A double-blind, placebo-controlled clinical trial to assess the effects of a combined nutraceutical on endothelial function in patients with mild-to-moderate hypercholesterolaemia.

INTRODUCTION: There is growing interest in lipid-lowering nutraceuticals; however, there are a relative scarcity of data on combined compounds. This study was aimed to assess the efficacy and tolerability of a combined nutraceutical (CARDIOL® Forte - CF) containing polyunsaturated fatty acids, hydroxytyrosol, Coenzyme Q10, folic acid, B12 and E vitamins, piperine, and red yeast rice in patients with mild-to-moderate hypercholesterolaemia.
MATERIAL AND METHODS: In this single-centre, double-blinded, placebo-controlled study enrolled subjects who were randomised to receive the tested combined nutraceutical for 16 weeks (CF group) or placebo (control group), in association with a low-fat diet. After 8 weeks of treatment, all patients underwent a 15-day washout period; then, a further 8 weeks of treatment was planned.
RESULTS: Of 80 enrolled subjects, 37 completed the study in the CF group and 38 in the control group. After 8 weeks of treatment, low-density lipoprotein cholesterol levels were reduced by 17% in the CF group and by 6.4% in the control group, compared to baseline (p = 0.0001); these changes were improved at the end of study. Total cholesterol and triglyceride levels significantly decreased during treatment; high-density lipoprotein cholesterol did not change. In the CF group, flow-mediated dilation increased by 18.8% after 8 weeks and by 39.3% at the end of treatment. No adverse events or musculoskeletal disorders were reported in either group.
CONCLUSIONS: The tested combined nutraceutical, in association with a controlled diet, can reduce cholesterol levels and improve endothelial function, thus reducing the cardiovascular risk in patients with mild-to-moderate hypercholesterolaemia.

Introduction

Although several programs and health initiatives have been started to diminish the prevalence of modifiable risk factors for cardiovascular disease (CVD), cardiovascular mortality remains the leading cause of death worldwide [1]. A correct diet and healthy life habits, which contribute to optimising the lipid profile, form the most effective intervention to limit the onset and progression of the atherosclerotic process [2]. However, they are often not enough to achieve low-density lipoprotein cholesterol (LDL-C) levels that are recommended as therapeutic targets, and pharmacological treatments often become necessary. Many molecules with antioxidant and anti-inflammatory effects acting on the atherosclerotic process are currently available on the market and contribute to reducing cardiovascular mortality and the burden of the disease [3, 4]. However, some of them, including statins, are associated with adverse events, which prevent their prescription or determine poor adherence to treatment [5].

In recent years, nutraceutical pharmacology has identified many food components with antioxidant and anti-inflammatory activity, with pleiotropic effects able to reduce plasma cholesterol levels and regulate lipid metabolism [6-8].

CARDIOL® Forte is dietary supplement containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), hydroxytyrosol polyphenol of olive oil, coenzyme Q10, folic acid, B12 and E vitamins, piperine, and monacolin K (10 mg) from red yeast rice (RYR), whose components have demonstrated to have lipid-lowering action and/or endothelial protective action [9-14].

Evidence from a meta-analysis including more than 9600 people indicated that different RYR reduced total cholesterol (TC), LDL-C, and triglycerides (TG) and increased high-density lipoprotein (HDL-C) levels. These effects were similar to those reported for statins: pravastatin, simvastatin, lovastatin, atorvastatin or fluvastatin. Furthermore, RYR monacolin K preparations were superior to nicotine and fish oils in controlling lipid metabolism, but comparable or inferior to fenofibrate and gemfibrozil [15]. Although some concerns on RYR safety in terms of musculoskeletal disorders (MuD) have been reported, a recent meta-analysis on 53 randomised studies did not evidence any association between RYR monacolin K and an increased risk of MuD, non- musculoskeletal disorders (non-MuD) and serious adverse events. Therefore, monacolin K could be considered as safe in patients with moderate hypercholesterolaemia [16, 17].

Reducing plasma cholesterol levels improves also the endothelial function, which is involved in development and progression of the atherosclerotic plaque [18-20]. Some evidence indicates that hydroxytyrosol and coenzyme Q10 improved endothelial function and reduced CV risk in hyperlipidaemic patients in primary prevention [21-24].

