Anti-neuroinflammatory sesquiterpenoids from Chloranthus henryi

Abstract Hupelactones A (1) and B (2), two new eudesmanolide-type enantiomers of the corresponding compounds, along with four mono- (3–6) and nine dimeric- (7–15) known sesquiterpenoids were isolated from the whole plant of Chloranthus henryi var. hupehensis (syn. C. henryi). The new structures including the absolute configurations were determined by comparison with previously reported enantiomers, extensive spectroscopic methods in combination with electronic circular dichroism (ECD) calculations. All the isolates were evaluated for their inhibitory activities against the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine BV-2 microglial cells. Among them, the dimeric lindenane sesquiterpenoids shizukaols F (8) and G (11) exhibited the most potent activities, with IC50 values of 2.65 and 4.60 μM, respectively. Graphical Abstract


Introduction
Chloranthus, predominantly distributing in eastern Asia, is a small genus belonging to Chloranthaceae family with only 13 species and 5 varieties endemic to China (Xia et al. 1999). The Chloranthus species, being well-documented to be rich in structurally diverse terpenoids, have attracted considerable attentions from the scientific communities of natural products and organic synthesis (Wang et al. 2015a;Chen et al. 2021). To date, more than 400 secondary metabolites have been reported from this genus, with mono-and di-sesquiterpenoids being the major components (accounting for about 75%) (Wang et al. 2015a;Chen et al. 2021). These Chloranthus-originated sesquiterpenoids possess diverse structural skeletons (e.g., eudesmane-, lindenane-, aromadendrane-, guaiane-, germacrene-, and opopanone-types) and exhibited broadspectrum bioactivities such as anti-tumor, anti-HIV, antimicrobial, and anti-inflammation (Wang et al. 2015a;Zhang et al. 2016;Chen et al. 2021). C. henryi var. hupehensis (Pamp.) K.F.Wu (Zhang et al. 2021a(Zhang et al. , 2021b, a synonym of C. henryi Hemsl. (The Plant List, 2013), is a perennial herbaceous plant endemic to China, mainly growing in damp, shady sites at an altitude of 800-2000 m in the mountainous areas of Hubei, Shaanxi, and Gansu provinces (Xia et al. 1999). This species, named 'Siyeqi' in Chinese Materia Medica, has long been applied for the treatment of traumatic injury and inflammatory swelling and for detoxification in traditional Chinese medicine (Editorial Committee of the Administration Bureau of Traditional Chinese Medicine. 1999). During our continuing effort to seek novel bioactive terpenoids from Chloranthus plants native to China (Xiong et al. 2013;Liu et al. 2014;Wang et al. 2014;Wang et al. 2015b;Xiong et al. 2015;Xiong et al. 2016;Wang et al. 2022), a number of sesquiterpenoids (1 À 15, Figure 1) were isolated and identified from C. henryi. We herein describe their isolation, structure elucidation, and anti-neuroinflammatory activities.
The positive-mode HRESIMS of hupelactone B (2) gave a protonated ion at m/z 267.1588 [M þ H] þ ; this in conjunction with the 13 C NMR data established a molecular formula of C 15 H 22 O 4 for 2, just with one CH 2 less than 1. Accordingly, the 1 H and 13 C NMR data of 2 highly resembled those of 1 (Table S1). The only difference is that, signals for the methoxy group at C-8 were absent, while instead, a hemiketal group was found for 2. Likewise, the same relative configuration as that of compound 1 was assigned for 2 by the closely similar coupling constants and analogous NOE correlations ( Figure S16). Such a structure assignment of 2 was found to be identical to that of 4a,8b-dihydroxy-5a(H)-eudesm-7(11)-en-12,8-olide, a known eudesmanolide isolated from C. spicatus (Xiao et al. 2010). Nevertheless, compound 2 possesses a positive specific rotation {[a] D 20 þ71 (c 0.1, MeOH)}, in contrast to a negative one {[a] D 20 À48 (c 0.3, MeOH)} reported for the known structure. Apparently, these two eudesmanolides are also a pair of enantiomers. In the case of 2, its absolute configuration was readily determined to be the same as the co-occurring isolates 1 and 3, which was evidenced from the similar pattern of Cotton effects in their ECD spectra ( Figure S2). In fact, the (8S)-configuration corresponding to a positive Cotton effect around 240 nm was in accord with the empirical rule for the a,b-unsaturated-c-lactone (Snatzke 1968). Consequently, the whole structure of hupelactone B (2) was identified as (4 R,5R,8S,10R)-4,8-dihydroxy-eudesm-7(11)-en-12,8-olide.
Considering the impact of human factors on the optical rotation measurement, compound 1 was subsequently analysed by HPLC on a Chiralpak IC column to check the optical purity. As expected, only a single chromatographic peak appeared on the chromatogram ( Figure S13). Meanwhile, compound 3 was also optically pure as evidenced by the X-ray crystallographic study (see Experimental). Compound 2 was not subjected to the chirality analysis due to the paucity of the material; Nevertheless, it is reasonable to assume that 2 is also optically pure from the biosynthetic perspective.
Inspired by the anti-neuroinflammatory activity of some Chloranthus origin terpenoids Wang et al. 2015b;Xiong et al. 2016;Wang et al. 2022), all the isolates were evaluated for their inhibitory effects against the over-production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated BV-2 cells. As shown in Table  S2, the lindenane dimeric sesquiterpenoids 7-11 showed inhibitory effects (IC 50 values of 16.09, 2.65, 11.53, 23.84, and 4.6 lM, respectively), without any cytotoxicity against BV-2 cells at concentrations up to 50 lM (cell viabilities ! 95%). L-NMMA (N G -monomethyl-L-arginine) was used as the positive control (IC 50 : 14.42 ± 1.23 lM). Notably, these five bioactive compounds feature a common methyl methacrylate unit (C7, C11$C13). In contrast, the co-occurring analogues (12-15) with a lactone unit were inactive, implying that the presence of methyl methacrylate unit would be essential for the anti-neuroinflammatory activities of lindenane dimers.

