Jatrolignans C and D: New Neolignan Epimers from Jatropha curcas

Two new neolignans jatrolignans, C (1) and D (2), a pair of epimers, were isolated from the whole plants of Jatropha curcas L. (Euphorbiaceae). Their structures were determined with HRESIMS, IR, and NMR data analysis, and electronic circular dichroism (ECD) experiments via a comparison of the experimental and the calculated ECD spectra. Their antichlamydial activity was evaluated in Chlamydia abortus. They both showed dose-dependent antichlamydial effects. Significant growth inhibitory effects were observed at a minimum concentration of 40 μM.


Introduction
Jatropha curcas L. has been used as a traditional medicine for the treatment of traumatic injury, fracture, itchy skin, eczema, and acute gastroenteritis [1]. Its extracts and monomeric compounds possess potential pharmacological activities, owing to the efficiency of clearing heat and detoxication scattered stasis detumescence [1]. Chemical constituent investigations of the roots, stems, and leaves of J. curcas led to the identification of structurally diverse diterpenoids [2], triterpenes [3], lignans [4], and coumarins [5], and many of these compounds exhibited promising cytotoxicity [6], antitumor [7], antimicrobial [8], cytopathic [9], anti-inflammatory [10], antioxidant [11], anticoagulant [12], insecticidal [13], and molluscicidal [14] activities. As a part of our ongoing research program for the discovery of potential pharmacological ingredients from natural products, we studied a methanol extract from the dried whole plant of J. curcas; and in the process, two undescribed neolignan epimers (compounds 1 and 2) were isolated. Structurally, 1 and 2 ( Figure 1) possessed the same planar structure, from which could be speculated a pair of epimers at either C-7 or C-8. The relative configurations of 1 and 2 were determined by comparing their coupling constants between H-7 and H-8, and the absolute configurations were deduced from the ECD spectra of 1 and 2. We, herein, report the details of the isolation and structural elucidation of 1 and 2, as well as their antichlamydial activity.
The absolute configuration of 1 was proposed as depicted, based on the calculated ECD curve, which agreed well with the experimental ECD data (Figure 3), allowing the The vicinal coupling constant of H-7/H-8 can be used to assign erythro versus threo relative configurations [18,19]. The open-china erythro isomer generally had smaller coupling constants than the open-china threo isomer in non-hydrogen bonding solvent. The coupling constant (8.8 Hz) of H-7/H-8 indicated a threo sterostructure for compound 1.
The absolute configuration of 1 was proposed as depicted, based on the calculated ECD curve, which agreed well with the experimental ECD data (Figure 3), allowing the absolute configuration of 1 to be defined as 7S and 8S. Hence, the structure of 1 was designated and named jatrolignan C [20].
olecules 2022, 27, x FOR PEER REVIEW 4 absolute configuration of 1 to be defined as 7S and 8S. Hence, the structure of 1 was ignated and named jatrolignan C [20]. Compound 2 was also obtained as an amorphous powder. The molecular form was established as C25H30O9 (9 degrees of unsaturation) from its HRESIMS (m/z 497.1 [M + Na] + ) (calcd for C25H30O9Na, m/z 497.1782). Its IR spectrum showed absorption b consistent with the presence of hydroxy (3436 cm −1 ), carbonyl (1738 cm −1 ), alkenyl ( cm −1 ), and aromatic ring (767 cm −1 ) functionalities. The 1 H and 13 C NMR signals (Tab of 2 were almost identical to those of 1. The discriminating coupling constants of H-8 and H2-9 indicated the relative stereochemistry of 2 was different from 1, thus 2 suggested to be the epimer of 1 at C-7 or C-8 and erythro sterostructure, which was firmed by the ECD spectra (Figure 3), indicating that 2 gave an exactly opposite Co effect at 220 nm compared with that of 1. The absolute configuration of 2 was propos depicted, based on the calculated ECD curve, where the calculated values of 7R an matched the experimental ECD curve (Figure 3), allowing the absolute configuration to be defined as 7R, 8S. Hence, the structure of 2 was designated and named jatroli D.
To determine whether compounds 1 and 2 were natural or artificial products MeOH extract of J. curcas was subjected to an MCI gel column with MeOH/H2O (8 applied to Sephadex LH-20 (MeOH), and then compared to the isolated compounds 1 2 using HPLC. The HPLC (Supporting information) showed that the preliminary ex contained compounds 1 and 2, indicating that the both 1 and 2 are a metabolites o plant. Compound 2 was also obtained as an amorphous powder. The molecular formula was established as C 25 H 30 O 9 (9 degrees of unsaturation) from its HRESIMS (m/z 497.1785, [M + Na] + ) (calcd for C 25 H 30 O 9 Na, m/z 497.1782). Its IR spectrum showed absorption bands consistent with the presence of hydroxy (3436 cm −1 ), carbonyl (1738 cm −1 ), alkenyl (1453 cm −1 ), and aromatic ring (767 cm −1 ) functionalities. The 1 H and 13 C NMR signals (Table 1) of 2 were almost identical to those of 1. The discriminating coupling constants of H-7, H-8 and H 2 -9 indicated the relative stereochemistry of 2 was different from 1, thus 2 was suggested to be the epimer of 1 at C-7 or C-8 and erythro sterostructure, which was confirmed by the ECD spectra (Figure 3), indicating that 2 gave an exactly opposite Cotton effect at 220 nm compared with that of 1. The absolute configuration of 2 was proposed as depicted, based on the calculated ECD curve, where the calculated values of 7R and 8S matched the experimental ECD curve (Figure 3), allowing the absolute configuration of 2 to be defined as 7R, 8S. Hence, the structure of 2 was designated and named jatrolignan D.
To determine whether compounds 1 and 2 were natural or artificial products, the MeOH extract of J. curcas was subjected to an MCI gel column with MeOH/H 2 O (80%), applied to Sephadex LH-20 (MeOH), and then compared to the isolated compounds 1 and

