A red yeast rice-olive extract supplement reduces biomarkers of oxidative stress, OxLDL and Lp-PLA₂, in subjects with metabolic syndrome: a randomised, double-blind, placebo-controlled trial

‘Syndrome X’, now called the metabolic syndrome’ (MetS), refers to the clustering of cardiovascular risk factors, including abdominal obesity, pre-diabetes, high blood pressure and dyslipidaemia. A combination of several of these conditions increases the risk for the development of chronic diseases such as type 2 diabetes mellitus and cardiovascular diseases. To date, the prevalence of MetS is increasing at a disturbing rate, and within the context of its proven association with cardiovascular disease, the leading cause of mortality in the modern world, more research is needed to unravel new therapeutic approaches [1].

Oxidative stress is involved in clinical manifestations related to MetS [2‐4]. Higher plasma concentrations of malondialdehyde (MDA) [5, 6] and 8-iso-prostaglandin F2α [7, 8] were associated with MetS. Also, increased oxidised low-density lipoprotein (OxLDL) levels—OxLDL is a very important biomarker of oxidative lipid damage [9], given its pro-inflammatory and pro-atherogenic activity [10]—are observed in MetS. [11, 12]. In recent years, there has also been growing interest in lipoprotein-associated phospholipase A₂ (Lp-PLA₂), a key enzyme that catalyses OxLDL hydrolysis, thus producing pro-inflammatory mediators such as lysophosphatidylcholine. Increased plasma Lp-PLA₂ activities have been reported in patients with MetS [9, 10], and Lp-PLA₂ is now identified as a good determinant of MetS [13]. Moreover, the association of this enzyme with coronary heart disease risk has been shown in different studies [14, 15]. Therefore, assessment of this biomarker is important in risk assessment of MetS as well as in the evaluation of a potential treatment. In early stages of type 2 diabetes mellitus, a positive correlation is observed between OxLDL and Lp-PLA₂ [16].

In cases of failure of lifestyle optimisation, treatment of MetS comprises a combination of drugs against particular aspects of MetS, including anti-hypertensive therapy and treatment of dyslipidaemia.

Novel health strategies are needed, however. In particular, anti-oxidant functional foods or food supplements may have beneficial effects on MetS. In this context, a double-blind, placebo-controlled, randomised trial of a commercially available food supplement combining red yeast rice (RYR) (Oryza sativa L. fermented by the yeast Monascus purpureus Went) and olive fruit (Olea europaea L.) extract was conducted in patients with MetS. The combination in this food supplement of red yeast rice and olive fruit extract (RYR-olive extract) was based on their health effects, which have been documented in the literature [17‐25]. This has led to the approval of health claims by the European Food Safety Authority (EFSA) for both RYR and polyphenols in olive oil [20, 26]. Although the anti-oxidant effect of olive oil has been proven in many studies [17‐21], which has led to the approval of the EFSA health claim, this claim has not been authorised yet for olive fruit extract.

Apart from the evaluation of the effect of this combination of olive fruit extract with RYR on clinical biomarkers of MetS, including waist circumference, lipid status and blood pressure, which have been published recently [27], this updated sub-analysis of the previous study was focused on its effect on biomarkers of oxidative stress. The effect on OxLDL levels has been reported previously [27] to correlate with the observed decreased LDL levels. Because MetS increases the risk of cardiovascular disease and a significant reduction of OxLDL has been observed, our extended study was focused on the assessment of specific biomarkers of oxidative damage to lipids and lipoproteins, including plasma MDA and Lp-PLA₂. Results for OxLDL have been linked to Lp-PLA₂ levels. In addition, 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of oxidative damage to DNA, was assessed in urine. To our knowledge, this is the first study including an assessment of the effect of RYR and olive extract on oxidative stress in patients with MetS.

Fifty participants complying with the inclusion criteria entered the study and were randomly allocated to the RYR-olive extract (n = 26) or placebo (n = 24) group [27]. The flow of patients through the study is shown in Fig. 1.

