with 544 in the actively treated arm and 536 in the control one.

Eligible studies were published between 2010 and 2020. Follow-up periods ranged between 4 weeks and 12 months. All selected trials were designed with parallel groups and were multicenter [29,35,37] or single-center [26–28,30–34,36] clinical studies. Enrolled subjects were patients in primary prevention for CVD [28–30,32,36], patients with documented coronary artery disease (CAD) [33], with a metabolic syndrome [26,30,34–36], or with a good status of health [28,37]. The baseline characteristics of the evaluated studies are summarized in Tables 1 and 2.





past three months


**Table 1.** *Cont*. Expressed as median (interquartile range); BMI = body mass index; CAD = coronary artery disease; CHD = coronary heart disease; CV = cardiovascular; CVD = cardiovascular disease; DBP = diastolic blood pressure; LDL-C = low-density lipoprotein cholesterol; NA = not available; PCI = percutaneous coronary intervention; SBP = systolic blood pressure; SD = standard deviation; TC = total cholesterol.


**Table 2.** Baseline lipids, fasting plasma glucose, and markers of insulin resistance.

*Nutrients* **2021**

, *13*, 638

assessment for insulin resistance; hs-CRP = high sensitivity C reactive protein; LDL-C = low-density lipoprotein cholesterol; NA = not available; SD = standard deviation; TC = total

cholesterol; TG = triglycerides.

#### *3.2. Risk of Bias Assessment*

Almost all of the included studies were characterized by sufficient information regarding sequence generation, allocation concealment, and personal and outcome assessments. All showed low risk of bias because of incomplete outcome data and selective outcome reporting. Details of the quality of bias assessment are reported in Table 3.

**Table 3.** Quality of bias assessment of the included studies according to the Cochrane guidelines.


H = High risk of bias; L = Low risk of bias; U = Unclear risk of bias.

#### *3.3. Effect of Armolipid Plus® on Anthropometric Measures, Blood Pressure, Serum Lipids, and Other Metabolic Parameters*

Meta-analysis of the data suggested that Armolipid Plus® supplementation exerted a significant effect on BMI (MD = −0.25 kg/m2, 95%CI(−0.43,−0.06) Kg/m2, *<sup>p</sup>* = 0.008; <sup>I</sup> <sup>2</sup> = 0%) (Figure 2) and serum levels of TC (MD = −25.07 mg/dL, 95%CI(−33.17,−16.97) mg/dL, *<sup>p</sup>* < 0.001; I<sup>2</sup> = 87%), TG (MD = −11.47 mg/dL, 95%CI(−17.85,−5.08) mg/dL, *<sup>p</sup>* < 0.001; I <sup>2</sup> = 34%), HDL-C (MD = 1.84 mg/dL, 95%CI(0.92,2.77) mg/dL, *p* < 0.001; I <sup>2</sup> = 0%), LDL-C (MD = −26.67 mg/dL, 95%CI(−33.76,−19.58) mg/dL, *<sup>p</sup>* < 0.001; I2 = 82%) (Figure 3), hs-CRP (MD = −0.61 mg/L, 95%CI(−1.13,−0.09) mg/L, *<sup>p</sup>* = 0.022; I<sup>2</sup> = 47%) (Figure 4), FPG (MD = −3.52 mg/dL, 95%CI(−5.1,−1.94) mg/dL, *<sup>p</sup>* < 0.001; I2 = 49%) (Figure 5), without affecting weight (MD = −0.89 kg, 95%CI(−4.60,2.82) kg, *p* = 0.638; I <sup>2</sup> = 0%), waist circumference (MD = −0.5 cm, 95%CI(−3.17,2.17) cm, *<sup>p</sup>* = 0.714; I2 = 0%) (Figure S1), SBP (MD =− 0.57 mmHg, 95%CI(−3.2,2.06) mmHg, *<sup>p</sup>* = 0.670; I2 = 12%), DBP (MD = −0.89 mmHg, 95%CI(−2.61,0.83) mmHg, *<sup>p</sup>* = 0.312; I2 = 0%) (Figure S2), FPI (MD = −0.58 mU/L, 95%CI(−1.24,0.09), *p* = 0.091; I <sup>2</sup> = 30%), and HOMA-IR (MD = −0.09, 95%CI(−0.44,0.26), *p* = 0.599; I2 = 61%) (Figure S3).


