Kadsindutalignans A–C: three new dibenzocyclooctadiene lignans from Kasura induta A.C.Sm. and their nitric oxide production inhibitory activities

Abstract Phytochemical study on the methanol extract of the stems and leaves of Kadsura induta led to the isolation of six dibenzocyclooctadiene lignans, including three new compounds named kadsindutalignans A–C (1–3), and three known ones, heteroclitalignan B (4), kadsuphilin C (5) and kadsulignan E (6). Their structures were elucidated based on extensive spectroscopic analyses, including HRESIMS, 1D- (1H NMR and 13C NMR), 2D-NMR (HSQC, HMBC, 1H-1H COSY and NOESY), and experimental circular dichroism (CD) spectra. All the isolates inhibited NO production in LPS-activated RAW264.7 cells with IC50 values in the range from 5.67 ± 0.54 µM to 38.19 ± 2.03 µM, compared to that of the positive control of NG-monomethyl-L-arginine acetate (L-NMMA) with an IC50 value of 8.90 ± 0.48 µM. Interestingly, the new compound 2 showed potential inhibition of NO production with an IC50 value of 5.67 ± 0.54 µM, which was higher than that of the positive control. Graphical Abstract


Introduction
The genus Kadsura (family Schisandraceae) comprises about 16 species, which is usually a climbing plant with separate male and female flowers growing on different plants.In Vietnam, there are six Kadsura species, K. chinensis, K. coccinea, K. hetroclita, K. longipedunculata, K. oblongifolia (Chi 2018), and K. induta A. C. Sm. a new record for the flora of Vietnam in 2012 (Thanh et al. 2012).In the traditional medicine of Vietnam, the leaves and stems of K. induta are used to treat arthritis, gastritis and duodenitis (Thanh et al. 2012).Previous phytochemical study on Kadsura species led to the isolation of lignans and triterpenes (Lu and Chen 2008;Liu et al. 2014;Su et al. 2014;Wang et al. 2021;Zhang et al. 2021;Tram et al. 2022).Notably, dibenzocyclooctadiene lignans, a typical substance of the genus Kasura, have been also found in K. induta and some of them exhibited antiviral and anti-HIV activities (Wenhui et al. 2007(Wenhui et al. , 2009;;Minh et al. 2014).In our continuing efforts toward discovering structurally interesting and biologically significant dibenzocyclooctadiene lignans from Kadsura species, six dibenzocyclooctadiene lignans (1-6) including three new ones (1-3) were isolated from the stem and leaves of this plant.All the isolates were found to inhibit NO production in LPS-activated RAW264.7 cells.Herein, we report the details of the isolation, structural elucidation, and the NO production inhibitory activities of these compounds.159.2, 151.0, 148.5, 141.3, 140.6, 136.2, 135.1, 130.9, 120.5, 120.2, 111.1 and 101.8] as similar to the most lignans found from the genus Kadsura (Li et al. 2006;Lu and Chen 2008;Liu et al. 2014;Minh et al. 2014).Of these, two carbons at d C 111.1 and 101.8 had HSQC cross peaks with the olefinic protons at d H 6.72 and 6.31, respectively.The abovementioned data suggested compound 1 was a dibenzocyclooctadiene lignan having one acetoxy, four methoxy, one dioxygenated methylene, and two hydroxy groups (Lu and Chen 2008;Minh et al. 2014).Detailed analysis of the NMR data (Table S1) showed that compound 1 was quite similar to Kadsuralignan B, which had two acetoxy groups at C-6 and C-9 (Li et al. 2006).In the HMBC spectrum, H-11  (Wang et al. 2021;Zhang et al. 2021) and further confirmed by HMBC spectrum as shown in Figure S1.The configuration of the biphenyl group of 1 was determined to be S, similar to all dibenzocyclooctadiene lignans found from the genus Kadsura, based on the positive cotton effects at 226 nm and negative cotton effects at 255 nm observed on the CD spectrum (Ikeya et al. 1988;Li et al. 2006;Wang et al. 2006;Shen et al. 2007).In the NOESY spectrum, the cross peaks between H-11 (d H 6.31) and H-8 (d H 1.94) indicated twist-boat-chair conformation of cyclooctadiene ring and H-8 adopted axial position (b-configuration) (Wang et al. 2006;Shen et al. 2007).Thus, the NOESY cross peaks between H-8 (d H 1.94) and H-9 (d H 4.84)/H-17 (d H 1.28) suggested the same b-configuration of H-9 and methyl group C-17.On the other hand, the NOESY cross peaks between H-4 (d H 6.72) and H-6 (d H 5.64) demonstrated for equatorial position (a-configuration) of H-6 (Figure S2).Thus, the chemical structure of compound 1 was determined as shown in Figure 1, a new compound named kadsindutalignan A.

