New compounds from Patrinia villosa Juss. and their anti-inflammatory activities

Abstract Five new iridoids, patriscabioins M-Q and a new monoterpene, eldanolide acid, together with three known iridoids, were isolated from the 95% aqueous EtOH extract whole plants of Patrinia villosa Juss. The structures were established by a variety of spectroscopic analysis, such as IR, 1 D and 2 D NMR spectra, MS, ECD and X-ray diffraction data. Bioactivity screening revealed the inhibitory effects on nitric oxide (NO) production of them in lipopolysaccharide-activated RAW264.7 cells with Aminoguanidine Hydrochloride as the positive control. Among them, patriscabioin M (1), patriscabioin N (2), patriscabioin P (4), patriscabioin Q (5), 8,9-didehydro-7-hydroxydolichodia (7) were found to markedly reduce LPS-induced NO production in murine macrophage cells with IC50 values of 18.14, 18.93, 22.00, 13.64, 26.48 μM, respectively. Graphical Abstract


Introduction
The genus Patrinia belongs to the family Valerianaceae and consists of approximately 20 species that distributes in northwestern North America and eastern to central Asia. In China, there are 10 species, 3 subspecies and 2 variations all over the country (Liu et al. 2017). Some of them are used as food and medicines. Patrinia villosa Juss., a perennial herb, commonly known as "bai hua bai jiang" in traditional Chinses mdeicine, has been used for the treatment of hepatitis, appendicitis, inflammation and cancer (Kim and Kang 2013). More than 200 compounds of secondary metabolites have been identified in Patrinia villosa Juss., including flavonoids, iridoids, lignans, triterpenoids, steroids and coumarins (Cui et al. 2016). In a further search for more compounds and related bioactivities, five new iridoids (Bianco 1990), patriscabioins M-Q (1-5) and one new monoterpene, eldanolide acid (6), together with three known iridoids (7-9), have been isolated and identified from the 95% aqueous EtOH extract of the whole plant of Patrinia villosa Juss. Their structures were elucidated by NMR, ECD, MS spectroscopic data and X-ray diffraction data. The known compounds were identified as 8,9-didehydro-7-hydroxydolichodia (7) (Schneider and Veith 1985), stenopterin E (8) (Dong et al. 2015), jatamanins A (9) (Lin et al. 2010). Herein, their isolation and structure elucidation, as well as their biological evaluations, are reported.

