Anti-Proliferative Effects of Iridoids from Valeriana fauriei on Cancer Stem Cells

We isolated seven new iridoid glucosides (valerianairidoids I–VII; 1–3, 6, 7, 9, and 12) and six known compounds from the methanol extract of the dried rhizomes and roots of Valeriana fauriei. Chemical and spectroscopic data were used to elucidate the chemical structures of the seven new iridoid glucosides, and their absolute configurations were determined by comparing their electronic circular dichroism (ECD) spectra with those determined experimentally. Aglycones 1a, 6a, and 9a, which were obtained by enzymatic hydrolysis of the isolated iridoid glucosides, exhibited anti-proliferative activities against cancer stem cells (CSCs) established by a sphere-formation assay using human breast cancer (MDA-MB-231) and human astrocytoma (U-251MG) cells. Interestingly, these iridoids selectively showed anti-proliferative activities against CSCs from MDA-MB-231 cells. These results suggest that the iridoids obtained in this study may have potency as a breast cancer treatment and as preventive agent via exterminating CSCs.


Introduction
Valeriana fauriei Briquet (Valerianaceae) is abundant in both Japan and China and has been used for centuries as a sedative and antispasmodic agent [1]. Iridoids bearing isovaleryl moieties [2] and cyclized guaiane-type sesquiterpenes [3], among others, have been isolated as V. fauriei constituents. As part of our ongoing research aimed at discovering new cancer-treatment and preventive agents [4][5][6][7], we found that cyclized guaiane-type sesquiterpenes and lignans from V. fauriei show cell-death-inducing activities against adriamycin-treated (ADR-treated) HeLa cells by inhibiting heat-shock protein (HSP), and through anti-proliferative effects against cancer stem cells (CSCs) and human astrocytoma cells [8]. As part of our continuing study, we isolated iridoid glycosides and evaluated their anti-proliferative activities against CSCs.
CSCs have been identified in many types of malignancy-including leukemia and breast, colorectal, and brain cancers [9]-which are leading causes of cancer recurrence following anti-cancer drug treatment because these cells are resistant to current anti-cancer drugs and radiation therapy, and play important roles in metastasis by acquiring mesenchymal properties, including improved motility and enhanced invasiveness [10]. Therefore, compounds that are anti-proliferative against CSCs are potentially useful in cancer treatment and as preventive agents. Pyranocoumarin [11], lignans [12], and sesquiterpenes [13] have been reported as naturally occurring agents that are anti-proliferative toward CSCs. In this report, we describe the isolation, structure determination, and anti-proliferative activities of isolated iridoid glycosides and their derivatives against CSCs obtained using a sphere-formation assay with MDA-MB-231 and U-251MG cells.
ti-proliferative toward CSCs. In this report, we describe the isolation, structure determination, and anti-proliferative activities of isolated iridoid glycosides and their derivatives against CSCs obtained using a sphere-formation assay with MDA-MB-231 and U-251MG cells.