**Figure 2.** Forest plot displaying mean differences and 95% confidence intervals for the impact of the supplementation with Armolipid Plus® on BMI.




**Figure 3.** *Cont*.


**Figure 3.** Forest plot displaying mean differences and 95% confidence intervals for the impact of the supplementation with Armolipid Plus® on serum levels of TC, TG, HDL-C, and LDL-C.


**Figure 4.** Forest plot displaying mean differences and 95% confidence intervals for the impact of the supplementation with Armolipid Plus® on serum levels of hs-CRP.


**Figure 5.** Forest plot displaying mean differences and 95% confidence intervals for the impact of the supplementation with Armolipid Plus® on FPG.

The effect sizes were robust in the leave-one-out sensitivity analysis and not mainly driven by a single study (data not shown).

A visual inspection of Begg's funnel plots did not show significant asymmetry, suggesting no potential publication bias for the effect of Armolipid Plus® on the efficacy outcomes (Figures S4–S7). This finding was confirmed by the results of Begg's rank correlation test and Egger's linear regression (Table 4).

The Duval and Tweedie trim-and-fill method identified three potentially missing studies on the left side of the funnel plot that resulted in the pooled effect size for DBP reaching statistical significance (Table 4).


**Table 4.** Assessment of publication bias on efficacy outcomes.

BMI = body mass index; DBP = diastolic blood pressure; FPG = fasting plasma glucose; FPI = fasting plasma insulin; HDL-C = high-density lipoprotein cholesterol; HOMA-IR = homeostatic model assessment for insulin resistance; LDL-C = low-density lipoprotein cholesterol; MD = mean difference; SBP = systolic blood pressure; TC = total cholesterol; TG = triglycerides.

#### *3.4. Safety Analysis*

Supplementation with Armolipid Plus® exerted a slight, though clinically insignificant, increase in serum levels of ALT (MD = 2.16 U/L, 95%CI(0.68,3.64) U/L, *p* = 0.004; I <sup>2</sup> = 0%) (Figure 6), without affecting AST (MD = 0.63 U/L, 95%CI(−0.96,2.21) U/L, *p* = 0.437; I <sup>2</sup> = 0%) or CPK (MD = 7.37 U/L, 95%CI(−1.20,15.93) U/L, *<sup>p</sup>* = 0.092; I<sup>2</sup> = 39%) (Figure S8).

**Figure 6.** Forest plot displaying mean differences and 95% confidence intervals for the impact of the supplementation with Armolipid Plus® on serum levels of ALT.

Moreover, supplementation with Armolipid Plus® was not associated with increased risk of either musculoskeletal disorders (OR = 0.78, 95%CI(0.29,2.11), *p* = 0.618; I<sup>2</sup> = 0%) or gastrointestinal disorders (OR = 1.19, 95%CI(0.35,4.06), P = 0.786; I2 = 0%) (Figure S9).

The effect sizes were robust in the leave-one-out sensitivity analysis and not mainly driven by a single study (data not shown).

A visual inspection of Begg's funnel plots did not show significant asymmetry, suggesting no potential publication bias for the effect of Armolipid Plus® on the safety outcomes (Figures S9–S13). This finding was confirmed by the results of Begg's rank correlation test and Egger's linear regression (Table 5).


**Table 5.** Assessment of publication bias on safety outcomes.

ALT = alanine aminotransferase; AST = aspartate aminotransferase; CPK = creatine phosphokinase; MD = mean difference; OR = odds ratio.