This double-blinded, placebo-controlled study aimed to assess the efficacy and tolerability of CARDIOL® Forte in patients with mild-to-moderate hypercholesterolaemia as primary prevention [25], to evaluate the effects on endothelial function, and to describe the safety profile of monacolin K supplementation.

Material and methods

A single-centre, double blinded, placebo-controlled study was performed at the Cardiovascular Prevention Unit – Hospital Department ASL Frosinone, Italy on consecutive patients with mild-to-moderate hypercholesterolaemia. Inclusion criteria were: i) age range from 18 to 70 years; ii) LDL-C levels 130–160 mg/dl, confirmed in two consecutive examinations. Exclusion criteria were: i) the presence of cardiovascular diseases; ii) triglyceride levels > 400 mg/dl; iii) use of lipid-lowering agents or drugs that modified lipid metabolism; known thyroid, liver, kidney or muscle disease; atrial fibrillation, bigeminal and trigeminal extra-systolic ventricular or supra-ventricular pulse. All patients were asked to sign an informed consent form before the enrolment. The study was approved by the local Ethics Committee (Comitato Etico Locale Lazio 2) and was performed according to the Helsinki Declaration.

During a screening phase, patients underwent a clinical-instrumental examination, in which anthropometric parameters, echocardiography, colour Doppler echocardiography, colour Doppler echocardiography on supra-aortic vessels, transaminases, creatine phosphokinase, renal function – creatinine and azotemia – HDL-C, LDL-C, triglycerides, fasting glycaemia, and glycated haemoglobin were recorded. At baseline before randomisation, flow-mediated dilatation (FMD) of the brachial artery was measured by echo-Doppler, as per the International Brachial Artery Reactivity Task Force [26, 27]. The measurement was performed in the morning, after 12-hour fasting, always by the same operator during the study.

After the enrolment, patients were randomised to receive CARDIOL® Forte for a total of 16 weeks (CF group) or placebo (control group) (both kindly furnished by U.G.A. Nutraceuticals, Monza, MB, Italy), in combination with a diet with low lipidic content (2000 kcal, 55% carbohydrates, 20% proteins, and 25% lipids): after 8 weeks of treatment, all patients underwent a 15-day wash-out period, then a further eight weeks of treatment were planned.

Blood samples were collected at baseline, after 8 weeks of treatment (visit 1), at the end of the wash-out period (visit 2), and at the end of the study (visit 3). During the scheduled visits, adherence to diet, treatment efficacy, and duration of response were evaluated in both groups; tolerability was monitored throughout the study, reporting all minimal changes in enzymatic levels that could suggest any eventual MuD and non-MuD occurrence.

Statistical analysis

Primary endpoints were to determine the change in total cholesterol, LDL-C, HDL-C, and triglycerides of nutraceutical supplement and to evaluate its effect on endothelial function. Hepatic, renal, and muscular tolerability was specifically evaluated [28, 29].

Considering an error α as 0.05 and an error β as 0.20, and a statistical power as 0080, at least 71 patients should be included in this analysis. Clinical and demographic data and measured variables were compared using Student’s test for unpaired data; the ANOVA test was used to estimate the response to different treatments (i.e. for comparing the endothelial function). Data were reported as mean and standard deviation and were considered as statistically significant when p < 0.05. Statistical analysis was performed with SSPS for Windows version 21.0.

Results

Study population

Of 80 enrolled patients, 75 completed the study (37 in the CF group and 38 in the control group), while 5 patients were excluded for protocol violations: 3 patients in the CF group for lack of adherence to treatment and 2 patients in the control group for lack of adherence to the diet. Clinical and demographic characteristics are summarised in Table I. In addition to hypercholesterolaemia, concomitant cardiovascular risk factors, including smoke, diabetes, and hypertension, were present in 64 patients: 58 patients had one more risk factor, and eight patients had two more risk factors. In the CF group, there were 24 patients with hypertension, 5 smokers, and 3 patients with diabetes; in the control group 22 patients had hypertension and one diabetes, while 11 were smokers.

