Elesesterpenes A–K: Lupane-type Triterpenoids From the Leaves of Eleutherococcus sessiliflorus

Elesesterpenes A–K (1–11), eleven new lupane-type triterpenoids, triterpenoid glycosides, and nortriterpenoid were isolated from the leaves of Eleutherococcus sessiliflorus. Their structures and relative configurations were completely elucidated by a combination of diverse methods including physical, spectroscopic data. The absolute configuration of elesesterpenes A–B (1–2) was defined by single-crystal X-ray diffraction. Meanwhile, all the isolates were evaluated for anti-inflammatory activities on lipopolysaccharide-induced nitric oxide production in BV2 microglial cells, and antiproliferative activities against human hepatoma (HepG2), human lung adenocarcinoma (A549), and human glioma cells (LN229) in vitro. It was found that some of them exhibited obvious anti-inflammatory activities and potent antiproliferative activities.


INTRODUCTION
Triterpenoids, a kind of structural diversity secondary metabolites in the plant kingdom, are derived from the cyclization of squalene through different pathways and are widely distributed in various plants (Salvador et al., 2012;Thimmappa et al., 2014). Most triterpenoids are tetracyclic or pentacyclic triterpenes; pentacyclic triterpenes can be roughly divided into four subtypes, including oleanane, ursane, lupane, and friedelane.
E. sessiliflorus is widely distributed in Northeast China. It is a type of folk herbal medicine which helps in nourishing liver and kidney, strengthening body and bones, and is used for treating cerebrovascular diseases, tumors, rheumatism, and arthralgia (Jung et al., 2005;Lee et al., 2012). Abundant secondary metabolites, mainly flavonoids and triterpenoids, have been isolated from E. sessiliflorus (Yoshizumi et al., 2006;Zhang et al., 2019). Among them, 3,4-seco-lupane-type triterpenoids are characteristic chemical constituents, which exhibit many pharmacological activities, such as antiproliferative, antidepressant, and hepatoprotective activities (Bian et al., 2017;Bian et al., 2018). In the current study, we describe six triterpenoids, four triterpenoid glycosides, and one nortriterpenoid ( Figure 1) from the leaves of E. sessiliflorus, including their isolation, structural elucidation, the antiinflammatory activity on BV2 cells, and antiproliferative activities on HepG2, A549, and LN229 cell lines.

Hydrolysis of Compounds 3, 4, 5, and 11
Each compound (1.0 mg) was hydrolyzed in 2 ml (2 mol/L) of HCL and incubated in water bath at 80°C for 4 h. The mixture was concentrated under vacuum, and the resulting residue was suspended in water and extracted with ethyl acetate (3 × 2.0 ml). And the aqueous layer was evaporated dry under reduced pressure. The residue was dissolved in dry pyridine (1 ml) with L-cysteine methyl ester hydrochloride (2 ml, 0.1 mol/L) and shaken at 60°C for 1h. Subsequently, N-trimethylsilylimidazole was added and heated at 60°C for 1 h. The reaction mixture was suspended in 1.0 ml H 2 O and extracted with n-hexane (3 × 1.0 ml). The layer of n-hexane was directly analyzed by Agilent GC-MS (7890A) using a DB-1701 column. Temperatures of both injector and detector were 250°C. The split ratio was 10:1. A temperature gradient system was used for the oven, starting at 220°C and increasing up to 270°C at a rate of 5°C/min, and held for 10 min at the final temperature. The absolute configurations of the sugar components were determined by comparison with the retention times of the authentic sugars (D-glucose, 10.72 min; L-rhamnose, 8.78 min).
Three human cancer cell lines (HepG2, A549, and LN229) were incubated at 2 × 10 5 cells/mL in a 96-well microplate for 12 h. Each tumor cell line was exposed to the test compounds at various concentrations (0,20,40,60,80, and 100 μM) for 24 h. Then the cells were tested with 0.5 mg/ml CCK-8 and incubated for 2 h at 37°C in 5% CO 2 .
BV2 microglial cells were cultured in a 96-well microplate, which was treated with 1 μg/ml LPS for 12 h. Then, they were treated with different concentrations of the compounds 1-11 (0, 20, 40, 60, 80, and 100 μM) for 12 h for the detection of nitric oxide (NO) content in the supernatant of cell culture medium. After the Griess reaction, they were incubated for 0.5 h at 37°C in 5% CO 2 . The absorbance of cells was determined at 490 nm by a microplate reader.