The Duval and Tweedie trim-and-fill method yielded one potentially missing study on the right side of the funnel plot, increasing the pooled effect size for ALT, and one potentially missing study on the right side of the funnel plot, increasing the pooled effect size for CPK. In addition, Duval and Tweedie's trim-and-fill method yielded one potentially missing study on the left side of the funnel plot, decreasing the pooled effect size for AST, and one potentially missing study on the left side of the funnel plot, decreasing the estimated risk of gastrointestinal disorders (Table 5).

#### **4. Discussion**

According to our findings, dietary supplementation with Armolipid Plus® exerts a significant effect on BMI and serum levels of TC, TG, HDL-C, LDL-C, hs-CRP, and FPG. Importantly, it is not associated with an increased risk of musculoskeletal symptoms and gastrointestinal disorders, though it results in a slight, though clinically insignificant, increase in ALT serum levels.

To our knowledge, this is the first systematic review and meta-analysis to comprehensively and critically evaluate the existing body of evidence for the use of Armolipid Plus® in daily clinical practice. As a matter of fact, previous meta-analyses on this topic are outdated and not PRISMA compliant [38,39]. Moreover, they included clinical trials that were not adequately controlled for Armolipid Plus® supplementation and clinical studies with an observational design that finally led to not fully reliable results [40–44].

Armolipid Plus® is a dietary supplement widely used in clinical practice and the only combined lipid-lowering nutraceutical recommended by the International Lipid Expert Panel (ILEP) for the management of hypercholesterolemia in statin-intolerant patients [45]. In effect, red yeast rice at the dosage contained in Armolipid Plus® has been shown to be safe also following a recent large meta-analysis of 53 randomized controlled clinical trials enrolling 8535 participants overall [46]. Dietary supplementation with Armolipid Plus® in statin-intolerant patients previously treated with ezetimibe resulted in reductions of approximately 35% in LDL-C and 25% in TG [47], which was similar to results reported for moderate-intensity statins, according to the latest guidelines from the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS) [48].

The lipid-lowering effect of red yeast rice (alone or combined with other lipid-lowering nutraceuticals) is well known, as it has been verified by several meta-analyses of random-

ized controlled clinical trials [49,50]. The interaction between red yeast rice and other natural products with different mechanisms of action, such as the other components of Armolipid Plus®, may have additive or synergistic lipid-lowering effects [51]. As a matter of fact, the inhibition of HMG-CoA reductase by monacolins contained in red yeast rice might be advantageously coupled with other nutraceuticals to enhance the hepatic uptake of cholesterol (berberine, soybean proteins), increase lipid excretion in the bowel (soluble fibers, plant sterols, glucomannan, probiotics), or induce LDL-C excretion (berberine, soy proteins, chlorogenic acid) [52,53]. Furthermore, several studies evaluated the efficacy and safety of red yeast rice in combination with policosanols, a mixture of aliphatic alcohols derived from purified sugar cane, even though the mechanism underlying their lipidlowering effect is still being discussed [54,55]. Policosanols, together with berberine, may also be responsible for the reduction in FPG levels observed after dietary supplementation with Armolipid Plus® [56].

Although there are no trials showing that Armolipid Plus® reduces the risk of ASCVD events, some studies have shown benefits in terms of improved vascular function, as demonstrated by flow-mediated dilation [27] and carotid-femoral pulse wave velocity [57]. Absolute and relative risk reduction (RRR) in CV events with Armolipid Plus® is challenging to estimate based on the available short-term data. There is a linear association between LDL-C reduction and a decrease in ASCVD events, as reported originally by the CTT's(Cholesterol Treatment Trialists') meta-analyses of the statin trials where a 1 mmol/L (~39 mg/dL) LDL-C reduction was associated with a 21–23% RRR in CV events over five years [58]. Robust and growing evidence highlights that this linear association is observed regardless of the LDL lowering approach adopted, i.e., low-fat diet, anion exchange resins, ezetimibe, etc. [59]. On the basis of our findings, it is therefore plausible to expect a 14–15% ASCVD event reduction after long-term dietary supplementation with Armolipid Plus®.