The Antichlamydial Activity of Compounds
Chlamydial infections in humans and animals are global health issues. Although chlamydial infections within the human population are currently manageable with the existing conventional therapies (antibiotics treatment), the extended exposure of Chlamydia to antibiotics provides greater opportunity for the development of antibiotic resistance in chlamydial species. Natural products show significant potential for treating chlamydial infections, which is expected to produce new antichlamydial treatment modalities. Neolignans have shown multiple activities, such as anticarcinoma, antioxidation, and anti-HIV effects. In order to find the new medicinal potential of neolignans, the antichlamydial activity of two novel neolignans, compounds 1 and 2, from the medicinal herb Jatropha curcas L. was evaluated in this study, which might reveal a new potential antichlamyidal agent for drug development.
Chlamydia spp. are a group of obligated intracellular bacteria associated with major diseases in humans and animals. In this study, the antibacterial activities were investigated in Chlamydia abortus, an important zoonotic chlamydial pathogen. Compounds 1 and 2 showed a similar antichlamydial effect on Chlamydia abortus, in a dose-dependent manner. As shown in Figure 4A, with the increasing concentration of compound 1, the intracellular chlamydial inclusions were smaller in size and less in number. At the highest concentration of 80 µM, inclusions were few and tiny, analogous to the positive control tetracycline (final concentration, 5 µM). A similar effect of compound 2 on chlamydial inclusions of Chlamydia abortus was also observed. The inclusion formation ratio was significantly reduced in cell cultures treated with compounds 1 and 2 at a concentration of 40 µM or more ( Figure 4B,C). Figure 4A, with the increasing concentration of compound 1, the intracellular chlamydial inclusions were smaller in size and less in number. At the highest concentration of 80 μM, inclusions were few and tiny, analogous to the positive control tetracycline (final concentration, 5 μM). A similar effect of compound 2 on chlamydial inclusions of Chlamydia abortus was also observed. The inclusion formation ratio was significantly reduced in cell cultures treated with compounds 1 and 2 at a concentration of 40 μM or more ( Figure 4B,C).

Plant Materials
Whole plants of Jatropha curcas L. were collected in October 2018 from Hainan Province, China, and identified by Associate Researcher Dao-Geng Yu of the Chinese Academy of Tropical Agricultural Science, with a voucher specimen (No. JA20181012) being deposited in the State Key Laboratory of Applied Organic Chemistry, Lanzhou University.

Extraction and Isolation
Air-dried whole plants of Jatropha curcas L.

ECD Calculation
The ECD calculations of 1 and 2 were carried out using previous methods. A detailed description of this section is provided in the Supplementary Materials.

Chlamydia Strains and Cell Line
The zoonotic intracellular bacterium Chlamydia abortus strain GN6 used in this study was cultured in the mouse embryonic fibroblast cell line McCoy, as described previously (PMID: 33065117).

Antichlamydial Activity Screening
To test the antichlamyidal activity, a concentration of 0 µM (0.5% DMSO as vehicle) to 80 µM of each compound was added in the medium. Tetracycline of 5 µM final concentration was used as a positive control. The Chlamyida inocula were incubated with 1 × 106 McCoy cells per well in a 6-well plate. After centrifugation, inocula were replaced with chlmaydial growth medium (RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS), 100 U/mL of kanamycine, 100 µg/mL of streptomycin, and 1 µg/mL of cycloheximide) with 0 µM to 80 µM of the tested compound added, and then incubated in a 5% CO 2 incubator at 37 • C for 48 h. Afterwards, the chlamydial inclusions were visualized by immunofluorescence staining, using a Chlamydia abortus specific mouse anti-MOMP monoclonal antibody as the primary antibody. The inclusion formation ratio (expressed as the number of inclusions/number of cells × 100%) was calculated in the cell cultures [21].

Conclusions
Jatrolignans C and D, two new neolignan epimers were isolated from the whole plants of Euphorbiaceae Jatropha curcas L. The absolute configurations of Jatrolignans C and D were accurately elucidated by means of spectroscopic techniques, especially an extensive NMR data analysis and ECD calculation. They exhibited weak antichlamydial activity compared to tetracycline, which was used as a positive control. To the best of our knowledge, this is the first report to evaluate the antichlamydial activity of neolignans. As components of Jatropha curcas L, more detailed chemical and biological investigations of the plant metabolites are required to determine their contribution to supporting and enhancing the application of herbal medicines.