Baseline characteristics of the study population are reported elsewhere [27], and there were no differences in clinical parameters or in dietary and physical habits between the RYR-olive extract intervention and placebo groups. Daily consumption of this RYR-olive extract, containing 10.82 ± 0.84 mg of monacolin K and 9.32 ± 0.54 mg of hydroxytyrosol, resulted in a 24% decrease of LDL levels. Plasma OxLDL was significantly reduced in the RYR-olive extract-treated group (absolute difference −19.35 ± 4.43 U/L) compared with the placebo group (absolute difference 3.65 ± 2.35 U/L) (p < 0.001). A relative reduction of OxLDL of 20% was observed in patients receiving RYR-olive extract (p < 0.001) [27]. In the present study, the effects on biomarkers of oxidative stress were assessed. Baseline oxidative stress levels are depicted in Table 1.

No significant difference was found in plasma MDA levels between the RYR-olive extract and placebo groups. Absolute and relative mean differences in the RYR-olive extract intervention group were 0.01 ± 0.02 μM and 2.35 ± 3.69%, respectively, and in the placebo group, the corresponding values were 0.07 ± 0.03 μM and 17.10 ± 6.81%, respectively (after 8 weeks). Figure 2 depicts mean MDA concentrations in both groups at baseline and after 8 weeks.

In 26 patients, baseline measurements of plasma Lp-PLA₂ were moderate to high (>151 nmol/ml/minute). After 8 weeks of the intervention, Lp-PLA₂ was specifically screened in this subgroup. Patients in the RYR-olive extract-treated group (n = 13) had significantly reduced Lp-PLA₂ levels after 8 weeks (−12.72 ± 5.44 nmol/ml/minute) of supplementation compared with those in the placebo group (n = 13; 40.16 ± 6.12 nmol/ml/minute) (p < 0.001). The mean reduction of Lp-PLA₂ in the intervention group was 6.69%. In the placebo group, no reduction of Lp-PLA₂ could be observed in any patient. The results of Lp-PLA2 screening are depicted in Fig. 3.

In the subgroup of patients with high Lp-PLA₂ (n = 26), a Pearson correlation test was conducted to investigate a possible correlation between before-and-after differences of OxLDL [27] and before-and-after differences in Lp-PLA₂. A positive correlation was observed between these two parameters (r = 0.740, n = 26, p < 0.001) (Fig. 4).

With regard to urinary 8-OHdG, a biomarker of oxidative DNA damage, no significant difference was observed. Figure 5 shows mean urinary 8-OHdG levels in both groups at baseline and after 8 weeks.

Current evidence suggests that oxidative stress may be an early manifestation which plays a central role in the development of MetS. Thus, therapeutic approaches which target oxidative stress may delay disease progression [4, 30‐32].

To our knowledge, this is the first study involving an investigation of the effect of a preparation of RYR and olive fruit extract on biomarkers of oxidative stress. Apart from a significant reduction of total cholesterol, LDL, triacylglycerol and OxLDL levels, which have been reported previously [27], the 2-month administration of this RYR-olive fruit extract supplement resulted in a significant decrease in plasma Lp-PLA₂, a biomarker of oxidative damage to lipoproteins. After 8 weeks of treatment, OxLDL was reduced by 20% and Lp-PLA₂ by 7% in the RYR-olive extract-treated group. In recent years, many studies have shown the contribution of these biomarkers to the inflammation process and the risk of coronary disease. OxLDL has been shown to be an important factor in atherosclerosis initiation and development [33, 34]. Other recent studies have shown that circulating Lp-PLA₂ activity is associated with risk of coronary disease and vascular mortality [14, 15, 35]. Recently, the association of OxLDL and Lp-PLA₂ has been proven both ex vivo in human atherosclerotic lesions and in vivo in patients with MetS [9, 33].

In this study, the reduction of Lp-PLA₂ was positively correlated with that of OxLDL [27] after 8-week treatment with the RYR-olive fruit extract in the subgroup of patients with high Lp-PLA₂ activity (>151 nmol/ml/minute). This is an interesting result which needs to be studied further in long-term follow-up trials. Given the recent associations of high Lp-PLA₂ activity in plasma and cardiovascular risk, supplementation of this anti-oxidant supplement in patients with MetS, specifically those with increased Lp-PLA₂ activity, might reduce the manifestation of severe cardiovascular events.

