New obovatol trimeric neolignans with NO inhibitory activity from the leaves of Magnolia officinalis var. biloba

New obovatol trimeric neolignans with NO inhibitory activity from the leaves of Magnolia officinalis var. biloba

Journal Pre-proofs New obovatol trimeric neolignans with NO inhibitory activity from the leaves of Magnolia officinalis var. biloba Van-Tuan Vu, Xiao-...

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Journal Pre-proofs New obovatol trimeric neolignans with NO inhibitory activity from the leaves of Magnolia officinalis var. biloba Van-Tuan Vu, Xiao-Qin Liu, Manh-Tuyen Nguyen, Yao-Lan Lin, Ling-Yi Kong, Jian-Guang Luo PII: DOI: Reference:

S0045-2068(19)31991-1 https://doi.org/10.1016/j.bioorg.2020.103586 YBIOO 103586

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

21 November 2019 13 January 2020 13 January 2020

Please cite this article as: V-T. Vu, X-Q. Liu, M-T. Nguyen, Y-L. Lin, L-Y. Kong, J-G. Luo, New obovatol trimeric neolignans with NO inhibitory activity from the leaves of Magnolia officinalis var. biloba, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103586

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New obovatol trimeric neolignans with NO inhibitory activity from the leaves of Magnolia officinalis var. biloba Van-Tuan Vua, Xiao-Qin Liua, Manh-Tuyen Nguyenb, Yao-Lan Lina, Ling-Yi Konga,*, JianGuang Luoa,*

Affiliations a

Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural

Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, People′s Republic of China. b

Department of Traditional Medicine, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoan Kiem,

Hanoi, Vietnam

Corresponding authors * Ling-Yi Kong: E-mail: [email protected] Tel/Fax: +86-25-8327-1405 * Jian-Guang Luo: E-mail: [email protected] Tel/Fax: +86-25-8327-1405

Abstract Six new obovatol trimeric neolignans, houpulignans A–F (1–6) were isolated from the leaves of Magnolia officinalis var. biloba. Their structures were determined on the basis of the interpretation of HRESIMS, NMR data, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are the first examples of neolignans derived from three units of obovatol bearing a rare 1,4-benzodioxepane moiety. Compound 3 possesses a benzodihydropyran ring, meanwhile three units of obovatol in 4–6 are connected by an alkyl chain. Compounds 1-3 inhibited NO production in LPS-stimulated RAW264.7 cells with IC50 values of 8.01, 20.21, and 4.05 µM, respectively.

Keywords: Magnolia officinalis var. biloba, obovatol trimeric neolignans, NO inhibitory activity

1. Introduction Magnoliae officinalis cortex described in the Chinese Pharmacopoeia is the bark of Magnolia officinalis Rehd. et Wils or Magnolia officinalis Rehd. et Wils var. biloba [1], which has been used as an important traditional medicine in China and many countries for the treatment of diseases, such as abdominal distension, vomiting, diarrhea, constipation, phlegm and fluid retention and cough resulting from asthma [1,2]. The chemical investigations of M. officinalis have been more studied. This plant was found to have neolignans, terpenoids, alkaloids, phenols, and steroids [2,3], which have been demonstrated to exhibit a wide range of biological effects, including anti-inflammatory [4,5], anticancer [6,7], anti-epileptic [8], antidepressant [9], and antibacterial [10] activities. The varietas, M. officinalis var. biloba is also known as “Aoye Houpu” in Chinese. Previous investigations of this species mainly focused on bark and fruits, resulting in the isolation of meroterpenoids [11,12], alkaloids [2], phenylethanoid glycosides [13]. To date, only one phytochemical study on M. officinalis var. biloba leaves has been reported, indicating neolignans as characteristic chemical constituents [14]. Neolignans, a group of natural products are formed by two C6–C3 units through a bond other than C-8 and C-8′. Magnolol, honokiol, obovatol are typical neolignans of Magnolia species [2]. It has reported that neolignans could be conjugated through C-C or ether linkages to form oligomeric neolignans, which are rare in natural resources [2,15]. In an effort to search for novel bioactive constituents, six new trimeric neolignans, houpulignans A-F (1–6) were isolated from the leaves of M. officinalis var. biloba. Herein, the isolation and structural elucidation of compounds 1–6 along with their inhibitory effect on NO production are presented. 2. Materials and methods 2.1. General experimental procedures