Despite its strengths, this systematic review and meta-analysis has some limitations. One limitation is the heterogeneity for the effect size on TC and LDL-C, which was moderately high, proving that additional evidence is needed to establish the extent of cholesterol reduction that can be achieved following supplementation with Armolipid Plus®. In addition, we had to exclude a relatively large number of clinical trials not compliant with the inclusion criteria for this meta-analysis. The sample size on some laboratory and clinical outcomes was consequently reduced. In particular, some non-significant results (e.g., changes in weight and waist circumference) might be related to a low statistical power.

Nonetheless, the observed results are in line also with a large trial carried out in a setting of the general population involving 1751 volunteers but not included in the meta-analysis, as it did not meet our pre-specified inclusion criteria [42].

Other lipid-lowering nutraceutical combinations could exert a relevant lipid-lowering effect, but the data on Armolipid Plus® are currently most robust.

#### **5. Conclusions**

Pooling data from the available randomized controlled clinical studies, the current systematic review and meta-analysis provides data in support of the use of Armolipid Plus® in clinical practice as add-on treatment to lifestyle modifications for hypercholesterolemia in order to promote improved cardiometabolic health. Further studies to identify a benefit in terms of CV outcomes are required.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2072-664 3/13/2/638/s1, Figure S1: Forest plot displaying mean differences and 95% confidence intervals for the impact of supplementation with Armolipid Plus® on weight and WC, Figure S2: Forest plot displaying mean differences and 95% confidence intervals for the impact of supplementation with Armolipid Plus® on SBP and DBP, Figure S3: Forest plot displaying mean differences and 95% confidence intervals for the impact of supplementation with Armolipid Plus® on FPI and HOMA-IR, Figure S4: Funnel plots detailing publication bias for the effect of supplementation with Armolipid Plus® on weight, BMI, and waist circumference, Figure S5: Funnel plots detailing publication bias for the effect of supplementation with Armolipid Plus® on blood pressure, Figure S6: Funnel plots

detailing publication bias for the effect of supplementation with Armolipid Plus® on serum lipid concentrations, Figure S7: Funnel plots detailing publication bias for the effect of supplementation with Armolipid Plus® on glycemia and markers of insulin resistance, Figure S8: Forest plot displaying mean differences and 95% confidence intervals for the impact of supplementation with Armolipid Plus® on AST and CPK, Figure S9: Forest plots displaying the risk of treatment-emergent adverse events during supplementation with Armolipid Plus®, Figure S10: Funnel plot detailing publication bias for the effect of supplementation with Armolipid Plus® on serum concentrations of ALT, Figure S11: Funnel plot detailing publication bias for the effect of supplementation with Armolipid Plus® on serum concentrations of AST, Figure S12: Funnel plot detailing publication bias for the effect of supplementation with Armolipid Plus® on serum concentrations of CPK, Figure S13: Funnel plot detailing publication bias for risk of treatment-emergent adverse events during supplementation with Armolipid Plus®.

**Author Contributions:** Conceptualization, F.F. and A.F.G.C.; methodology, F.F. and A.F.G.C.; software, F.F.; validation, F.F. and A.F.G.C.; formal analysis, F.F.; investigation, F.F., M.B., P.P.T., and A.F.G.C.; data curation, F.F., M.B., and A.F.G.C.; writing—original draft preparation, F.F. and A.F.G.C.; writing—review and editing, C.K., T.K., M.B., C.M.G.G., A.Š., and P.P.T.; supervision, A.F.G.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, due to the study design (meta-analysis).

**Informed Consent Statement:** Patient consent was waived due to the study design (meta-analysis).

**Data Availability Statement:** Data supporting findings of this analysis are available from the Corresponding Authors upon reasonable request.

**Conflicts of Interest:** A.F.G.C. served as consultant to Meda-Mylan and Sharper; F.F. served as consultant to Meda-Mylan and Neopharmed Gentili s.p.a. The other authors declare no conflict of interest.

#### **References**


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