New dibenzocyclooctadiene lignans from Kadsura induta with their anti-inflammatory activity

Five new dibenzocyclooctadiene lignans, named kadsuindutains A–E (1–5), and three known ones schizanrin F (6), schizanrin O (7), and schisantherin J (8) were isolated from the stems of Kadsura induta. Their structures were determined by analyses of HR-ESI-MS, NMR, and ECD spectra. Compounds 1–5 contain a 2′,4′-dioxygenated-2′,3′-dimethylbutyryl moiety which is rarely reported for dibenzocyclooctadiene lignans. Molecular docking predicted that compounds 1–8 displayed good binding affinity to the active site of iNOS and TNF-α proteins but unstable binding to the active site of COX-2 protein. Additionally, in vitro experiments showed that compounds 1–8 inhibited NO production in LPS-activated RAW264.7 cells with IC50 values from 10.7 μM to 34.0 μM, compared to the positive control L-NMMA (IC50 = 31.2 μM).


Introduction
The genus Kadsura (Schisandraceae family) comprises 16 species which are widely distributed in Asian countries. 1 The stems, roots, and leaves of Kadsura species have been used in traditional medicines to treat arthritis and gastritis, promote the circulatory system, and relieve pain. 2 Recently, a lot of phytochemical studies have been focused on Kadsura species due to being a rich source of highly oxygenated dibenzocyclooctadiene lignans and triterpenoids. 2,3 Not only interesting in chemical structure, lignans and triterpenoids from Kadsura species also exerted various benecial biological activities such as anti-inammatory and cytotoxic activities, as well as being potent antiviral agents. [4][5][6][7][8][9] Dibenzocyclooctadiene lignans have been suggested to be important sources for development of potent anti-hepatitis drugs. 9 With the aim to nd bioactive dibenzocyclooctadiene lignans, K. induta was selected for investigation and lead to isolation of ve new and three known dibenzocyclooctadiene lignans. We describe, herein, the isolation and structural elucidation of those compounds. In addition, the anti-inammatory activities of the isolated compounds were studied by molecular docking on iNOS, TNF-a, COX-2 proteins, and in vitro evaluated on the NO production in LPSactivated RAW264.7 cells.

Results and discussion
The methanol extract from the stems of K. induta was separated between dichloromethane and water. The organic layer was subjected to ash column chromatography on silica gel or ODS reversed phase, and further puried by semipreparative HPLC to afford eight C 18 -dibenzocyclooctadiene lignans (1)(2)(3)(4)(5)(6)(7)(8).

General experimental procedures
Optical rotation was measured on a Jasco P2000 polarimeter. ECD spectrum was measured on a Chirascan spectrometer. NMR spectra were obtained on a Bruker AvanceIII 500 MHz FT-NMR spectrometer. HR-ESI-MS was analysed on an Agilent 6530 Accurate-Mass QTOF. Semi-preparative HPLC was performed on an Agilent 1260 innity II system including binary pump, autosampler, DAD detector, and YMC J'sphere ODS-H80 (20 Â 250 mm, 4 mm) HPLC column. Mobile phase was an isocratic system of methanol/water or acetonitrile/water at ow rate of 3 mL min À1 . Thin layer chromatography was performed on precoated plates with silica gel or reversed phase C18 (RP-18). Column chromatography was performed using silica gel (40-63 mm) or RP-18 (150 mm) as adsorbents.

Plant material
Plant samples were collected in February 2022 at Sapa, Lao Cai province, Vietnam and taxonomically identied to be Kadsura induta A. C.$Sm. by Dr Bui Van Thanh and Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. Voucher specimen (IMBC-NCXS-02) was kept at the Institute of Marine Biochemistry, VAST.

Extraction and isolation
Dried powdered K. induta stems (6 kg) was ultrasonically macerated with methanol at room temperature for three times (15 L and 2 h each time) to give methanolic residue. The obtained residue (150 g) was well suspended with 3.0 L of water and then successively partitioned with dichloromethane and ethyl acetate to give dichloromethane, ethyl acetate, and watersoluble extracts. The dichloromethane extract (52 g) was separated on a silica gel column, eluting with gradient solvent system of n-hexane/acetone (1/0-0/1, v/v) to give six fractions, KID1-KID6. Fraction KID3 (17 g) was continuously chromatographed on a silica gel column, eluting with n-hexane/acetone (7/1, v/v) to give three fractions, KID3A-KID3C. Fraction KID3A was puried by semi-preparative HPLC (SemiPre-HPLC) using acetonitrile/water (3/1, v/v) to yield compounds 5 (12.5 mg, t R 61.3 min) and 8 (14.1 mg, t R 68.1 min). Fraction KID4 (12 g) was subjected to a silica gel column and eluted with n-hexane/ acetone (

Molecular docking
Molecular docking was carried out using AutoDock v4.2 program as previously described. [21][22][23][24] The crystal structure of inducible nitric oxide synthase with inhibitor (S71), TNF-a with inhibitor (307), and COX-2 with inhibitor (celecoxib) were obtained from the RCSB Protein Database Bank (PDB ID: 4CX7, 2AZ5, and 3LN1, respectively). The 3D structures of compounds 1-8 were obtained by Spartan18 soware. Conformational search was started with the MMFF molecular mechanics set and then minimized energy by PM3 semi-empirical condition. A conformer with lowest energy was used as a ligand for molecular docking. Receptor was prepared as following steps: delete water molecules, remove initial ligands, repair the missing residues, add polar hydrogen atoms, add and spread Kollman charges over all macro-molecule. The grid box dimensions were 40 Â 40 Â 40Å with 0.5Å grid spacing and center coordinates at original inhibitor to locate active site of the receptors (x: À15.0, y: À66.0, z: 17.0 for iNOS 4CX7; x: À9.3, y: 68.7, z: 20.2 for TNFa 2AZ5, and x: 29.8, y: À21.3, z: À17.4 for COX-2 3LN1). Auto-Dock calculations were run by Lamarckian genetic algorithm and set at running times of 50, population sizes of 300, and default setting for other miscellaneous parameters. Docked complex with the lowest binding energy was selected to represent the most favourable interaction between the ligand and receptor. Results were re-rendered using Chimera v1.16.

Conclusions
Our phytochemical investigation on the stems of K. induta resulted eight dibenzocyclooctadiene lignans comprising ve new (1-5) and three known (6-8) ones. Compounds 1-5 possessed unique structure and rarely found in the nature. Molecular docking suggested that compounds 1-8 are good binding affinity with anti-inammatory mediators including iNOS and TNF-a. Additionally, compounds 1-8 inhibited NO production in LPS activated RAW 264.7 cells with IC 50 values from 10.7 mM to 34.0 mM which are almost smaller than IC 50 value of positive control, L-NMMA.

Author contributions
PV Kiem, BH Tai, NX Nhiem, contributed to research idea and writing; BV Thanh, NT Cuong contributed to sample collection and taxonomical identication. NH Hoang, PTT Huong, NV Dung, NA Bang contributed to isolation; PH Yen, NX Nhiem, PV Kiem, BH Tai contributed to structure elucidation; BH Tai, NX Nhiem contributed to molecular docking and NO assay.

Conflicts of interest
There are no conicts to declare.