Nine Unique Iridoids and Iridoid Glycosides From Patrinia scabiosaefolia

Patrinia scabiosaefolia is a medical and edible Chinese herb with high nutritional and medicinal value. The continuing study of its chemical constituents led to the discovery of nine unique iridoids and iridoid glycosides, including three new iridoids (1-3) and six previously unknown irioid glycosides (5-10), and one known compound (4). Among them, compound 1 was a deformed iridoid, while compounds 3, 5-7, and 10 formed a new ring in their skeletons which was uncommon in this genus. For compound 3, the new ring existed between C-3 and C-10, while a 1,3-dioxane appeared between C-7 and C-10 in compounds 5-7 and 10. Moreover, compound 10 was a bis-iridoid glycoside, which was the first reported in P. scabiosaefolia. And the sugar of irioid glycosides (5-10) was glucose at C-11, except in 9 which had a 5-deoxyglucose moiety. All their structures were confirmed based on the extensive spectroscopic analysis, including IR, UV, HR-ESI-MS, ECD, and 1D- and 2D-NMR experiments. Their cytotoxic activities against HL-60, A-549, SMMC-7721, MCF-7, SW480 were also tested.


INTRODUCTION
There are about 400 species in 13 genera of Valerianaceae, mostly distributed in northern temperate zones and the subtropics or frigid zones. In China, this family comprises three genera, Nardostachys, Valeriana, and Patrinia, which can be found all over the country (Delectis Flora Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita., 1986). Plants from the genus Patrinia, such as P. scabiosaefolia, P. villosa, and P. heterophylla, have a long history of use as a traditional Chinese medicine for detoxification, swelling, empyema, liver protection, and cholagogue,. Some species are also edible as wild herbs, for example P. scabiosaefolia, P. villosa, P. punctiflora, and P. angustifolia (Xiao et al., 2007).
Among them, P. scabiosaefolia is a medical and edible Chinese herb, first recorded in "Sheng Nong's Herbal Classic." It is of great nutritional value and is enriched with amino acids, vitamins, β-carotene, and trace elements. The content of vitamin C per 100 grams is 42.65 mg, which is higher than that in some vegetables and fruits. Also, there is 14995.69 mg of amino acids in it, including eight kinds of essential amino acids which account for 36.06% of the total amount of amino acids (Zhong et al., 2001). It was also verified to have effects on the initial stages of edema, appendicitis, endometriosis, and inflammation (Delectis Flora Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita., 1986).

GRAPHICAL ABSTRACT | Nine unique iridoids and iridoid glycosides from Patrinia scabiosaefolia.
Phytochemical research showed that different kinds of compounds existed in the Patrinia genus, including triterpenes, iridoids, flavones, lignans, and their glycosides (Kim and Kang, 2013). Among these, iridoids are considered as the main components with diverse structures and various activities, which attract our attention to further explore the plant P. scabiosaefolia. As a result, we found a series of iridoids from ethyl acetate extract of P. scabiosaefolia, including three bis-iridoids which were first reported (Liu et al., 2017a,b;Liu et al., 2019).
In the continuing study, chemical constituents on n-butanol extract of P. scabiosaefolia led to the discovery of nine unique iridoids and iridoid glycosides, including three new iridoids (1)(2)(3) and six novel irioid glycosides (5-10), and one known compound (4) (Figure 1, Graphical Abstract). Among them, compound 1 was a deformed iridoid, compound 3 formed a cycle between C-3 and C-10, compounds 5-7 with a 1,3-dioxane between C-7 and C-10, and compound 10 was a bis-iridoid glycoside, which was the first reported in P. scabiosaefolia. Herein, we discussed their isolation, structure elucidation, and their cytotoxic activities.

