New ent-kaurane diterpenoid acids from Nouelia insignis Franch and their anti-inflammatory activity

Eleven undescribed ent-kaurane-type diterpenoid acids, namely noueinsiancins A–K (1–11), together with sixteen related known analogs (12–27) were isolated from Nouelia insignis Franch. The chemical structures and absolute configurations of the new compounds were confirmed by the extensive spectroscopic data, electronic circular dichroism (ECD) data analysis and single crystal X-ray diffraction. Additionally, the anti-inflammatory assay was applied to estimate the nitric oxide (NO) inhibitory activities of all compounds by using lipopolysaccharide (LPS)-induced RAW 264.7 cells in vitro. The results revealed that 4–7 and 13–17 significantly inhibited NO production at the concentrations of 2.5 μM, 5.0 μM and 10.0 μM. Meanwhile, compounds 6 and 7 were found to down-regulate the protein expression levels of IL-6 and TNF-α in RAW 264.7 cells induced by LPS in a dose-dependent manner. In conclusion, these findings provided the reference values for exploring the new chemicals with biological activities from this genus.


Introduction
Nouelia insignis Franch belonging to the genus Nouelia is one of the infrequent woody plants from the Asteraceae family. It is widely distributed at an altitude of 1000-2500 meters in the mountainous areas of the Yunnan and Sichuan Provinces, especially in Panzhihua City in China. It is an endemic and endangered woody remnant species in our country due to the limited distribution and human deforestation. [1][2][3][4] Meanwhile, the roots and leaves of Nouelia insignis Franch have been widely applied in traditional Chinese medicine (TCM), and have a long history of use to treat acute icteric hepatitis, chronic hepatitis, bacterial infections, inammation, etc. 5,6 At present, we nd that there are few studies on the chemical ingredients of this plant based on the previous literature and querying a database (CA). Consequently, only a few biological activity studies were reported. In this manuscript, our phytochemical investigation of the MeOH extract of Nouelia insignis Franch led to the isolation and the identication of 27 ent-kaurane diterpenoid acids, including eleven new ones (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), together with sixteen related known analogs (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Herein, the details of the isolation, structural elucidation and anti-inammatory activities of the new compounds are reported (Fig. 1).

Cell culture and cytotoxicity assay
The RAW 264.7 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10% FBS, 100 U mL À1 penicillin and 100 mg mL À1 streptomycin at 37 C, 5% CO 2 . All the test compounds were dissolved in dimethyl sulfoxide (DMSO) and freshly prepared each time before use. Then, RAW 264.7 cells were seed at 96-well plates at a density of 5 Â 10 4 cells per well cultured overnight. The cells were treated with the test compounds (2.5, 5.0 and 10.0 mM) and LPS (1 mg mL À1 ), respectively. Aer 24 h incubation, 10 mL CCK-8 solution was added to each well and incubated at 37 C for 30 min. Finally, the optical OD-value was measured at 450 nm through a microplate reader, respectively (BioTek Instruments, Inc.).

Nitric oxide production inhibitory activity assay
The nitric oxide (NO) level was determined through measuring the concentration of nitrite in culture medium using Griess assay. RAW 264.7 cells were seeded in 96-well plates (5 Â 10 4 cells per well) for 24 h, then, the cells were treated with or without the test compounds (2.5, 5.0 and 10.0 mM) for 2 h prior to LPS (1 mg mL À1 ). Aer incubation for 24 h, 100 mL supernatant mixed with 100 mL Griess reagent and incubated for 20 min at 37 C in the dark. Finally, the absorbance was measured at 540 nm through a microplate reader, respectively (BioTek Instruments, Inc.).

The detection of cytokines production
RAW 264.7 cells were seeded in 12-well plates (2 Â 10 5 cells per well) for 24 h and the cells were also treated with or without the test compounds at the concentration of 2.5, 5.0 and 10.0 mM for 2 h before LPS (1 mg mL À1 ) stimulation. Aer incubation for 24 h, the culture media were collected. Then, the levels of TNFa and IL-6 were measured using the ELISA kits (Beyotime Institute of Biotechnology, Nantong, China) according to the manufacturer's instruction.