Structure Elucidation of Compounds
Elesesterpene D (4), a yellow amorphous solid, has a molecular formula of C 49 H 78 O 18 as established by the HR-ESI-MS ion at m/ z 972.5525 ([M + NH 4 ] + , calculated for C 49 H 82 NO 18 , 972.5532). It was discovered to be almost consistent with sessiloside isolated from Acanthopanax sessiliflorus by comparing the 1 H and 13 C NMR data of 4 (Yoshizumi et al., 2006). The HMBC correlation ( Figure 2) of H 3 -1′′′′/C-3 showed that the extra methyl ester (δ C 51.3, δ H 3.65) was connected to C-3 in 4. The relative configuration of 4 was assigned by the NOESY experiment to be the same as those of 3 ( Figure 6).
Elesesterpene E (5) was also purified as a yellow amorphous solid. Its molecular formula was determined to be C 50 H 80 O 18 by the HR-ESI-MS ion at m/z 986.5679 [(M + NH 4 ) + , calculated for C 50 H 84 NO 18 , 986.5688]. Through comparison of the 1 H and 13 C NMR data between 5 and 4, it was obvious that the methyl ester at C-3 was replaced by an ethyl ester (δ C 60.3 and 14.3); this judgment was confirmed by several correlations of H 2 -1′′′′/H 3 -2′′′′; H 2 -1′′′′/C-3 in the 1 H-1 H COSY and HMBC (Figure 2). The relative configuration of 5 was entirely consistent with that of 4 ( Figure 6).
Elesesterpene F (6) was obtained as a colorless amorphous solid with a molecular formula of C 30 H 44 O 7 based on the HR-ESI-MS ion at m/z 517.3179 ([M + H] + , calculated for C 30 H 45 O 7 , 517.3165). The 1 H-NMR and 13 C-NMR data of 6 were similar to those of chiisanogenin obtained previously from Acanthopanax divaricatus, and the only change was that the Δ 20 (30) double bond was replaced by a carboxyl group . It was supported by correlations of H-20, H 3 -29/C-30 in HMBC (Figure 2). NOESY correlations (Figure 3) between H-1/H 3 -25; H-5/H-9/H 3 -27 were observed. Therefore, H-1 and H-5 were inferred as βand α-orientation, respectively.
Elesesterpene G (7)   close similarity between the structures, except for the existence of a ketone carbonyl signal at δ C 211.1. The 13 C NMR and DEPT spectra of 7 resolved 29 carbon signals; these suggested that 7 was a nortriterpenoid. Further analysis of the correlations (H-18, H 3 -29/C-20) established that the ketone carbonyl was straightforwardly assigned to C-19. The relative configuration of 7 was entirely consistent with that of 6 ( Figure 3). Elesesterpene H (8) was obtained as a yellow amorphous solid. The molecular formula of the compound was C 31 H 48 O 7 as established by HR-ESI-MS data [(M + H) + , 533.3467; calculated for C 31 H 49 O 7 , 533.3478]. The 1 H-NMR and 13 C-NMR data ( Table 3) of 8 were similar to those of acanthosessilioside C obtained from the fruits of Acanthopanax sessiliflorus, and it was found to lack a sugar moiety at the C-28 position by contrast (Lee et al., 2012). The correlation of H-1/C-4 in the HMBC (Figure 2) suggested that C-1 and C-4 were connected by an oxygen bridge. Furthermore, the coupling constant between H-22 and H 2 -21 was 5.3 Hz, indicating that H-22 is β-orientation (Shirasuna et al., 1997). The NOESY correlations of H-1/H-11/H 3 -25 ( Figure 3) indicated that H-1 and H-11 were designated to be of β-orientation.
Elesesterpene I (9) was obtained as a yellow amorphous solid with a molecular formula of C 32 H 50 O 7 as determined by the HR-ESI-MS [(M + H) + , 547.3627; calculated for C 32 H 51 O 7 , 547.3635]. The 1 H and 13 C-NMR data of 9 (Table 3) were closely related to those of 8. Through the 1 H-1 H COSY correlation of H 2 -1′′′′/H 3 -2′′′′ and the HMBC correlation of H 2 -1′′′′/C-3, it was determined that C-3 was connected to ethyl ester rather than methyl ester. The relative configurations of 9 were established to be the same as those of 8 by the NOESY experiment ( Figure 3).
Elesesterpene J (10) was isolated as a white amorphous powder. Its molecular formula was determined as C 30 H 46 O 6 on the basis of HR-ESI-MS data [(M + H) + , 503.3366; calculated for C 30 H 47 O 6 , 503.3373]. Comparison of the NMR data (Tables 3) of 10 with the data of 8 showed the absence of a methoxy signal and a hydroxyl signal. The correctness of the inference was confirmed by the correlation signals of H-9/H 2 -11/H 2 -12 in 1 H-1 H COSY spectra ( Figure 2). As with compound 8, H-1 and H-22 were defined as β-orientation.
Elesesterpene K (11) was isolated as a yellow amorphous solid. The molecular formula of 11 was established to be C 50 H 80 O 20 on the basis of its HR-ESI-MS data at m/z 1,001.5293 [(M + H) + , calculated for C 50 H 81 O 20 , 1,001.5321]. The 1 H and 13 C-NMR data (Tables 3) were similar to those for 9, with the main difference being the absence of a hydroxyl group and the presence of three sugar units. Acid hydrolysis of 11 released D-glucose and L-rhamnose, which were identified by GC analysis after derivatization. The sugar moieties (δ C 95.2, 74.0, 78.5, 70.7, 77.9, 69.3; δ C 104.9, 75.2, 76.3, 78.1, 77.0, 61.2; δ C 102.6, 72.4, 72.6, 73.8, 70.2, 18.4) were assigned via comparison of the experimental and reported NMR data. The correlations of H-18, H 2 -22/C-28 were observed from the HMBC spectrum. The correlation peaks of H-1/H-11/H 3 -25 in the NOESY spectrum showed that H-1 and H-11 have uniform β-orientation in space.