Table I

Main baseline demographic and clinical characteristics of two groups of treatment

Parameter	CF group(N = 37; 23 women)		Control group(N = 38; 21 women)		P-value
	Mean	SD	Mean	SD
Age [years]	57.3	9.3	54.3	9.8	0.178
BMI [kg/m2]	32.8	5.1	28.5	4.1	0.561
TC [mg/dl]	217.6	14.1	221.6	14.1	0.222
HDL-C [mg/dl]	58.7	14.3	58.3	13.8	0.899
LDL-C [mg/dl]	156.3	30.8	153.5	32.2	0.064
TG [mg/dl]	130.8	65.3	117.8	33.8	0.286
Glucose [mg/dl]	93.9	13.1	97.3	11.3	0.479
Glycated hemoglobin (%)	5.4	0.3	5.4	0.4	1.000

BMI – body mass index, TC – total cholesterol, HDL-C – high-density lipoprotein-cholesterol, LDL-C – low-density lipoprotein-cholesterol, TG – triglycerides.

Lipid profile

After 8 weeks of treatment (visit 1), LDL-C levels were reduced by 17% in the CF group and by 6.3% in the control group compared to baseline (p = 0.002). These changes were persistent until the end of the wash out period (visit 2), with further reduction by 23.5% in the CF group and by 14.1% in the control. At the end of the study (visit 3), LDL-C in the CF group was reduced by 34.3%, while in the control group by 22.6% (p = 0.013). Furthermore, TC and TG levels were significantly decreased throughout the study in the CF group, while in the control group TC was reduced by 8.5 ±5.18%, and no change was observed in TG levels (p = 0.522) (Figure 1). HDL-C did not change in both groups (Table II).

Figure 1

Change in total cholesterol, low-density lipoprotein – cholesterol and triglycerides in CF and control groups at the end of the study (visit 3) *P < 0.005.

Table II

Total cholesterol, HDL-C, LDL-C and triglycerides levels in CF and control groups and change from baseline to each study visit

Parameter		CF group	Control group	Change % in CF groups vs. baseline	Change % in Control group vs. baseline	P-value
TC:
	T0	217.6 ±14.1	221.6 ±14.1			0.223
	T1	182.9 ±24.7	188.0 ±20.0	16.1	15.1	0.0001
	T2	177.5 ±22.7	184.8 ±21.4	18.4	16.6	0.038
	T3	171.1 ±21.4	202.8 ±20.0	21.3	8.5	0.007
HDL-C:
	T0	58.7 ±14.2	58.3 ±13.8			0.899
	T1	57.7 ±11.9	55.1 ±10.9	1.7	5.5	0.334
	T2	55.8 ±12.1	53.6 ±10.5	4.9	8.1	0.401
	T3	55.1 ±9.9	59.1 ±11.5	6.3	-1.4	0.104
LDL-C:
	T0	156.5 ±32.2	152.3 ±30.8			0.060
	T1	129.8 ±11.6	142.7 ±10.8	17.0	6.3	0.002
	T2	119.7 ±16.9	130.7 ±15.3	23.5	14.1	0.004
	T3	102.7 ±14.1	117.8 ±13.8	34.3	22.6	0.013
Triglycerides:
	T0	130.7 ±65.3	117.8 ±33.8			0.284
	T1	115.5 ±48.8	108.7 ±31.6	11.6	7.7	0.476
	T2	110.9 ±44.7	110.7 ±33.9	15.1	6.0	0.983
	T3	103.7 ±45.2	112.4 ±37.9	20.6	4.6	0.368

Flow-mediated dilatation

In the CF group, FMD increased by 18.8 ±3.2% after 8 weeks of treatment, and a further increase was observed on visit 2 by 22.9 ±4.4% and on visit 3 by 39.3 ±5.2%; in the control group, FMD increased by 10.9 ±4.2% on visit 1 and by 17.1 ±5.2% on visit 3. The increases in FMD recorded within each group are statistically significant (Table III).

Table III

Baseline flow-mediated dilatation and change after treatment

FMD	CF group	Change %	P-value	Control group	Change %	P-value
T0	12.21 ±0.03			11.12 ±0.04
T1	14.53 ±0.04	18.8 ±3.2		12.35 ±0.04	10.9 ±4.2
T2	15.02 ±0.04	22.9 ±4.4		13.14 ±0.04	18.0 ±4.1
T3	17.06 ±0.05	39.3 ±5.2	< 0.001	13.05 ±0.03	17.1 ±5.2	0.004

Tolerability

No occurrence of MuD, non-NuD, or serious adverse events were reported during the study. In the CF group, CPK levels decreased by 7.8% after eight weeks of treatment on visit 1 and by 13.2% on visit 2; a small increase was observed on visit 3 (by 6.2%) compared to visit 2. There was no significant difference between the two groups in all visits (Table IV). AST and ALT were reduced in both groups, while creatinine did not change (Table V).

