Liquidambarines A − C, three new abietane diterpenoids from Liquidambar formosana Hance and their anti-inflammatory activities

Abstract Liquidambarines A − C (1−3), three new abietane-type diterpenoids, together with five known compounds (4−8) were isolated from the resin of Liquidambar formosana Hance. Their structures were elucidated by the combination of spectroscopic and computational methods. We explored their anti-inflammatory potential by analyzing the protein expression of iNOS and COX-2. Compounds 1 and 3 exhibit significant anti-inflammatory activities without cytotoxicity. These experimental studies suggest these new abietane-type diterpenoids have the potential to be candidates for inflammation-associated diseases. Graphical Abstract


Introduction
Liquidambaris Resina, an adhesive gum harvested from Liquidambar formosana Hance (Altingiaceae Horan), is in irregular block shape, pale yellow or yellow brown in color and glossy in cross section and light in smell, which is known as Chinese sweet gum. Burning the resins with fire, there is a smoky flame with a specific aroma. Liquidambar formosana Hance, is an ornamental tree, mainly distributed in Southern China (Yunnan, Guizhou, Taiwan, Hainan), Laos, Vietnam and North Korea (Yu et al. 2012). Essential oils extracted from the resins of L. formosana Hance have been widely used in cosmetics (Ouyang et al. 2016). Furthermore, the resins have also been used as a traditional Chinese medicine for the treatment of inflammation, fall injury, carbuncle swelling, hematemesis, and trauma hemorrhage (Pharmacopoeia of the People's Republic of China 2020). The ethyl acetate (EtOAc) extract of the Liquidambaris Resina could affect the vascular active factor of the mice (Cheng et al. 2011). Previous phytochemical studies on L. formosana resins have shown different class of terpenoids such as pentacyclic tritepenoids (oleane, lupane and ursane type) (Pham and Vien 1975;Iankov et al. 1980;Yankov et al. 1980;Liu et al. 1991Liu et al. , 1995Dat et al. 2004;Yang et al. 2011) and diterpenoids (abietane and sempervirol type) (Shang et al. 2014). These terpenoids exhibit promising bioactivities including cytotoxic (Yang et al. 2011), antibacterial (Shimozu et al. 2017), antioxidant , anti-inflammatory (Man et al. 2020) and antiangiogenic (Zhu et al. 2021). Abietane-type diterpenoids are a class of compounds constructed with a hydrophenanthrene ring system (Sun 2012). Our efforts to discover bioactive diterpenoids from the EtOAc extract of L. formosana Hance resin led to the isolation of three new and five known abietane-type diterpenoids. Herein, we describe the isolation, structure identification, and biological evaluation of all the new metabolites.
The relative configuration of compound 1 was determined by the interpretation of ROESY experiment ( Figure S3). Cross peaks of CH 3 -20/CH 3 -18 suggest that CH 3 -20 and CH 3 -18 are in same direction of the ring. As per the relative configuration of 1, the electronic circular dichroism (ECD) calculations were performed. The calculated ECD curve of (4 R,5R,10S)-1 ( Figure S4) is in well agreement with the experimental curve of 1, and it reveals the absolute configuration of compound 1 is 4 R,5R,10S. Herein, the structure of 1 was assigned and named Liquidambarine A.
Abietane-type diterpenoids derivatives have been identified to have therapeutic potential for inflammation (Feng et al. 2017;Huang et al. 2022). Based on the traditional medicinal properties of L. formosana Hance, we investigated the anti-inflammatory effects of compounds 1À3. As shown in Figure S4, the cells viability of compounds 1, 2 and 3 are 93.5%, 93% and 86% respectively. The results indicate that all compounds exhibit low toxicity on RAW 264.7 cells. Compounds 1 and 3 significantly suppress LPS-induced iNOS expression in RAW264.7 cells. Meanwhile, all compounds do not affect the COX-2 expression induced by LPS. The human iNOS (NOS2) and COX-2 (MT-CO2) are two important mediators for inflammatory disease. iNOS is coded by gene that located on chromosome 17. It is regulated mainly on transcriptional level and is independent of intracellular calcium concentration. And the COX-2 is coded by MT-CO2 that located on mitochondrial. Compounds 1 and 3 only suppress the protein expression of iNOS, which may indicate these compounds exhibit antiinflammatory effects by influencing the transcriptional process. Consequently, compounds 1 and 3 exhibit promising remedy for treatment of inflammation. This structural class of abietane compounds may indicate a room for further structural modifications to enhance anti-inflammation activities.