Statistical analysis
Prism 8 (GraphPad, San Diego, CA, USA) were used for statistical analysis. Dates of three separate studies were analysed by the one-way ANOVA test and the Tukey post hoc test. Meanwhile, all date expressed as mean AE standard deviation (SD), which collected from three independent experiments. The signicant difference was determined with P < 0.05.  -16]. The remaining four ones exhibited the existence of a tetracyclic system in 1. 7 Consequently, the four-ring was inferred to be a 6/6/6/5 fused rings A, B, C and D, a characteristic ent-kaurane diterpenoid skeleton, 8-10 based on the 2D NMR experiments (Fig. 2) 3 -19 to C-3, C-4, C-5 and C-18; from H 3 -20 to C-1, C-5, C-9 and C-10; from H 2 -7 to C-6 and C-8; from H-9 to C-5, C-8, C-10, C-11 and C-12; from H 2 -14 to C-8, C-9, C-13, C-15 and C-16; and from H 3 -17 to C-13, C-15 and C-16. In addition, the occurrence of a 11,16-epoxy motif in 1 was determined by the HMBC correlations of H-11 with C-16. Thus, the planar structure of 1 was determined.

Structural identication
The relative conguration of 1 was determined through the NOESY spectrum (Fig. 3). The NOESY correlations of H 3 -19/H-5/ H-9 indicated they were assigned the b-orientation. Meanwhile, H 3 -20, H-12 and H-11, H-13, H-15 were shown to be in a aorientation according to the NOESY interactions from H 3 -20 to H-12 and H 3 -20 to H-11, H-13 and H-15. In order to determine its structure and absolute conguration, an appropriate crystal of 1 was obtained from a solvent system of MeOH/H 2 O aer many single crystal cultures. Therefore, the X-ray crystallographic analysis with Cu Ka radiation unambiguously conrmed the absolute conguration of 1 was 4R, 5S, 8R, 9S, 10R, 11R, 12S, 13S, 15S, 16R (Fig. 4). Finally, the structure of 1 was ascertained completely and named noueinsiancin A.
Compound 2, obtained as colorless needle crystals, possessed the same molecular formula of C 20 H 30 O 5 as 1 based on the ion peak at m/z 373.1963 ([M + Na] + , calcd for C 20 H 30 O 5 Na, 373.1985) in the positive HRESIMS spectrum. Overall analysis of the 1D NMR (Tables 1 and 2) and 2D NMR ( 1 H-1 H COSY and HMBC) spectroscopic spectrum indicated that the gross structure of 2 was nearly close to that of 1. In fact, the only difference between them was the presence of a hydroxy group at C-3 (d C 71.5) position in 2 instead of the hydroxy group at C-12 (d C 79.8) position in 1. This was veried by the 1 (Fig. 2). The analysis of NOESY data revealed that the molecular conformation of 2 was identical to that of 1 (Fig. 3). Similarly, to further determine the structure and absolute conguration of 2, a suitable crystal of 2 for the single-crystal X-ray diffraction experiment (Cu Ka) was obtained. Finally, its absolute conguration was conrmed as 3S, 4S, 5S, 8R, 9S, 10R, 11S, 13S, 15S, 16R (Fig. 4). Hence, the structure of 2 was dened and named noueinsiancin B.
Compound 3 was isolated as a white amorphous powder and showed a molecular formula of C 20 H 30 O 5 according to the positive HRESIMS data: 373.1960 ([M + Na] + , calcd for C 20 H 30 O 5 Na, 373.1985). Detailed comparison of the 1 H and 13 C NMR spectra of 3 and 1 (Tables 1 and 2) revealed that the structure of 3 was similar to that of 1. The main difference was that the absence of a hydroxy group at C-12 position in 3 and the replacement of the methyl group in 1 at C-16 position by a hydroxy-methylene group in 3. This conclusion was supported by the hydroxyl-methylene signal at d C 61.8 (C-17) and the HMBC correlations from H 2 -17 (d H 3.59) to C-13 (d C 38.3), C-16 (d C 86.8) and C-15 (d C 77.7) (Fig. 2). Additionally, detailed analysis of the NOESY data of 3 indicated its relative conguration was also the same as 1. Aim to elucidated its absolute conguration, ECD calculations, a method regarded as a powerful and effective method to conrm the absolute conguration of the natural products, 11,12 was applied. Subsequently, its absolute conguration was established as 4R, 5S, 8R, 9S, 10R, 11S, 13S, 15S, 16S by comparing the calculated and experimental ECD spectrum (Fig. 5). Consequently, the structure of 3 was obtained and named noueinsiancin C.
Compound 4 was initially puried as colorless needle crystals and assigned a molecular formula of C 20 H 30 O 4 from the [M + Na] + ion peak at m/z 357.2023 (calcd for C 20 H 30 O 4 Na, 357.2036) in positive HRESIMS. The 1D NMR data of 4 (Tables 1 and 2) were closely similar to those of 1, except for the absence of the 11,16-epoxy group in 4 and the substitution of C-15 position (the hydroxy group for the former, and the carbonyl group for the latter). These above deductions were revealed by the 1 (Fig. 2). In the NOESY spectrum (Fig. 3), the correlations of H 3 -19/H-5/H-9 and H 3 -20/H-12/H-16/H 2 -14 demonstrated the b-conguration and a-conguration of these protons, respectively. Finally, by means of the single-crystal Xray diffraction analysis with Cu Ka radiation (Fig. 4), the absolute conguration of 4 was assigned as 4R, 5S, 8R, 9S, 10S, 12R,    13R, 16R. Consequently, the structure of 4 was determined named noueinsiancin D.