Results and discussion
The molecular formula of compound 2 was elucidated as C 31 H 33 NO 11 by the exhibition of HR-ESI-MS ion peak at m/z 596.2136  43.4), between H-4 (d H 6.84) and C-6, and between H-6 (d H 5.88) and C-11.The CD spectrum of 2 showed the Cotton effects at (þ) 228 and (À) 249 nm indicating S-configuration of the biphenyl groups (Li et al. 2006;Ikeya et al. 1988).In the NOESY spectrum, the cross peaks between H-8 (d H 2.26) and H-11 (d H 6.53)/H-9 (d H 5.74)/H-17 (d H 1.40), H-4 (d H 6.84) and H-6 (d H 5.88) demonstrated the same stereochemistry of dibenzo cyclooctadiene moiety between 2 and 1 (Figure S2).Therefore, the chemical structure of compound 2 was determined as shown in Figure 1, a new compound named kadsindutalignan B.
Compound Comparing the NMR data of 3 with those of 1 and 2 showed that the lower carbon chemical shifts of C-3 (d C 148.6) and C-12 (d C 148.7) together with the absence of the dioxygenated methylene signals in 3 suggested two hydroxy groups were at C-3 and C-12 (Li et al. 2006).The other hydroxy group at C-9 confirmed by the 1 H-1 H COSY correlations of H-6/H-7/H-8/H-9, H-7/H-17, H-8/H-18, and by HMBC correlations from H-9 to C-11 and from H-11 to C-9 as shown in Figure S1.Four methoxy groups were at C-1, C-2, C-13 and C-14 determined by HMBC spectrum.The CD spectrum of 3 showed the Cotton effects at (þ) 215 and (À) 250 nm indicating S-configuration of the biphenyl groups (Li et al. 2006 andIkeya et al. 1988).The NOESY cross peaks between H-17 (d H 0.93) and H-4 (d H 6.67)/H-18 (d H 1.17), H-8 (d H 1.92) and H-7 (d H 2.08)/H-9 (d H 4.62)/H-11 (d H 6.49), H-11 and H-9 were observed evidencing these protons were close in proximity, indicating H-7, H-8, and H-9 groups in the same side of the molecule (Figure S2) (Wang et al. 2006;Shen et al. 2007).Thus, the chemical structure of compound 3 was determined as shown in Figure 1, a new compound named kadsindutalignan C.
The dibenzocyclooctadiene lignans have been reported for their potential antiinflammatory activity (Li et al. 2006;Wang et al. 2021).Therefore, compounds 1-6 were evaluated for anti-inflammatory activity by their ability to inhibit NO production in LPS stimulated RAW 264.7 cells (Supporting information).At a concentration of 100 mM, compounds 1-6 did not significantly show cytotoxic activity by MTT assay (data not shown).Therefore, the levels of NO production in the cell medium were measured in the presence of 1-6 at serial diluted concentrations (0-100 mM).As shown in Table S3, all the tested compounds exhibited NO inhibitory activity with IC 50 values in the range from 5.67 ± 0.54 to 38.19 ± 2.03 mM compared to that of the positive control L-NMMA (IC 50 8.90 ± 0.48 mM).Regarding structure activity relationship, our results suggested that the dibenzocyclooctadiene lignans which had a dioxygenated methylene group at C-12 and C-13 may have significant NO inhibitory activity and the compounds having benzyl or pyridinecarboxyl groups showed higher NO inhibitory activity than that of the other tested compounds.This result is well in agreement with those reported in literature (Li et al. 2006;Wang et al. 2021).

General
Optical rotation was measured on a Jasco P2000 polarimeter.The CD spectra were measured on a Chirascan spectrometer (Applied Photophysics, Surrey, UK).The HR-ESI-MS was taken on an Agilent 6530 Accurate Mass Q-TOF LC/MS.The NMR spectra were recorded on a Bruker 500 MHz spectrometer using TMS as an internal Standard.Preparative HPLC were run on an Agilent 1100 system including quaternary pump, autosampler, DAD detector, and preparative HPLC column YMC J'sphere ODS-H80 (4 mm, 20 Â 250 mm).Isocratic mobile phase with the flow rate of 3.0 mL/min was used in pre-HPLC.The compound was monitored at wavelengths of 205, 230, 254 and 280 nm.Flash column chromatographies were performed using silica gel (60, 70-230 mesh and 230-400 mesh, Merck, Darmstadt, Germany) or reversed phase C-18 (YMC, Kyoto, Japan) as stationary phase.Thin layer chromatographies (TLC) were carried out on pre-coated silica gel 60 F 254 and RP-18 F 254S plates.The spots were detected by spraying with aqueous solution of H 2 SO 4 5% followed by heating with a heat gun.

Plant material
The stems and leaves of Kadsura induta A.C.Sm.were collected from Sapa, Lao Cai, Vietnam, in April 2022 and authenticated by Dr Nguyen Van Thanh, Institute of Ecology and Biological Resources, VAST.A voucher specimen (code NCCT-P149) is kept at the Herbarium, Institute of Ecology and Biological Resources, Hanoi, Vietnam.