Optical rotations were recorded on a JASCO P-1020 polarimeter in MeOH at room temperature. UV spectra were performed on a UV−2450 spectrophotometer. IR spectra were recorded in KBr disc on a Bruker Tensor 27 spectrometer. A JASCO J–810 spectropolarimeter (Jasco, Tokyo, Japan) was used to collect electronic circular dichroism (ECD) spectra. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVIII600 NMR instrument (1H: 600 MHz,

13C:

150 MHz) equipped with CryoProbe (Bruker,

Karlsruhe, Germany). High-resolution electrospray ionization (HRESIMS) spectra were measured on an Agilent 6529B Q-TOF instrument (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC was carried out on a Shimadzu LC-6A system (Shimadzu, Tokyo, Japan) equipped with a Shim-pack RP-C18 column (200 × 20 mm; 10 μm). 2.2. Plant material The leaves of M. officinalis var. biloba were collected in the Botanical garden of China Pharmaceutical University in October 2018 and authenticated by Prof. Minjian Qin of the Research Department of Pharmacognosy, China Pharmaceutical University. A voucher specimen was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University (accession number 2018-MOL). 2.3. Extraction and isolation The dried leaves of M. officinalis var. biloba (3.5 kg) were extracted using EtOH 95% (12 L × 3 times × 4 hours at room temperature) with ultrasonic assistance. The EtOH extract was filtered and evaporated under reduced pressure to yield a green residue (486 g). The crude extract was suspended in distilled H2O (2 L) and successively partitioned with CH2Cl2 and EtOAc. The CH2Cl2 extract (80 g) was initially fractionated by normal-phase silica gel chromatography column (CC) eluted with a gradient of petroleum ether-EtOAc (1:0, 10:1, 4:1, 2:1, 1:1 v/v) to give six fractions (A-F). Fraction C (10.2 g) was chromatographed on an ODS CC eluted with a gradient system of MeOH-H2O (60:40 to 100:0, v/v) to give eight

subfractions (C1-C8). Subfraction C4 was subjected to preparative RP-HPLC (MeOH-H2O, 85:15, v/v) to give 1 (6 mg) and 3 (4 mg). Compound 2 (11 mg) was obtained from subfraction C5 by using the preparative RP-HPLC (MeOH-H2O, 87:13, v/v). Compounds 4 (6 mg) and 5 (4 mg) were obtained from subfraction C3 using the preparative RP-HPLC (MeOH-H2O, 85:15, v/v, and MeOH-H2O, 82:18, v/v, respectively). Subfraction C2 was purified by the preparative RP-HPLC (MeOH-H2O, 80:20, v/v) to afford compound 6 (5 mg). 2.4. Spectroscopic data 20

Houpulignan A (1): white amorphous powder; [α] D +10.4 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 207 (5.07), 276 (4.18) nm; ECD (MeOH) λ (Δε) 227 (+10.30); IR (KBr) νmax 3423, 2925, 1596, 1504, 1441, 1216 cm−1; 1H NMR and

13C

NMR data, see Table 1;

HRESIMS m/z 843.3521 [M + H]+ (calcd for C54H51O9, 843.3528). 20

Houpulignan B (2): white amorphous powder; [α] D +12.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 210 (5.04), 276 (4.17) nm; ECD (MeOH) λ (Δε) 228 (+7.53); IR (KBr) νmax 3447, 2977, 1606, 1504, 1448, 1220, 1167 cm−1; 1H NMR and

13C

NMR data, see Table 1;

HRESIMS m/z 865.3348 [M + Na]+ (calcd for C54H50O9Na, 865.3347). 20

Houpulignan C (3): white amorphous powder; [α] D −6.8 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 207 (5.05), 275 (4.18) nm; ECD (MeOH) λ (Δε) 210 (+0.66), 216 (−2.14), 226 (+5.11); IR (KBr) νmax 3453, 2975, 1504, 1439, 1215, 1167 cm−1; 1H NMR and

13C

NMR

data, see Table 1; HRESIMS m/z 843.3529 [M + H]+ (calcd for C54H51O9, 843.3528). Houpulignan D (4): white amorphous powder;

20

[α] D +13.4 (c 0.16, MeOH); UV

(MeOH) λmax (log ε) 206 (5.07), 275 (4.19) nm; ECD (MeOH) λ (Δε) 207 (+2.75), 223 (+7.45), 229 (+4.47); IR (KBr) νmax 3421, 2923, 1611, 1504, 1434, 1215, 1168 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 897.3606 [M + Na]+ (calcd for C55H54O10Na, 897.3609).