Bioactive Activity
Finally, the antiproliferative activities of these compounds were evaluated against HepG2, A-549, and LN229. And most of the compounds showed significant inhibitory effects. Further analysis of the data showed that compounds 5, 7, 8, and 10 exhibited more extensive and potent effects with the IC 50 values ranging from 1.05 to 8.60 μM ( Table 4).
The bioactivities of isolated metabolites were also evaluated for the anti-inflammatory assay in vitro. All of them exhibited different degrees of suppression on NO production in LPSactivated BV2 microglial cells (Table 4), and compound 3 was the best (IC 50 2.33 ± 0.31 μM).

CONCLUSION
E. sessiliflorus was a kind of medicinal and edible herbal medicine. Numerous new compounds were isolated from the leaves in our study, including ten triterpenoids (1-6 and 8-11) and one nortriterpenoid (7). These were worth mentioning; the C-30 of compounds 6 and 7 had a rare change from the double bond to carboxyl and ketone carbonyl groups, respectively; in compound 2, C-1 and C-11 were linked to form a rare five-membered oxygen ring. All of these changes were reported for the first time. In addition, compounds 3, Frontiers in Chemistry | www.frontiersin.org January 2022 | Volume 9 | Article 813764 8

5, 9,
and 11 all had an extra segment (-OCH 2 CH 3 ) in C-3 compared with known triterpenoids. Thus, 3, 5, 9, and 11 were possible to be the artifacts. A possibility was proposed: some esterification reactions occurred during the refluxing extraction of ethanol. To further avoid the introduction of 3-OCH 2 CH 3 , a lot of methods are currently being carried out in our studies, and the results will be reported in due course.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.