The protective effects of olive oil polyphenols against oxidative damage to lipids, particularly oxidation of LDL, have been shown in different recent studies, and dose-effect relationships have been established [17‐19, 21]. Because OxLDL plasma levels and Lp-PLA₂ plasma activity in the food supplement-treated group decreased, the anti-oxidant effects of this supplement are confirmed in the present study. Although the observed anti-oxidant effects were at least in part due to the hydroxytyrosol-rich olive fruit extract, it was not possible with the present study design to unambiguously assign the anti-oxidant activity to the olive fruit extract. The product also contained RYR delivering 10.84 ± 0.84 mg of monacolin K (of which 5.88 ± 0.46 mg was in lovastatin lactone form). This is an important aspect because researchers in recent studies also reported plasma OxLDL level- and Lp-PLA2 activity-reducing effects of certain statins, such as simvastatin and pravastatin [14, 34]. Although those trials were longer and the doses of the synthetic statins administered were higher than the monacolin K delivered by the RYR in the present study, it is possible that the monacolin K contributed to the anti-oxidant effects observed here. To dedicate the biologic effects to the specific components in the food supplement, and to investigate possible synergistic effects of combining both, a trial should be conducted to compare the effect of an RYR extract on one hand and an olive fruit extract (both with comparable composition to the extracts tested in this trial) on the other, on these biomarkers of oxidative stress.

The short follow-up period of 8 weeks is also a limitation of the study. A follow-up study over a longer period should be conducted to draw definite conclusions on the effect of the reduction of these biomarkers of oxidative stress on the reduction of cardiovascular disease occurrence.

MDA, another biomarker of oxidative damage to lipids, did not change significantly, which has been observed regularly in early oxidative stress-related disease stages. In general, MDA values obtained tend to differ in function of the experimental set-up in animal studies, as well as in the severity of the observed pathology and analytical technique used for MDA quantification. This is apparent both in observational studies comparing disease and control groups and in interventional trials investigating a potential therapy. Armutcu and co-workers, using a spectrophotometric method, did observe significantly higher MDA values in subjects with MetS than in a healthy control group [5]. Our previous research, however, demonstrated that MDA quantification by HPLC is preferable, thus avoiding possible interfering factors [29].

In animal models, a clearly developed pathology was found to be important to observing significant differences in plasma MDA because a diabetic rat model and an atherosclerotic rabbit model did show increased plasma MDA values (indicating increased systemic oxidative lipid damage), whereas this was not seen in vitamin E-deficient rats [36].

Other biomarkers of oxidative lipid damage have been reported to differ in patients with MetS. Thiobarbituric acid reactive substances (TBARS) have been found to be increased in obese patients with MetS (mean BMI 33 kg/m²) [6]. In that previous study, BMI (33 kg/m²) was more elevated than in our population (BMI 27.5–27.8 kg/m²), and TBARS levels were determined spectrophotometrically. Plasma F2-isoprostanes were positively associated with most components of MetS in a recent report by Black et al. [37]. In that same report, however, 8-OHdG, a biomarker of oxidative damage to DNA, did not show significant changes. In our study, a significant change in 8-OHdG was also not observed. Parameters of oxidative lipid damage are more likely to be affected in MetS, involving altered lipid metabolism, than biomarkers of DNA damage. This reflects two distinct pathways of oxidative damage that are not necessarily closely inter-related and confirms the importance of measuring multiple markers of oxidative stress [37].

Assessment of both biomarkers MDA and 8-OHdG after supplementation over a longer period merits attention to draw definite conclusions regarding these parameters. Given the increased risk of major cardiovascular events among patients with MetS, the present data regarding the anti-oxidant effects of an RYR-olive fruit extract, as well as its effects on blood lipid parameters and blood pressure as reported in our recent publication [27], clearly demonstrate the value of this food supplement in preventing deterioration of metabolic abnormalities.

After 8 weeks of treatment of subjects with MetS in a double-blind, placebo-controlled, randomised trial, daily intake of a RYR-olive fruit extract supplement containing 10.82 ± 0.84 mg of monacolin K and 9.32 ± 0.54 mg of hydroxytyrosol as bioactive compounds resulted in a 7% decrease of Lp-PLA₂ and correlated with a 20% decrease of plasma OxLDL. The reduction in these oxidative stress biomarkers may lead to a reduced risk of cardiovascular disease in patients with MetS in the longer term. Future investigations should be focused on the long-term anti-oxidant effects of this supplement and the prevention of cardiovascular events.