20

Houpulignan E (5): white amorphous powder; [α] D +11.6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 206 (5.10), 274 (4.16) nm; ECD (MeOH) λ (Δε) 209 (+2.18), 224 (+6.13); IR (KBr) νmax 3423, 2976, 1604, 1503, 1438, 1216, 1168 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 897.3604 [M + Na]+ (calcd for C55H54O10Na, 897.3609). 20

Houpulignan F (6): white amorphous powder; [α] D −8.9 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 207 (5.11), 275 (4.16) nm; ECD (MeOH) λ (Δε) 205 (+0.71), 214 (−3.88), 227 (+5.03); IR (KBr) νmax 3457, 2972, 1605, 1505, 1447, 1219, 1166 cm−1; 1H NMR and

13C

NMR data, see Table 1; HRESIMS m/z 897.3612 [M + Na]+ (calcd for C55H54O10Na, 897.3609). 2.5. Quantum chemical ECD calculation The 3D structures of selected compounds were subjected to Schrödinger MacroModel 9.1 (Schrödinger. LLC, USA) to perform conformational analysis using the MMFF force field in gas phase. The conformers with Boltzmann-population of over 5% were chosen, and then the conformers were initially optimized at B3LYP/6-31g (d, p) level in gas phase by Gaussian 09 program package. The theoretical calculation of ECD was conducted in MeOH using Time-dependent Density functional theory (TD-DFT) at the B3LYP/6-31g (d, p) level for all optimized stable conformers. Rotatory strengths for a total of 60 excited states were calculated. ECD spectra were generated using the program SpecDis 1.7.1 with UV correction and a half-bandwidth of σ = 0.20 eV [16]. 2.6. NO inhibitory assay The RAW264.7 cell line was purchased from the Chinese Academic of Sciences. The cells were cultured in DMEM containing 10% FBS with penicillin (100 U/mL) and streptomycin (100 U/mL) at 37ºC in a humidified atmosphere with 5% CO2. The cells were allowed to grow in 96-well plates with 1 × 105 cells/well to treat test compounds. After being incubated for 2 h, the cells were treated with 100 ng/mL of LPS for 18 h. Nitrite in culture

media was measured to assess NO production using Griess reagent. The absorbance at 540 nm was measured on a microplate reader. N-monomethyl-L-arginine was used as the positive control. Cytotoxicity was determined by the MTT method, after 48 h incubation with test compounds. All the experiments were performed in three independent replicates [17]. 3. Results and discussion Compound 1 was isolated as white amorphous powder. Its molecular formula was determined to be C54H50O9 on the basis of HRESIMS (m/z 843.3521 [M + H]+, calcd for C54H51O9, 843.3528), with 30 degrees of unsaturation. The IR spectrum showed absorption bands at 3423, 1596, 1504, 1441 cm-1, suggesting the presence of hydroxyl and aromatic ring functionalities. The 1H NMR data (Table 1) showed 12 aromatic methine proton signals at δH 6.58 (2H, d, J = 8.4 Hz), 6.63 (4H, d, J = 8.4 Hz), 6.86 (2H, d, J = 8.4 Hz), 7.04 (2H, d, J = 8.4 Hz), and 7.10 (2H, d, J = 8.4 Hz) of three para-substituted benzene rings; four metacoupled aromatic methine proton resonances at δH 5.98 (1H, br s), 6.36 (1H, br s), 6.56 (1H, br s), and 6.61 (1H, br s) from two tetrasubstituted benzene rings; a singlet signal of proton at δH 6.57 due to one pentasubstituted aromatic ring; one oxygenated methine proton at δH 4.86 (1H, d, J = 9.6 Hz); one oxygenated methylene group at δH 3.92 (1H, dd, J = 12.0, 9.0 Hz), 4.21 (1H, dd, J = 12.0, 3.2 Hz); one methine proton at δH 3.72 (1H, m). In addition, the signals of five allyl groups including five overlapping olefin methine protons at δH 5.75 (1H, m), 5.95 (4H, m); five exo-methylene proton resonances at δH 4.89 (1H, m), 5.09 (9H, m); five methylene proton signals at δH 2.96 (1H, dd, J = 15.6; 6.0 Hz), 3.16 (1H, dd, J = 15.6; 6.0 Hz); 3.27 (2H, d, J = 6.6 Hz), 3.35 (2H, d, J = 6.6 Hz), 3.37 (4H, d, J = 6.6 Hz) were also observed in 1H NMR spectrum of 1. On the basis of the DEPT and HSQC data, the 54 carbon signals in