General experimental procedures
CD spectra and UV spectra were recorded on a Jasco J-815 circular dichroism spectrometer (JASCO, Japan). Optical rotation was obtained on an Anton Paar MCP-100 digital polarimeter. HRESIMS spectral data were collected by a Shimazu LC-20 CE AB SCIEX triple TOF X500R MS spectrometer with a dual spray TurboV ESI source (Shimadzu Corporation, Tokyo, Japan). The injection experiment was carried out at the syringe flow rate of 0.3 mL/min in positive ionization, with source parameters as follows: injection volume 2.0 mL, TOF start mass 100 Da, TOF stop mass 2000 Da, accumulation time 0.25 s, declustering potential À80 V, collision energy À10 V, time bins to sum 4, column oven temperature 30 C. Data acquisition, data handling, and instrument control were performed by SCIEX OS Software. The 1 D and 2 D NMR spectra were measured on Bruker Avance 500 and Avance III 600 MHz spectrometer (Bruker, Karlsruhe, Germany) instrument operating at 9.4 T and 298 K, with TMS as an internal standard. Generally, chemical shifts (d) were expressed in ppm with reference to the solvent signals. Among them, the spectral width of the heteronuclear single quantum coherence (HSQC) experiment was 16 ppm for the proton nucleus and 230 ppm for the carbon proton nucleus. The relaxation time was 1.5 s and the data matrix set was 8 K Â 256 points. The heteronuclear multi-bond correlation (HMBC) experiment used the same spectral width as the HSQC experiment, the relaxation time was 1.5 s and a data matrix of 12 K Â 256 points. The semi-preparative LC apparatus is Saipuruisi (SEP) Beijing Technology Co., Ltd., model: LC52. A Saipuruisi (SEP) chromatograph with an Agilent ZORBAX SB-C18 column (250 mm Â 4.6 mm, i.d., 5 lm) or a YMC-Pack ODS-A column (250 mm Â 9.4 mm, i.d., 5 lm) was employed for semi-preparative HPLC and the wavelength of UV detector was 254 nm. Column chromatography was performed on silica gel (200 À 300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), MCI gel CHP 20P (75 À 150 lm, Mitsubishi Chemical Industries, Tokyo, Japan), (40 À 60 mm, Daiso Co., Tokyo, Japan). The chromogen was 10% ethanol sulfate; Ultrapure water was used for the column chromatography; Wahaha Purified water was used for liquid phase; Methanol and acetonitrile were used for chromatography, which were purchased from Energy Chemical.

Plant resins
The resins of L. formosana Hance were purchased from local people of Weixi Lisu Autonomous County, Diqing Tibetan Autonomous Prefecture, Yunnan Province, China (North latitude 26 53 0 ～28 02 0 , East longitude 98 54 0 ～99 34 0 ), collected at same place in July 2018 and identified by Prof. Bin Qiu (Yunnan University of Traditional Chinese Medicine) via morphological comparison with authentic samples. A voucher specimen (CHYX0671) was deposited at School of Pharmaceutical Sciences, Shenzhen University Health Science Center, China.

Quantum chemical computations
Molecular Merck force field (MMFF) conformational search and DFT/TD-DFT calculations were performed by the Spartan'14 (wavefunction Inc., Irvine) and Gaussian 09 program (Frisch et al. 2013). To select low-energy conformers within 10 kcal/mol, conformational search was applied in CONFLEX 7.0. The major conformers were optimized at the B3LYP/6-311G (d, p) level by DFT calculations, using the PCM solvent model, in which MeOH was solvent. B3LYP/6-31G (d, p) was further implemented for ECD calculations with PCM in MeOH. The tested ECD curves were fitted with the experimental CD spectra in SpecDis 1.62 software.
Cell viability assay: RAW264.7 cells were seeded into 96-well plates with completed DMEM at an initial density of 105 cells/well. After incubation with the compounds (40 lM) for 2 h and then stimulated with LPS (1 lg/mL) for additional 24 h. 10 lL Cell Count Kit-8 (CCK-8, C0037, Beyotime, Shanghai, China) was added into each well for 1 h at 37 C. Cell viability was obtained by scanning with a microplate reader (BioTek, USA) at 450 nm.
Western blot: RAW264.7 cells (4 Â 10 5 cell/well) were seeded in 12-well plates. After overnight culture, cells were pre-treated with compounds (40 lM) or DMSO for 2 h and then stimulated with LPS (1 lg/mL) for additional 24 h. The cells were lysed with RIPA lusis buffer (Beyotime). The protein concentration of cells was detected by Pierce BCA Protein Assay kit (Beyotime). The mixtures were boiled at 100 C for 5 min. Protein samples were isolated by 8% SDS-PAGE and transferred to the PVDF membrane. The PVDF membrane was blocked by 5% BSA at room temperature for 1 h, and incubated at 4 C with primary antibodies, and then incubated with the secondary antibodies. The final results were performed by chemiluminescence gel imaging system (BIO-RAD).

Conclusion
In summary, three new abietane-type diterpenoids, Liquidambarines A À C (1À3) and five known diterpenoids were isolated from the ethyl acetate extract of L. formosana Hance resins. The absolute configurations of all the isolates were clarified by both spectroscopic and computational methods. Compounds 1 and 3 could suppress LPSinduced iNOS expression in RAW264.7 cells, indicating 1 and 3 have anti-inflammatory potential.