General
Optical rotations were determined on a Rudolf Autopol IV automatic polarimeter (Rudolph Research Analytical, USA) at 20 C. IR and UV spectra were recorded on a Nicolet Is5 FTIR infrared spectrometer (Thermo Fisher Scientific, USA) with KBr disks and a Hitachi U-2900E spectrophotometer (Hitachi High Technologies, Japan), respectively. ECD spectra were acquired by a JASCO-810 CD spectrometer (JASCO, Japan). Xray crystallographic data were measured on a Bruker Apex Duo Diffractometer (Ga Ka). NMR data were obtained on a Bruker Avance III 400 MHz or 600 MHz instrument (Bruker, USA). HR-ESIMS data were performed on an AB Sciex Triple TOF 5600 spectrometer (AB Sciex Pte Ltd., USA). Column chromatography (CC) was conducted using MCI gel CHP20P (75-150 lm, Mitsubishi Chemical Industries, Japan), silica gel (100-200 mesh, Qingdao Marine Chemical Co. Ltd., China), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). Analytical and semi-preparative HPLC were carried out on a Waters e2695 system equipped with a 2998 photodiode array detector (Waters, USA), or a Shimadzu LC-20AT system with an SPD-M20A diode array detector (Shimadzu, Japan). A Diacel Chiralpak IC column (5 lm, 4.6 Â 250 mm, flow rate: 0.8 mL/min) was used for chiral analysis, while an X-Bridge C 18 column (5 lm, 10 Â 250 mm, flow rate: 3.0 mL/min) was utilised for HPLC semi-preparation. Fractions were monitored by TLC (GF 254 , Qingdao Marine Chemical Co. Ltd., China).

Extraction and isolation
The air-dried and powdered whole plant of C. henryi (9.2 kg) was extracted five times with 90% MeOH (each 12 L, 24 h) at ambient temperature. After removal of the solvents under reduced pressure, a dark-brown extract (904.3 g, semi-dry) was obtained, which was then dispersed in 2.5 L water and partitioned with petroleum ether (PE), ethyl acetate (EtOAc) and n-butanol (BuOH) in the same volume (2.5 L) three times, sequentially. Subsequent analyses of the three fractions by a combination of TLC, HPLC, and 1 H NMR revealed that the EtOAc-soluble fraction was rich in terpenoids. Thus, the entire EtOAc-soluble portion (102.4 g, semi-dry) was subjected to passage over a silica gel (100-200 mesh) CC with a stepwise gradient solvent system (neat PE, PE/EtOAc 20:1!10:1!5:1!2:1!1:1, neat EtOAc, EtOAc/MeOH 1:1, neat MeOH), giving six fractions (Fr. A À Fr. F).

ECD calculation of compound 1
Monte Carlo conformational searches were carried out by means of the Spartan's 14 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmannpopulation of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31g level in gas. The theoretical calculation of ECD was conducted in MeOH using TDDFT at the B3LYP/6-31g (d, p) level for all conformers of compound 1. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of W€ urzburg, W€ urzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma ¼ 0.3 eV.

Anti-neuroinflammation assay
The anti-neuroinflammatory activities of all samples were tested according to a previously described protocol (Wang et al. 2022).

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

Funding
This work was supported by the National Natural Science Foundation of China (NSFC) (grants No. 21772025 and 21937002).