13C

NMR spectrum of 1 were assigned for 11 methylenes, 24 methines, and 19

quaternary carbons. From the above analyzed data, compound 1 was determined to be a trimer of obovatol, a 3-O-4′ diphenyl ether-type neolignan previously isolated from Magnolia

species [2,18]. The structure of 1 was further elucidated based on the detailed analysis of its 1D and 2D NMR spectra. The COSY correlations revealed a coupling network of H-7/H8/H2-9 of subunit A (Fig. 2). The ROESY interaction of H-2 (subunit A) with H-3′/H-5′ (subunit B) indicated that subunit A was connected to subunit B via an aryl ether linkage at C3. The HMBC correlations of H-2 (subunit A, δH 5.98), H-6 (subunit A, δH 6.36) with C-7 (subunit A, δC 84.5); of H-8 (subunit A, δH 3.72) with C-1 (subunit C, δC 131.9), C-2 (subunit C, δC 122.0), C-3 (subunit C, δC 140.7); of H-6 (subunit C, δH 6.57) with C-1 (subunit C, δC 131.9), C-2 (subunit C, δC 122.0) (Fig. 2) suggested the direct linkage between C-8 of subunit A and C-2 of subunit C. The absence of ROESY correlation from H-6 of subunit C to H-3′/H5′ of subunit D revealed that subunit C was linked to subunit D through an ether bond at C-3. The existence of six aromatic rings and five allyl groups in the structure of 1 accounted for 29 degrees of unsaturation, hence one remaining degree of unsaturation must be contributive to a ring formation. The HMBC correlations from H-7 (subunit A, δH 4.86), H-2 (subunit E, δH 6.61), H-6 (subunit E, δH 6.56) to C-4 (subunit E, δC 140.9); from H-9 (subunit A, δH 3.92, 4.21), H-6 (subunit E, δH 6.56) to C-5 (subunit E, δC 154.0) (Fig. 2) indicated that C-7 and C9 of subunit A were connected to C-4 and C-5 of subunit E, respectively, through the ether linkages, which led to the formation of a 1,4-benzodioxepane moiety. The chiral carbons C-7 and C-8 of subunit A were defined to be a trans configuration based on the large coupling constant value between H-7 and H-8 (J = 9.6 Hz). The absolute configuration of 1 was determined by comparison of its experimental and theoretical ECD spectra. As shown in Fig. 3, the similarity between the experimental and calculated ECD spectra indicated the R,R configurations of C-7 and C-8 of subunit A, respectively. Therefore, the structure of 1 was elucidated as shown in Fig. 1, and named houpulignan A. Houpulignan B (2), a white amorphous powder, had the same molecular formula as 1, C54H50O9, which was established from HRESIMS data. The UV, IR, and NMR spectra of 2