Compound 5, isolated as colorless needle crystals, provided the same molecular formula as that of 4 by its positive HRE-SIMS. Comparison of the 1D NMR spectra ( 1 H, 13 C and DEPT) of 5 (Tables 1 and 2) with those of 4, followed by the detailed analysis the 2D NMR spectra ( 1 H-1 H COSY and HMBC) established that the structure of 5 was close to that of 4. The main difference between 5 and 4 was in the positioning of a hydroxy group, which in 5 was at C-2 position and in 4 was at C-12 position. This ratiocination was corroborated by the 1 (Fig. 2). Moreover, the crucial NOESY interactions of H-2/H 3 -20 and H 3 -20/H-12a/H 2 -14/H-16 (Fig. 3) conrmed that these protons were both a-oriented and the conguration of 2-OH was b-oriented. Its absolute conguration was 2S, 4R, 5S, 8R, 9S, 10S, 13R, 16R as consolidated by the single crystal X-ray diffraction with Cu Ka radiation Then, the absolute conguration of 6 was conrmed as 2S, 4R, 5S, 8R, 9S, 10S, 13R on the basis of the single crystal X-ray diffraction experiment with Cu Ka radiation (Fig. 4). Hence, the structure of 6 was obtained and named noueinsiancin F.
Compound 7 was assigned an equal molecular formula (C 20 H 28 O 4 ) to that of 6 from the ion peak at m/z 355.1860 ([M + Na] + , calcd for C 20 H 28 O 4 Na, 355.1880) in the positive HRESIMS spectrum and 13 C NMR data. Detailed interpretation of its 1D NMR (Tables 1 and 3) and 2D NMR data indicated that the structure of 6 was highly resembled that of 5 with different positioning of a hydroxy group (the hydroxy group at the C-3 (d C 72.0) position for 7, and the hydroxy group at the C-2 (d C 64.8) position in 6). This deduction was determined by the 1 1.24, 3H) to C-3 (d C 72.0), C-4 (d C 45.0) and C-5 (d C 49.6). Additionally, the NOESY spectrum (Fig. 3) suggested that 7 had the same relative conguration as 6, where the H-3 and H 3 -20 assigned the aorientation by the NOESY correlations from H-3 to H 2 -2 and H 3 -20. Its absolute conguration was determined to be 3S, 4S, 5S, 8R, 9S, 10S, 13R based on the comparison of the calculated and  experimental ECD spectra (Fig. 5). Thus, the structure of compound 7 was established and named noueinsiancin G.  (Fig. 2). Similarly, the NOESY data was also showed their same relative conguration, and the OH-15 was assigned as b-oriented veried by the NOESY cross-peaks from H 3 -20 to H-12a, H-13 and H-15 (Fig. 3). The absolute conguration of 8 was determined as 3S, 4S, 5S, 8R, 9S, 10S, 13R, 15R by the single crystal X-ray diffraction experiment with Cu Ka radiation (Fig. 4). Consequently, its structure was dened and named noueinsiancin H.
Compound 9 was obtained as colorless needle crystals, with a molecular formula of C 20 H 32 O 5 based on the quasi-molecular ion peak at m/z 375.2123 ([M + Na] + , calcd for C 20 H 32 O 5 Na, 375.2142) in its positive HRESIMS data. A detailed analysis revealed the 1 H and 13 C NMR spectra (Tables 1 and 3 (Fig. 2). In the NOESY spectrum (Fig. 3), the correlations of H 3 -19/H-5/H-9 and H 3 -20/H-12a/H-13 veried the band aorientation of these protons. However, the relative conguration of the remaining chiral carbons could not be determined due to the lack of NOESY correlations. Then, a suitable crystal of 9 was obtained followed by the single-crystal X-ray diffraction experiment using the Cu Ka radiation (Fig. 4). Finally, its absolute conguration was conrmed as 4R, 5S, 8R, 9S, 10S, 13R, 15S, 16R. Consequently, the structure of 9 was dened and named as noueinsiancin I.
The molecular formula of 10 (C 20 H 32 O 5 ) was possessed to be identical to that of 9 on the basis of the (+)-HRESIMS ion peak at m/z 375.2121 ([M + Na] + , calcd for C 20 H 32 O 5 Na, 375.2142). The planar structure of 10 was conjectured to be the same as 9 by the detailed analysis its NMR data (1D and 2D) (Fig. 2). However, further upon comparison of their 13 (Fig. 3), suggesting these protons were on the same face and conrmed as a-orientation. Therefore, compound 10 was deduced as the 16-epimer of compound 9. By comparing the calculated and experimental ECD spectra (Fig. 5), the absolute conguration of 10 was conrmed as 4R, 5S, 8R, 9S, 10S, 13R, 15S, 16S. Hence, its structure was established and named noueinsiancin J.
Compound 11 was obtain as white amorphous powder and possessed a ion peak at m/z 373.  (Fig. 3). In addition, the NOESY spectrum suggested that the relative conguration of 11 was identical with 7. H-16 was assigned to be a-orientation based on the NOESY correlations from H 3 -20 to H-12a, H 2 -14 and H-16 (Fig. 3). The absolute conguration of 11 was assigned to be 3S, 4S, 5S, 8R, 9S, 10S, 13R, 16S based on by the high agreement between the calculated and experimental ECD curves (Fig. 5), accordingly, its structure was determined and named noueinsiancin K.

