Terpenoid Glucosides from Gentiana macrophylla That Attenuate TNF-α Induced Pulmonary Inflammation in A549 Cells

Four previously undescribed terpenoid glucosides, including one sesquiterpenoid di-glucoside (1), two new iridoid glucosides (2, 3), and a new triterpenoid tri-glucoside (4), were isolated from a 70% ethanol extract of the root of Gentiana macrophylla (Gentianaceae), along with eight known terpenoids. Their structures were determined by spectroscopic techniques, including 1D, 2D NMR, and HRMS (ESI), as well as chemical methods. The absolute configuration of compound 1 was determined by quantum chemical calculation of its theoretical electronic circular dichroism (ECD) spectrum. The sugar moieties of all the new compounds were confirmed to be D-glucose by GC analysis after acid hydrolysis and acetylation. Anti-pulmonary inflammation activity of the iridoids were evaluated on a TNF-α induced inflammation model in A549 cells. Compound 2 could significantly alleviate the release of proinflammatory cytokines IL-1β and IL-8 and increase the expression of anti-inflammatory cytokine IL-10.


Introduction
The dry root of Gentiana macrophylla (Qin Jiao), a perennial herb that widely distributed in western and northern China, Mongolia, and Russia, was applied in traditional Chinese medicine for the treatment of rheumatic arthralgia and jaundice about two thousand years ago [1].Modern pharmacological studies of the extracts demonstrated inhibitory effects on rat model of rheumatoid arthritis [2], anti-inflammatory and analgesic effects [3], as well as alleviating systemic lupus erythematosus [4], osteoporosis [5], and liver injury [6].
In the current research, a rarely occurring sesquiterpenoid di-glucoside (1), two new iridoids (2, 3), and a new triterpenoid tri-glucoside (4), together with eight known terpenoids, were isolated from the root of G. macrophylla (Figure 1).Their structures were determined by spectroscopic methods, chemical methods, and quantum chemical calculations.All of the iridoids (2, 3, 5-8) were subjected to anti-inflammatory activity evaluation on TNF-α induced pulmonary inflammation in A549 cells.could induce G0/G1 arrest and pro-death autophagy in human non-small cell lung cancer cell lines [27].
In the current research, a rarely occurring sesquiterpenoid di-glucoside (1), two new iridoids (2,3), and a new triterpenoid tri-glucoside (4), together with eight known terpenoids, were isolated from the root of G. macrophylla (Figure 1).Their structures were determined by spectroscopic methods, chemical methods, and quantum chemical calculations.All of the iridoids (2, 3, 5-8) were subjected to anti-inflammatory activity evaluation on TNF-α induced pulmonary inflammation in A549 cells.

Anti-Pulmonary Inflammation Activity of Iridoids
In order to evaluate the protective effect of iridoids on lung cell inflammation, a nonlethal concentration (10 ng/mL) of TNF-α was applied to induce an inflammatory response in A549 cells.Expression of the proinflammatory cytokine interleukin-1β (IL-1β) and interleukin-8 (IL-8), as well as the anti-inflammatory cytokine interleukin-10 (IL-10), were detected by enzyme-linked immunosorbent assay (ELISA).As shown in Figure 5, TNF-α stimulation very significantly upregulated the production of IL-1β (p < 0.01) and IL-8 (p < 0.001), while the expression of IL-10 was also increased via inverse feedback of living cells.Compounds 2, 3, and 6 could strongly decrease the content of IL-1β and IL-8 at 10 μM.Compound 2 could also upregulate the expression of the anti-inflammatory cytokine IL-10 significantly.The above data showed that the iridoids, especially 2′-Ocaffeoylgentiopicroside (2), could effectively attenuate the inflammatory response in lung cells and might be a potential lead compound for the development of anti-pneumonia drug.

Anti-Pulmonary Inflammation Activity of Iridoids
In order to evaluate the protective effect of iridoids on lung cell inflammation, a non-lethal concentration (10 ng/mL) of TNF-α was applied to induce an inflammatory response in A549 cells.Expression of the proinflammatory cytokine interleukin-1β (IL-1β) and interleukin-8 (IL-8), as well as the anti-inflammatory cytokine interleukin-10 (IL-10), were detected by enzyme-linked immunosorbent assay (ELISA).As shown in Figure 5

General Experimental Procedures
Optical rotations were determined on an Anton Paar MCP 200 automatic polarimeter (Graz, Austria).UV Spectra were recorded on a Shimadzu UV-2600 UV-Visible spectrometer.IR spectra were acquired on a Thermo Scientific Fourier Transform

Plant Material
Dry root of G. macrophylla Pall.was purchased from Dali city, Yunnan province of P. R.China and was authenticated by Prof. Youkai Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.A voucher specimen (GM2021A) was deposited in the School of Biotechnology and Health Sciences, Wuyi University (Jiangmen, China).

Extraction and Isolation
The air-dried roots of G. macrophylla (10 kg) were powdered and extracted with 70% EtOH (3 × 45 L, 7 days for each time) at room temperature.The solvent was evaporated under reduced pressure to obtain a crude extract (4.6 kg), which was then suspended in H 2 O (2.5 L) and successively partitioned with petroleum ether (PE, 4 × 2.5 L), EtOAc (4 × 2.5 L), and n-BuOH (4 × 2.5 L).The PE and EtOAc extracts were combined (100.1 g) and then the n-BuOH fraction (771.5 g) was obtained.
Analysis condition of GC-MS: the temperature of the injector is 250 • C; Injection volume 1.0 µL; Split ratio 41.667:1; Carrier gas helium, flow rate 1.

Quantum Chemical Calculation of ECD Spectrum of 1
The absolute configurations of 1 were determined by quantum chemical calculations of its theoretical ECD spectrum.The theoretical CD spectra of both 6R-1 and 6S-1 were studied.Conformational analyses were first carried out via Monte Carlo searching using molecular mechanisms with MMFF force field in the Spartan 18 program [38].The results showed 7 lowest energy conformers for 6R-1 and 5 ones for 6S-1 within an energy window of 2.0 Kcal/mol.Those conformers were then reoptimized using DFT at the B3LYP/6-31G(d) level using the Gaussian 09 program [39].The B3LYP/6-31G(d) harmonic vibrational frequencies were further calculated to confirm their stability.Six stable conformers for 6R-1 and three ones for 6S-1 (∆G within 2.0 Kcal/mol) with no imaginary frequencies were, respectively refined and considered for the next steps.The electronic excitation energies and rotatory strengths (velocity) of the first 60 excited states of these conformers were calculated using the TDDFT methodology at the M062X/TZVP level in the gas phase.The ECD spectra were simulated by the overlapping Gaussian function [40] (σ = 0.48 eV), in which velocity rotatory strengths of all the excited states were adopted.To get the final ECD spectra, the simulated spectra of the lowest energy conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (∆G) (see Supplementary Figures S37 and S38 and Tables S1-S4).
Cytotoxicity of compounds was tested by the MTT method according to the manufacturer's instructions.A549 cells were seeded (5 × 10 3 /well) into 96-well plates for 24 h.In addition to an untreated control group, compounds (10,20,40,80, or 160 µM) were added to cells and TNF-α (final concentration,10 ng/mL) was added in after 2 h of incubation.Each group had 3 duplicate wells.A total of 24 h later and 10 µL MTT (final concentration, 0.5 mg/mL) was added and incubated at 37 • C for 4h.Optical density (OD) values were detected with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 550 nm.