Determining the Structures of Valerianairidoids
In addition, H-5/H-9, H-5/H-6β, and H-9/H-10 NOESY cross-peaks suggest that H-5, H-6β, H-9, and H-10 are located on the same side. We obtained aglycone 1a by enzymatically hydrolyzing 1. While 1a was identified to be a patrinoside-aglucone by NMR spectroscopy and MS, its absolute configuration was not previously discussed in [16]. Therefore, we determined the absolute configuration of 1a by calculating its electronic circular dichroism (ECD) spectrum. The calculated ECD data for 1S,5S,7S,8S,9S-configured 1a are in good agreement with the experimental data, whereas the calculated ECD spectrum of ent-1a (1R,5R,7R,8R,9R) is essentially the mirror image of that acquired experimentally ( Figure 4). Finally, 1 was subjected to acid hydrolysis using 20% aqueous H2SO4 in 1,4-dioxane, which yielded D-glucose by HPLC separation of its diastereomeric tolylthiocarbamoyl thiazolidine derivative [17]. The coupling constants (J = 7.6 Hz) for the anomeric position of the two glucoses suggested that they have β-configurations at the glycosidic bonds. We conclude that the chemical structure of valerianairidoid I (1) is as shown in Figure 1, based on the evidence provided above.  . Their molecular formulas (C 32 H 52 O 17 ) were determined using HRMS and 13 C NMR spectroscopy. A comparison of the NMR data for 1, 2, and 3 reveals that they contain the same aglycone, a glucose moiety attached at C-11, and an isovaleryl moiety attached at C-1. Moreover, 2 and 3 each contain an additional glucose unit and an additional isovaleryl substituent. The HMBC correlations [2: H-6 /C-1 and H-6 /C-1 ] and [3: H-1 /C-10 and H-6 /C-1 ] and total correlation spectroscopy (TOCSY) correlation [3: H-1 /H-6 ] suggest that the additional glucose moiety is attached at C-6 in 2 and C-10 in 3, and that the additional isovaleryl moiety is attached at C-6 in 2 and C-6 in 3 ( Figure 2). Enzymatic hydrolysis of 2 gave 1a, which suggests that 1 and 2 share the same absolute configuration, and the absolute configuration of 3 was deduced to be identical to that of 1 and 2. Based on the above data, we conclude that valerianairidoids II and III (2 and 3, respectively) have the chemical structures shown in Figure 1. . Their molecular formulas (C32H52O17) were determined using HRMS and 13 C NMR spectroscopy. A comparison of the NMR data for 1, 2, and 3 reveals that they contain the same aglycone, a glucose moiety attached at C-11, and an isovaleryl moiety attached at C-1. Moreover, 2 and 3 each contain an additional glucose unit and an additional isovaleryl substituent. The HMBC correlations [2: H-6″/C-1′′′′ and H-6′/C-1″] and [3: H-1″/C-10 and H-6′/C-1′′′′] and total correlation spectroscopy (TOCSY) correlation [3: H-1′/H-6′] suggest that the additional glucose moiety is attached at C-6′ in 2 and C-10 in 3, and that the additional isovaleryl moiety is attached at C-6″ in 2 and C-6′ in 3 ( Figure 2). Enzymatic hydrolysis of 2 gave 1a, which suggests that 1 and 2 share the same absolute configuration, and the absolute configuration of 3 was deduced to be identical to that of 1 and 2. Based on the above data, we conclude that valerianairidoids II and III (2 and 3, respectively) have the chemical structures shown in Figure 1.
Valerianairidoids IV and V (6 and 7) were isolated as amorphous solids with negative optical rotations  Figure 2. A comparison of the NMR data for 6 and 7 reveals that 7 contains one more glucose moiety attached at C-6′ compared to 6. The same aglycone (6a) was obtained when 6 and 7 were enzymatically hydrolyzed using  Figure 2. A comparison of the NMR data for 6 and 7 reveals that 7 contains one more glucose moiety attached at C-6 compared to 6. The same aglycone (6a) was obtained when 6 and 7 were enzymatically hydrolyzed using β-glucosidase. The planar chemical structure and relative configuration of 6a were elucidated from the NMR and MS data ( Table 2). The absolute configuration of 6a was determined to be 1S,5S,7S,8S,9S based on the experimental and calculated ECD spectra, in a similar manner to 1a (Figure 4). We conclude that the chemical structures of valerianairidoids IV and V (6 and 7, respectively) are as shown in Figure 1.
Valerianairidoid VI (9) was isolated as an amorphous solid with a negative optical rotation ([α] 25 D −15.4 in MeOH), and its molecular formula (C 32 H 50 O 18 ) was determined by HRESIMS and 13 C NMR spectroscopy. The 1 H and 13 C NMR (CD 3 OD) spectra suggest that 9 contains an iridoid moiety, a gentiobiose moiety, and two isovaleryl moieties (Table 3); their positions were determined by DQF COSY and HMBC NMR spectroscopy (Figure 2), and the relative stereochemistry of 9 was determined by NOESY spectroscopy (Figure 3). Aglycone 9a was obtained by the enzymatic hydrolysis of 9 using cellulase and identified as the aglycone of kanokoside A [18]; however, its absolute configuration was not determined previously. The absolute configuration of 9 was further established as 1S,5S,6S,7S,8R,9S by comparing the experimental and calculated ECD spectra of 9a. We conclude that the chemical structure of valerianairidoid VI (9) is as shown in Figure 1. Valerianairidoid VII (12) was isolated as an amorphous powder with a negative optical rotation ([α] 25 D −47.8 in MeOH); its molecular formula (C 23 H 34 O 13 ) was determined by HRMS and 13 C NMR spectroscopy, and its 1 H and 13 C NMR (CD 3 OD) spectra suggest that 12 contains iridoid and glucose moieties in a similar manner to 9. In addition, 12 contains isovaleryl and acetyl moieties {a methyl [δ H 2.05 (s, H-2 ) and an ester [δ C 172.4 (C-1 )]} ( Table 3). The positions of the isovaleryl and acetyl moieties were determined by DQF COSY and HMBC NMR spectroscopy (Figure 2). The absolute configuration of the iridoid moiety was determined to be that same as in 9. We conclude that the chemical structure of valerianairidoid VII (12) is as shown in Figure 1. Valerianairidoid VI (9) was isolated as an amorphous solid with a negative optical rotation ([α] 25 D −15.4 in MeOH), and its molecular formula (C32H50O18) was determined by HRESIMS and 13 C NMR spectroscopy. The 1 H and 13 C NMR (CD3OD) spectra suggest that 9 contains an iridoid moiety, a gentiobiose moiety, and two isovaleryl moieties (Table 3); their positions were determined by DQF COSY and HMBC NMR spectroscopy (Figure 2), and the relative stereochemistry of 9 was determined by NOESY spectroscopy (Figure 3). Aglycone 9a was obtained by the enzymatic hydrolysis of 9 using cellulase and identified as the aglycone of kanokoside A [18]; however, its absolute configuration was not determined previously. The absolute configuration of 9 was further established as 1S,5S,6S,7S,8R,9S by comparing the experimental and calculated ECD spectra of 9a. We conclude that the chemical structure of valerianairidoid VI (9) is as shown in Figure 1.

Plant Material
The dried rhizomes and roots of V. fauriei from Hokkaido (Japan) were purchased from Tochimoto Tenkaido (Osaka Prefecture, Japan) in August 2020.