Plant Material
The whole plants of Patrinia scabiosaefolia were collected in October 2010 from Shucheng county, Anhui Province, People's Republic of China, and were stored in a cool and dry place at room temperature. The material was identified by Prof. Shou-Jin Liu in Anhui University of Chinese Medicine and a voucher specimen (Wan1295) was deposited in Anhui University of Chinese Medicine. The plants of P. scabiosaefolia are common in the local area and the collection was permitted. We also ensured that the local population of P. scabiosaefolia was not destroyed through the means of collection at different locations.

Extraction and Isolation
The air-dried and powdered whole plants (29 kg) of P. scabiosaefolia were extracted with 95% ethanol (3 × 75 L) under room temperature and concentrated under reduced pressure. Then the residue (3 kg) was dissolved in water and partitioned successively with n-butanol to yield n-butanol extract (0.85 kg) after concentration. The n-butanol extract was subjected to silica gel column chromatography eluted with a gradient of CHCl 3 -MeOH (8:1→ 0:1, v/v) to obtain five fractions 1-5 by TLC plate analysis.

Cytotoxicity Assays
The following human tumor cell lines were used: HL-60, SMMC-7721, A-549, MCF-7, and SW-480. These were obtained from ATCC (Manassas, VA, USA). All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone) at 37 • C in a humidified atmosphere with 5% CO 2 . Cell viability was assessed by conducting colorimetric measurements of the amount of TABLE 2 | The 1 H and 13 C NMR data of 5-9 (CD 3 OD, δ in ppm, J in Hz).  insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO, USA) (Monks et al., 1991). In brief, 100 µL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 10 5 cells/mL in 100 µL medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 µg) was added to each well, and the incubation continued for 4 h at 37 • C. The cells were lysed with 100 µL of 20% SDS-50% DMF after removal of 100 µL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by the Reed and Muench method (1938).