Table IV

Changes of creatinine phosphokinase levels

Parameter	NC group			Control group
	Mean	SD	Change %	Mean	SD	Change %
T0	113.60	55.13		114.32	36.79
T1	104.72	37.02	7.81	104.13	44.21	8.91
T2	98.61	33.91	13.19	102.46	43.82	10.37
T3	104.95	43.95	7.61	110.15	43.02	–3.64

Table V

Renal and hepatic tolerability

Parameter	CF group			Control group
	Baseline (T0)	End of treatment (T3)	P-value	Baseline (T0)	End of treatment (T3)	P-value
AST	19.83 ±4.11	17.69 ±2.96	0.012	21.17 ±5.76	17.54 ±2.89	0.001
ALT	19.91 ±6.12	17.74 ±5.31	0.109	22.97 ±7.96	18.21 ±4.47	0.003
Creatinine	0.78 ±0.11	0.79 ±0.12	0.699	0.85 ±0.13	0.82 ±0.17	0.345

AST – aspartate aminotransferase, ALT – alanine aminotransferase.

Discussion

There is growing interest in lipid-lowering nutraceuticals; however, there are a relative scarcity of data on combined compounds and on their effect on vascular function [30].

This double-blinded, placebo-controlled study showed that the tested combined product had superior efficacy to placebo in reducing LDL-C levels in patients with mild-to-moderate hypercholesterolaemia in primary prevention after 16 weeks of treatment. Our results were consistent with literature evidence [31, 32].

An early LDL-C reduction was observed after 8 weeks of treatment, and it was enhanced at the end of washout period, thus indicating a persistent and durable response compared to diet alone. The tested combined product improved also the endothelial function, as documented by a significant FMD increase, in all study visits. This is of particular interest because a meta-analysis of 35 studies including 17,280 subjects showed that each 1% increase in FMD is associated with a 12% reduction in predicted cardiovascular events (relative risk = 0.88, 95% CI: 0.84–0.91, p < 0.001) [33, 34].

Overall, these results were mainly related to pharmacokinetic and pharmacodynamic characteristics of nutraceutical supplement. Indeed, the tested combined product contains molecules that have different mechanisms of actions and synergistically act to globally reduce the cardiovascular risk in the CF group. Monacolin K inhibits HMG-CoA reductase, omega-3 EPA and DHA fatty acids reduce triglyceride synthesis, and the concomitant administration of vitamin E, coenzyme Q10, and hydroxytyrosol significantly reduces cholesterol levels and LDL-C oxidation, thus ameliorating endothelial-dependent vasodilation in the CF group compared to controls.

RYR alone or in combination with monacolin K demonstrated a favourable safety profile; in their meta-analysis, Fogacci et al. did not find statistical increased probability of the occurrence of adverse events [17]. Consistently, our data showed that monacolin K did not determine any MuD or non-MuD, as suggested by normal levels of CPK and normal hepatic and renal functioning in both study groups.

The study has some limitations. The population size was relatively small but adequately powered. Moreover, the study design – randomised and double-blinded – and treatment duration allowed us to describe a difference among treatment and control groups. Some patients included in the analysis were under anti-hypertensive therapy, which could affect endothelial function. However, in all cases the therapy was well established and ongoing before the enrolment; therefore, it could not be considered as a relevant cofounding factor in FMD assessment. Finally, the test of a combined compound does not allow us to draw conclusions about the responsibility of a single component as regards the observed effects. However, data literature already confirmed the effect of the single nutraceutical components, while we aimed to test the efficacy and tolerability of the tested combined product.

In conclusion, a nutraceutical supplement containing lipid-lowering and antioxidant components, in association with a lifestyle change and a controlled diet, can significantly reduce cholesterol levels and improve endothelial function, thus reducing the cardiovascular risk in patients with mild-to-moderate hypercholesterolaemia.