2.4.1. About Monacolin Labelling of RYR DS

Only 18 of the 31 RYR DS (58%) tested specified a monacolin(s) content on their label: 14 indicated a level of MK, 1 the sum MK + MKA, and the label was imprecise for 3 DS, 1 indicating "*Monascus* *purpureus*" and 2 "monacolin" (Table 6). The amount of MK measured was in the range 90%–110% of declared amount for only 3 of the 14 DS mentioning a quantity of MK (DS **13**, **23** and **27**). If we hypothesize that the label "MK" includes MK + MKA, three additional DS (**15**, **22** and **24**) were in the range 90%–110%. If we consider that the claimed dose corresponds to TotalM for the three DS indicating "*Monascus purpureus*" (DS **4**) or "monacolin" (DS **25** and **30**), the measured amount was between 90% and 110% (1H-NMR and UHPLC mean amount for DS **25**) (Table 6). So, 50% of the formulations analyzed contained less than the declared amount of monacolin(s). It is also interesting to note that three DS (**10**, **11** and **12**) contain a very small amount of MK (or MK + MKA) compared to the advertised dose (Table 6). It can thus be concluded that the monacolin label information is not reliable for many RYR products. This deviation between labelled and measured contents has already been reported by Mornar et al. [12] in five DS.

#### 2.4.2. About the Variability of Monacolins Consumed Per Day in RYR DS

There is great variability in the daily consumption of monacolin amounts in terms of MK, MK + MKA, and TotalM (or TotalM-DiMK) calculated from the UHPLC and 1H-NMR data taking into account the recommended serving(s) per day indicated on the labels of the RYR products (Table 6). The amounts of MK, MK + MKA and TotalM consumed daily ranged respectively between 0.08 and 46.2 mg, 0.08 and 47.6 mg, 0.08 and 47.7 mg (UHPLC values) and 0.6 (the very low monacolin content in 3 samples (DS **11**, **12** and **29**) being undetected) and 47.8 mg (NMR values). Although the range of monacolins consumed is the same considering MK, MK + MKA or TotalM, this is not true for all formulations. Indeed, MK represented less than 60% of TotalM in 45% (14/31) of the formulations analyzed and MK + MKA accounted for less than 80% of TotalM in ≈40%, emphasizing the helpfulness of the NMR method which allows the content in all monacolins to be easily determined.

About half of the RYR DS analyzed (48%, 15/31) contained more than 7 mg of MK + MKA or 8 mg of TotalM per recommended daily serving, whereas for ≈45% (14/31), the daily intake of MK + MKA or of TotalM was less than ≈ 3 or 4 mg respectively. Although only single batch formulations have been analyzed and the monacolin content may vary from batch to batch, the question of how effective these levels of monacolin can be for lowering/regulating cholesterol levels can be raised. In 2011, the EFSA concluded that a cause and effect relationship has been established between the consumption of 10 mg per day of MK (sum of lactone and hydroxyl acid forms) from RYR DS and the maintenance of normal low-density lipoproteins-cholesterol (LDL-C) levels [38]. However, several clinical trials using RYR products at daily MK doses well below 10 mg (around 5 mg and even 3 mg) without any other cholesterol-lowering agents such as berberine, policosanol or garlic, showed a statistically significant reduction in LDL-C compared to placebo but these low values either corresponded to MK in the sole lactone form [40,42] or it was not indicated whether they included the hydroxyl acid form of MK [3,4,46]. Nevertheless, when the RYR composition in monacolins was available, the amounts of MK + MKA and TotalM were always at least around 7 mg and 10 mg respectively [40,42]. From these data, it can be concluded that a daily dietary intake of ≈7 mg of MK + MKA or ≈8 mg of TotalM is sufficient to induce a cholesterol-lowering/regulating effect. On the other hand, the very low daily intake of monacolins found in ≈ 45% of the RYR DS analyzed might not lead per se to a significant reduction/regulation of cholesterol levels.



#### *Molecules* **2020** , *25*, 317