Computational Study
The CHARMM force field and DFT/TDDFT calculations were performed with Discovery Studio 4.0 and Gaussian09 program package, respectively. Conflex conformational search generated low-energy conformers within a 20 kcal/mol energy window and were subjected to geometry optimization using DFT method without imposing any symmetry constraints at the B3LYP/6-31G(d) level. Frequency calculations were carried out using the same level to verify that the molecular structures were true minimum. The calculated ECD spectra were generated by the program SpecDis2 using a Gaussian band shape with 0.3eV exponential half-width from dipole-length dipolar and rotational strengths (Bruhn et al., 2013). , and one oxygenated methylene at δ C 66.6 (t). These data were similar to those of 8,9-didehydro-7-hydroxydolichodial (Veith et al., 1986), except for the disappearance of conjugated aldehyde and double bonds, and the appearance of two methines and one oxygenated methane, especially the change of methyl at C-10 to oxygenated methylene. Therefore, we speculated that the methyl at C-10 of 8,9-didehydro-7-hydroxydolichodial was oxidized to oxygenated methylene, and then underwent a hemiacetal formation with the aldehyde group at C-1 to form the deformed iridoid-compound 1 with 5/5 rings, which occurred simultaneously to the hydrogenation at C-8 and C-9, and finally the C-1 was methylated (Figure 2). This conclusion was confirmed by HMBC spectrum with the key correlations from H-1 [δ H 4.89 (s)] to C-10 (δ C 66.6), C-8 (δ C 48.6), and C-9 (δ C 57.1), and OMe (δ C 54.5) were observed, and the correlations of H-1/H-9 and H-9/H-5 in the ROESY spectrum (Figure 3). The absolute configurations of H-5 and H-7 were both S deduced from the comparison of chemical shift value to the known compound 8,9-didehydro-7-hydroxydolichodial (Veith et al., 1986). The α-orientation of H-8 was elucidated by the correlation of H-7 with H-8 in ROESY (Figure 3). The β-orientations of H-1 and H-9 were confirmed by the ROESY correlations from H-5 to H-1 and H-9 (Figure 2). And the R-configuration of the new formed acetal methine at C-1 was further confirmed by the comparison of experimental and calculated ECD spectra, which the positive Cotton effect near 200 nm in calculated ECD spectra of R-configuration at C-1  agreed with the experimental ECD spectra (Figure 4). Hence, the structure of compound 1 was defined as (1R,5S,7S,8S,9S)-1,10-epoxy-7,10-dihydroxy-dolichodial, named Patriscabioin M.
Compound 3 was found to have a molecular formula of C 14 H 24 O 5, determined by its positive HRESIMS data ([M + Na] + , m/z 295.1515, calcd. for 295.1516) and 13 C-NMR spectrum ( Table 1), with 3 degrees of unsaturation. From the NMR data, compound 3 showed spectral characteristics of iridoid at δ H 5.19 (d, J = 2.8 Hz, H-1) and δ C 99.5 (d, C-1), with a n-butanol group [δ C 67.5 (t), 32.2 (t), 19.7 (t), 14.0 (q)]) located in C-1, which was confirmed by the correlation from H-1 to C-1' in HMBC (Figure 6). However, the biggest difference from other iridoids' skeletons (such as compounds 2, 5-9) was the added 1 unsaturation, of which the former's degree was 2, and the latter was 3. Meanwhile, the NMR spectra exhibited the disappeared double bonds and the appearance of two methines, among which the chemical shift was δ C 94.7 (d, C-3), but there was no other unsaturated group. Therefore, it was presumed that there was a ring that existed  in the skeleton of compound 3. The new ring was formed by the ether bond between C-3 and C-10, which was further verified by the fragment of H-3-H-4-H-5-H-9-H-8-H-10 in COSY, and the correlation from H-3 to C-10 in HMBC (Figure 7). According to the S-configuration of H-5 and H-9 and R-configuration of H-1, the configuration of H-4 was determined as R-configuration and the configurations of H-3 and H-8 were determined as S-configuration by the correlations of H-4/H-5, H-3/H-11, and H-7/H-8 in ROESY (Figure 6). There was good agreement between experimental and calculated ECD spectra, which was further verified these configurations (Figure 7). Hence, the structure of 3 was established as (1R,3S,4R,5S,7S,8S,9S)-1-n-butoxy-3,10epoxy-7-hydroxy-3,4,5,6-tetrahydrovaltrate hydrinm, named patriscabioin O.
The relative configuration of 5 was similar to patriscabioins A-L and patriscabiobisins A and B (Liu et al., 2017a,b) from P. scabiosaefolia through comparison with their ROESY and CD spectrum (Figure 11). The ROESY cross peaks of H-1"'/H-8 and 2"'-Me/H-1"' suggested the α-orientation of H-1"' and 2"'-Me. Thus, the structure of 5 was identified as shown and named patrinoside B.
