2.1.1. Qualitative 1H-NMR Analysis

Four characteristic spectra are illustrated in Figure 1 (the 1H-NMR spectra of all RYR DS analyzed are shown in Figure S1). The monacolin resonances were identified by comparing the 1H-NMR spectra of RYR DS with those of standard monacolins whose chemical structures are respectively characteristic of monacolins in lactone form (MK and compactin (CP)), monacolins in hydroxyl acid form (MKA), dehydromonacolins (DeMK) and dihydromonacolins (DiMK), the main monacolin derivatives found in RYR (see Figure 2 for chemical structures) [10,12,13]. Their 1H-NMR assignments are given in Table 2. A complete one-dimensional (1D) and two-dimensional (2D) description of the 1H and 13C-NMR signals of standard MK, MKA, CP and DiMK is presented in Table S1.

The resonances of the hexahydronaphthalene ring ethylenic protons (H5, H6, H4 at ≈ 6.01, 5.84 and 5.56 ppm, respectively) are representative of all the monacolins usually found in RYR, except dihydromonacolins as the δ of the ethylenic protons H5 and H6 of DiMK resonated at 5.42 and 5.69 ppm, respectively (Figures 1 and 3, Table 2). The H1 signal (q) at ≈ 5.33 ppm is characteristic of monacolins with a hexahydronaphthalene ring and an ester group (O-CO-R) in position 1. Indeed, for the monacolins with an OH (monacolin J (MJ) and derivatives) or H substituent (monacolin L (ML) and derivatives) instead of ester (Figure 2), the H1 proton(s) resonated respectively at ≈ 4.24 ppm or 1.17 and 1.77 ppm [1,19]. The H20 multiplet at ≈ 4.60 ppm is characteristic of all the monacolins in lactone form including dihydromonacolins but not of monacolins in hydroxyl acid form and dehydromonacolins (Table 2, Figure 3). The H22 signal at ≈ 4.25 ppm is also characteristic of all the monacolins in lactone form (Table 2, Figure 3).

All these monacolin resonances mainly arise from MK, the main monacolin present in RYR DS [1] but other characteristic signals of monacolins were also identified: dehydromonacolins (H22 at 7.03 ppm), dihydromonacolins (H6 at 5.69 ppm) and monacolins in hydroxyl acid form (H22 at 4.05 ppm and H20 at 3.63 ppm) (Figure 1B for some signals).

The singlet of the H1 of citrinin at 5.94 ppm and 8.45 ppm (see footnote 4 of Table 2 for explanation) was never observed, which is not surprising due to the expected very low amount of this compound even if it could have been present [12,13].

Beside the assignments of the different monacolin families, 1H characteristic signals of many other compounds mentioned or not on the label of the RYR DS were detected (Table 2). Fatty acids, both saturated (SFA) and non-conjugated unsaturated (UFA), were found in all the formulations (Figure 1 and Table 3) as it has been reported that they represent ≈ 3% of RYR extracts, each group in approximately identical proportion (≈1.4%) [1]. The presence of glycerol and glucose was observed in 25 and 24 samples, respectively. The singlet at ≈5.50 ppm characteristic of the H4 of monascin and/or other pigments with the same skeleton (ankaflavin, monascuspiloin, monaphilones A or B) (Figure 2) was observed in 20 formulations. Some other compounds, generally mentioned on the label of the RYR DS, were detected in few samples: sorbitol and piperine in two samples as well as carnitine, vitamin B3, vitamin C, chlorogenic acid and isopropyl alcohol in one sample (Tables 2 and 3).


**Table 1.** Red Yeast Rice (RYR) dietary supplements investigated in this study.

<sup>1</sup> Dietary supplements bought on internet web sites (I) or in health food stores (HFS).

**Figure 1.** 1H-NMR spectra of selected RYR dietary supplements (DS) recorded in CD3CN:D2O (80:20). Entire spectrum of DS **15** (**A**) and enlarged downfield region (4–9 ppm) (**B**) of the DS **3**, **15**, **20** and **23**. DeM: dehydromonacolins, Monacolins lactone: monacolins in lactone form, Monacolins OHAc: monacolins in hydroxyl acid form, Mo: monascin and other pigments with the same skeleton, FA: fatty acids (saturated and unsaturated), -: non-conjugated unsaturated fatty acids, C: carnitine, Vit: vitamin, TSP: sodium 2,2,3,3-tetradeutero-3-(trimethylsilyl) propanoate, set at 0 ppm.

**Figure 2.** *Cont*.

**Figure 2.** Chemical structures of monacolins and pigments discussed in this study.


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solution at physiological

are observed. \* From the literature data, it was not possible to assign the resonances of H9, H10 and H11 to a specific form. 5 As a model of

 pH, one

diastereoisomer

 being major and the other minor. As the solvent used in the present study is a CD3CN:D2O

 (80:20) mixture, the two forms (I) and (II)

non-conjugated

 unsaturated fatty acids.

**Figure 3.** 1H-NMR spectra of standards of monacolin K in lactone form (MK) and in hydroxyl acid form (MKA), dehydromonacolin K (DeMK), dihydromonacolin K (DiMK) and monascin recorded in CD3CN:D2O (80:20). The chemical structures of all the compounds and their protons numbering are given in Table 2. (\*) The signal of H11 of monascin disappears with time due to exchange with D2O.