Results and discussion
Compound 1 was isolated as pale yellow solid and its molecular formula was determined by analysis of the [M þ H] -11 (d H 9.18) to C-3 (d C 167.5), C-4 (d C 126.1) and C-5 (d C 31.0) and from H-1 and H-5 to C-3 established the planar structure of 1. NOESY experiment exhibited clear correlations between H-5, H-7, H-10 and H-9. Thus, the relative configuration of 1 was revealed. The computational electronic circular dichroism (ECD) spectra of two isomers of the relative configuration was predicted by the timedependent density functional theory (TDDFT) method, and the experimental curve of 1 ( Figure S10) was compared with the two calculated ones and matched very well with that of the (5S, 7 R, 8 R, 9S)-isomer. The absolute configuration of 1 was thus established as shown in Figure 1. Herein, the structure of 1 was elucidated as (5S, 7 R, 8 R, 9S)-7-hydroxy-8-methyl-1, 5, 6, 7, 8, 9-hexahydro-cyclopenta[c]pyran-5-carbaldehyde, and named as patriscabioin M.
Compound 2 was also obtained as pale yellow solid and its molecular formula was determined on the basis of analysis of the [M þ H] peak at m/z 199.0968 (calcd for C þ 10 H 15 O 4 þ , 199.0965) in the HRESIMS spectrum. The 1 H and 13 C NMR data of 2 were highly similar to those of 1, except for the C-11 and H-11 (d C 193.5 and d H 9.18 for 1 Vs. d C 171.5 and absence of corresponding H signal for 2), indicating that 2 was a iridoid similar to 1, with the only difference being the 11-carboxyl group in 2 instead of 11-aldehyde group in 1. Therefore, the planar structure of compound 2 was elucidated as shown in Figure 1. Comparisons of the NOESY NMR data of 2 with those of 1 allowed the relative configurations at C-5, C-7, C-8, and C-9 to be identically assigned. Because of biogenetic considerations and similar experimental ECD curves of 2 ( Figure  S20) and 1, the absolute configurations at C-5, C-7, C-8, and C-9 of 2 were assumed to be identical to those of 1. On the basis of the above evidences, the structure of 2 was determined as (5S, 7 R, 8 R, 9S)-6-hydroxy-7-methyl-1, 5, 6, 7, 8, 9-hexahydro- (Lee et al. 2018). This was confirmed by the analysis of HMBC correlations for 3. Expect the isovalerate group, the remaining 10 carbon signals, including those for two methyl groups (d C 21.7, 11.9), two methylenes (d C 65.0, 31.5), four methine carbons (d C 80.8, 45.6, 38.5, 36.4), an oxygenated tertiary carbon (d C 79.9) and a lactone carbon (d C 175.0). The 1 HÀ 1 H COSY spectrum showed the connectivities of the proton coupling sequence for the (C-1)- fragments. HMBC correlations from H-1, H-4, H-5 and H-11 to C-3, from H-5 to C-4 and C-11, from H-1, H-6, H-7, H-9 and H-10 to C-8, and from H-7 and H-9 to C-10 were observed. Analysis of the 1 H and 13 C correlations, exhibited in the 1 HÀ 1 H COSY, HMQC, and HMBC spectra, allowed the establishment of an iridomyrmecin-type iridolactone structure of 3. The HMBC experiment on 3 showed a long-range correlation between H-7 (d H 4.84) and the C-1 0 ester (d C 172.9) of the isovalerate group, confirming the isovalerate group to be located at C-7. NOESY correlations between H-4, H-5, H-9 and H-10 were observed, but no correlations were observed between H-7 and H-4 or H-5 or H-9 or H-10. Thus, the relative configuration of 3 was revealed. The singlecrystal X-ray crystallographic diffraction experiment was then undertaken, which unambiguously confirmed the structure and provided the absolute configuration of 3 as 4S, 5S, 7S, 8S, 9 R. Therefore, the structure of 3 was elucidated as (4S, 5S, 7S, 8S, 9 R)-4-methyl-butyric acid 5, degrees of unsaturation. The 1 H, 13 C spectra data for 4 revealed the presence of a methyl, a methine, two hydroxymethyl groups, a ketone carbon, a terminal double bond, and a tetrasubstituted double bond. The above information showed that three degrees of unsaturation were attributed to one carbonyl group and two double bonds, and the remaining one degree of unsaturation required one ring. Thus, 4 should be a monocyclic iridoid formed by the breaking of ether bond between C-1 and C-3. This was supported by the absence of HMBC correlations between H-3 and C-1 or between H-1 and C-3 in the HMBC spectrum of 4. The presence of a cyclopentenone ring in 4 was established via the HMBC cross-peaks from H-10 to C-7, C-8 and C-9, from H-1 to C-8, C-9 and C-5, and from H-6 to C-7 and C-8. Likewise, the 1 HÀ 1 H COSY spectrum permitted the correlation of H-5 and H-6. HMBC correlations from H-11 to C-3, C-4 and C-5, from H-6 to C-4 established the planar structure of 4. The calculated ECD spectrum of the (5 R) model of 4 was in agreement with the experimental version ( Figure S39), suggesting the absolute configuration of 4 to be (5 R). Consequently, compound 4 was identified as (R)-5-(1-hydroxymethyl-vinyl)-8-methylcyclopent-9-hydroxymethyl-8-enone, and named as patriscabioin P. Compound 5 was obtained as pale yellow oil and had the same molecular formula as that of 4, deduced from the HRESIMS ions at m/z [M þ H] 183.1013 (calcd for 183.1016). The analysis of its þ1 H and 13 C spectroscopic data indicated that compound 5 was similar to 4 with the difference being the aldehyde group at C-1 in 5 instead of hydroxymethyl group in 4 and the oxygenated methine at C-7 in 5 instead of ketone group in 4. Analysis of the 1 HÀ 1 H and 1 HÀ 13 C correlations, exhibited in the 1 HÀ 1 H COSY, HMQC, and HMBC spectra, allowed the establishment of structure for 5. NOESY correlations between H-5 and H-10 were observed, no NOESY correlations were observed between H-7 and H-5 or H-10, suggesting H-5 and H-7 were in different orientations. The experimental ECD curve of 5 ( Figure S49, Figure  S50) was compared with the four calculated ones and matched very well with that of the (5 R,7S)-isomer. The absolute configurations of 5 were thus established as shown in Figure 1. Accordingly, the structure of 5 was proposed as (5 R, 7S)-5-(1hydroxymethyl-vinyl)-7-hydroxy-8-methyl-cyclopent-9-enecarbaldehyde, and named as patriscabioin Q.
All of isolated compounds were tested for their inhibitory effects on NO production in LPS-activated RAW 264.7 macrophages with Aminoguanidine Hydrochloride as the positive control at nontoxic concentrations (Table S1). Compounds 1, 2, 4, 5, 7 showed significant activities against RAW 264.7 cells at nontoxic concentrations with IC 50 values of 18.14 lM, 18.93 lM, 22.00 lM, 13.64 lM, 26.48 lM, respectively, while the IC 50 value of Aminoguanidine Hydrochloride is 14.25 lM. The EC50 values of the cytoxicity of all compounds are more than 100 lM.

Conclusion
In summary, phytochemical investigation into the whole plant of Patrinia villosa Juss. lead to the isolation and identification of five new iridoids (1-5), one new monoterpene (6) and three known iridoids (7-9). The possible pathway for the transformation of these isolated iridoids was elaborated as shown in Figure 2 (Biswanath 2019). Compound 1-5, 8, 9 was synthesized from compound 7 through a series of oxidation, reduction, cyclization and rearrangement reactions. The potential cytotoxicity and antiinflammatory activity of these iridoids were also discussed. Compounds 1, 2, 4, 5, 7 markedly decreased LPS-induced NO production in a concentration-dependent manner. These findings could shed new light on further phytochemical studies of Patrinia species.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Mega-project for Innovative Drugs (2019ZX09735-002).