Compound 6 had the molecular formula C 25 H 38 O 11 based on its quasimolecular ion peak at m/z 537.2302 [M + Na] + (calcd for 537.2306) in HR-ESI-MS spectrum and 13 C-NMR data ( Table 2). Comparison of the 13 C NMR and DEPT spectra of 6 with those of 5 revealed that 6 shared the same aglycone, a glucose at C-11, and the 3-methylcrotonyl group at C-1 as compound 5, and the significant difference between them was the disappearance of two methylenes, one methine and one methyl in the high field region, and the molecular weight of 6 was 56 less than that of 5. This indicated that the fragment of 2-methyl-heptaldehyde in compound 5 was changed to n-butyraldehyde in compound 6, which was further verified by the correlations from H-1"' (δ H 4.52) to C-2"' (δ C 38.2), and H-4"' (δ H 0.90) to C-2"' (δ C 38.2), C-3"' (δ C 18.2) in HMBC. Therefore, the structure of 6 was deduced as shown and named patrinoside C.
The molecular formula of 7 was identified as C 26 H 40 O 11 from HR-ESI-MS at m/z 551.2467 [M + Na] + (calcd for 551.2463). Comparing 1 H-NMR and 13 C-NMR data of 7 with those of 6, it was found that they shared similar NMR data, and the obvious distinction between them was the appearance of a methylene (δ C 34.8) and the change in chemical shift of methyl signal of C-4' (δ C 27.6 → 12.3). It indicated that a 3-methylcrotonyl group at C-1 in 6 was replaced by the 3,4-dimethylcrotonyl group in 7. This was ultimately confirmed by 1D and 2D NMR spectra. In HMBC spectrum, the correlations between H-2' (δ H 5.68) and C-4' (δ C 34.8) and C-5' (δ C 12.3) provided solid evidence for the existence of the 3,4-dimethylcrotonyl group. Finally, the structure of 7 was elucidated as shown and named patrinoside D.
Compound 8  (calcd for 497.1993), whose molecular weight is 54 less than that of 7. Careful analysis of its NMR data found that it had the same aglycone as compound 7, as well as a glucose at C-11 and a 3,4-dimethylcrotonyl group at C-1, but lacked an oxymethine at δ C 102.0, two methylenes and a methyl, which suggested that it did not have a 1,3-dioxaneformed at C-7 and C-10. This could be further verified by the chemical shifts at C-7 and C-10 which were shifted up field from δ C 79.2 to 72.7 and form δ C 67.1 to 62.1, respectively, compared with compound 7. Finally, the structure of compound 8 was characterized as (1S,5S,7S,8S,9S)-1-O-(3,4-dimethylcrotonyl)-7,10-dihydroxy-11-β-D-glucose-5,6-dihydrovaltrate hydrin and named patrinoside E.
Thus, the structure of compound 10 was characterized as shown, namely patriscabiobisin C.
In conclusion, compounds 1-10 were a series of 5,dihydrovaltrate hydrins with characteristic substituents, such as 3-methylcrotonyl group or 3,4-dimethylcrotonyl group in the Valerianaceae family, belonging to iridoids derived from iridodial. Iridoial was enoxylated and then subjected to intramolecular hemiacetal formation to form iridoid. And iridoid suffered a series of chemical changes to achieve diverse compounds (1-10) (Figure 10). Iridodial was oxidized and then underwent the hemiacetal formation between C-1 and C-10 to form the deformed iridoid-compound 1 with 5/5 rings. Iridoid was oxidized and combined with different substituents to constitute compounds 2, 4, 8-9, and when it was dehydration condensed between C-10 and C-3 to generate a new ring, like compound 3. However, when the hydroxyl of C-7 and C-10 of iridoid combined with the aldehyde compounds, a 1,3-dioxane group would be formed, such as compounds 5-7. Furthermore, if the aldehyde group came from an iridoid, then it would generate a bis-iridoid, like compound 10. And the configurations of compounds 1-10 were further confirmed by ECD (Figure 11).
Finally, the cytotoxic activities of these compounds were evaluated against five human cancer cell lines (HL-60, A-549, SMMC-7721, . Unfortunately, none of the compounds showed significant cytotoxicities at 40 µM, except for compound 5 which showed the cell inhibition of 102.42, 95.13, 73.07, and 80.93% against HL-60, SMMC-7721, MCF-7, and SW-480 respectively (Figure 12). CONCLUSION P. scabiosaefolia is a medical and edible Chinese herb with high nutritional value and a wide range of biological activities. Research showed plants in the Patrinia genus are rich in iridoids and terpenoids. In our ongoing study, we found a series of iridoids and iridoid glycosides, including three new iridoids (1-3) and six novel irioid glycosides (5-10), and one known compound (4). Among them, compound 1 was a deformed iridoid, compound 3 formed a cycle between C-3 and C-10, compounds 5-7 with a 1,3-dioxane between C-7 and C-10, and compound 10 was a bis-iridoid glycoside, which was the first reported in P. scabiosaefolia. Cytotoxicity assays found compound 5 showed good cell inhibition against HL-60, SMMC-7721, MCF-7, and SW-480. All these results enriched the study on the chemical constituents and activities of Patrinia genus. However, the activities of these compounds were not thorough, just the evaluation of the cytotoxicity assays, and the other activities and their mechanisms are worth further exploration.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.