Nitric oxide production inhibitory activity
All the isolated compounds (1-27) were assayed for their inhibitory activities against NO production in LPS-stimulated RAW 264.7 cells. Compared with the LPS group, compounds 4-7 and 13-17 evidently decreased the NO production in LPSinduced RAW 264.7 cells (Fig. 6A). And, they possessed no signicant cytotoxicity at their effective concentration for the inhibition of NO production (Fig. 6B). Notably, the new compounds 6 and 7 showed stronger inhibitory activities at 2.5, 5.0 and 10.0 mM dose concentrations. Thus, we chose 6 and 7 as the material to further study on the anti-inammatory activity. The preliminary analysis of the structure-activity relationship on inhibitory activities against NO production in LPS-induced RAW 264.7 cells were discussed as follows: in all the tested compounds, we found that the ve compounds (6, 7, 15-17) possessed the amethylene-g-carbonyl group at C-16 and C-15 positions, displaying much better inhibitory activities than others. Additionally, in compounds 4, 5, 13 and 14, the methyl group instead of the amethylene group at C-16 position would weaken the activity (6, 7, 15-17 > 4, 5, 13, 14). Consequently, we inferred that the a-methylene-g-carbonyl group was a functional structure for inhibiting NO production in LPS-induced RAW 264.7 cells.
3.3 6 and 7 decreased the protein levels of TNF-a and IL-6 in LPS-induced RAW 264.7 cells Due to the TNF-a and IL-6 were the important factors of inammation. 14-16 Therefore, the effects of 6 and 7 on the protein levels of IL-6 and TNF-a in LPS-induced RAW 264.7 cells were examined. Aer RAW 264.7 cells were induced by LPS (1 mg mL À1 ) for 24 h with or without 6 and 7, the culture media were collected. Then, the levels of TNF-a and IL-6 were measured using the ELISA kits. As shown in Fig. 7A-D. Compared with the normal group, treated LPS could evidently up-regulated the protein expressions of TNF-a and IL-6. Then, compounds 6 and 7 treatment led to an obvious downregulation in the abovementioned protein expressions in a dose-dependent manner, comparing to the LPS-stimulated group.

Conclusions
In conclusion, noueinsiancins A-K (1-11), eleven new entkaurane diterpenoid acids, together with sixteen related known analogs (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) were isolated from Nouelia insignis Franch. The chemical structures including absolute congurations of the eleven new compounds were identied by the analyses of their NMR spectra, HRESIMS spectra, ECD calculation and single crystal X-ray diffraction. Moreover, the anti-inammatory activity evaluation research revealed that compounds 4-7 and 13-17 signicantly inhibited NO production at the concentration of 2.5 mM, 5.0 mM and 10.0 mM in LPS-stimulated RAW 264.7 cells. At the same time, compounds 6 and 7 evidently reduced the protein expression levels of IL-6 and TNF-a in RAW 264.7 cells induced by LPS. Especially, the analysis of the structure-activity relationship indicated that the a-methyleneg-carbonyl group was a functional structure for anti-inammatory activity. Above all, our ndings provide the idea that the administration of Nouelia insignis Franch may be benecial for the treatment of inammation disease.

Conflicts of interest
The authors declare no conicts of interest in this paper.