were identical to that of 1 suggesting that 2 was also a trimer of obovatol, possessing a 1,4benzodioxepane unit. The main differences between 2 and 1 were that C-7 and C-9 of subunit A were linked via the ether bonds to C-5 and C-4 of subunit E in 2, respectively, instead of being linked to C-4 and C-5 of subunit E as in 1, respectively. This was proven by HMBC cross-peaks from H-7 (subunit A, δH 5.12), H-6 (subunit E, δH 6.64) to C-5 (subunit E, δC 152.3); from H-9 (subunit A, δH 3.82, 4.06), H-2 (subunit E, δH 6.48), H-6 (subunit E, δH 6.64) to C-4 (subunit E, δC 142.8). The trans position between H-7 and H-8 of subunit A was verified from the large coupling constant (J = 9.6 Hz). The CD spectrum of 2 was in good agreement with that of 1 assigning the absolute configurations of C-7 and C-8 of subunit A in 2 to be R,R, respectively (Fig. S2.10). Thus, compound 2 was established as displayed in Fig. 1. Houpulignan C (3), was obtained as white amorphous powder. The molecular formula of 3 was assigned to be C54H50O9 on the basis of ion peak at m/z 843.3529 [M + H]+, (calcd for C54H51O9, 843.3528), and its 13C NMR data, corresponding to 30 degrees of unsaturation. The detailed analysis of NMR data indicated that 3 was also an obovatol trimer. The connectivities of subunits A/B, subunits C/D, and subunits A/C in 3 resembled those of 1 and 2. The appearance of one singlet aromatic methine proton of subunit E in 3 instead of two meta-coupled aromatic methine protons in 1 and 2 as well as the replacement of one oxygenated methylene group in 1 and 2 by one methylene group (H-9 subunit A) in 3 implied that C-7 and C-9 of subunit A were linked to subunit E through the ether and C-C bonds, respectively, to form a benzodihydropyran unit. In the HMBC spectrum, the correlations of H-7 (subunit A, δH 5.39) to C-3 (subunit E, δC 143.6); H-9 (subunit A, δH 2.67, 2.80), H-6 (subunit E, δH 6.34), H-7 (subunit E, δH 2.90) to C-2 (subunit E, δC 117.2) indicated the linkages of C-7 (subunit A) to C-3 (subunit E) and of C-9 (subunit A) to C-2 (subunit E). The trans stereochemistry of C-7 and C-8 of subunit A was deduced from the large coupling

constant (J = 9.6 Hz). The absolute configurations of C-7 and C-8 of subunit A of 3 were determined to be R,S, respectively on the basis of comparing experimental and calculated ECD curves (Fig. 3). Accordingly, the structure of 3 was assigned as shown. Houpulignan D (4) was obtained as white amorphous powder that gave a [M + Na]+ peak at m/z 897.3606 in the HRESIMS, consistent with a molecular formula of C55H54O10 (calcd for C55H54O10Na, 897.3609), accounting for 29 degrees of unsaturation. The resemblance in the NMR data of 4 and 1–3 suggested that 4 was also a neolignan, which was composed from three obovatols. The presence of methoxy group (δH 3.11; δC 56.9) and its HMBC correlation to C-7 (δC 84.4) of subunit A indicated that C-7 of subunit A in 4 was connected to a methoxy group instead of ring E via ether bond to form a ring as in 1–3, which was in accordance with 29 degrees of unsaturation of 4 as compared to 30 ones of 1–3. Subunit C was determined to associate to C-8 of subunit A through the HMBC correlations of H-8 (subunit A, δH 3.59) with C-1 (subunit C, δC 132.7), C-2 (subunit C, δC 122.5), C-3 (subunit C, δC 140.6); of H-6 (subunit C, δH 6.54) with C-1 (subunit C, δC 132.7), C-2 (subunit C, δC 122.5). The HMBC interactions of H-9 (subunit A, δH 4.08, 4.48), H-6 (subunit E, δH 6.26) to C-5 (subunit E, δC 147.1) suggested that C-9 of subunit A was linked to C-5 of subunit E by an ether bond. The attachment of subunit E to subunit F at C-3 via an aryl ether linkage was demonstrated by the correlation from H-2 of subunit E to H-3′/H-5′ of subunit F in the ROESY spectrum. The coupling constant of H-7 and H-8 of subunit A (J = 9.6 Hz) in the 1H NMR of 4 implied a threo configuration between the two chiral centers C-7 and C-8. The experimental ECD spectrum was in agreement with calculated one assigning the absolute configurations of C-7 and C-8 of subunit A to be R,R, respectively (Fig. 3). Thus, the structure of 4 was elucidated and named houpulignan D. The molecular formula of houpulignan E (5) was deduced to be same as that of 4 based on its

13C

NMR and HRESIMS spectra. The connection of subunits A/B, subunits C/D,

subunits E/F, and subunits A/C in 5 were similar to that of 4. The only difference between 5 and 4 was the linkage of subunit A to subunit E. The HMBC correlations of H-9 (subunit A, δH 4.13, 4.28), H-2 (subunit E, δH 6.22), H-6 (subunit E, δH 6.58) to C-4 (subunit E, δC 135.5) indicated that C-9 of subunit A was linked to C-4 of subunit E in 5 instead of C-5 of subunit E as in 4. The relative and absolute configurations of 5 were found to be identical to those of 4 from analysis of their closely related J-coupling constant between two chiral centers and their CD spectra (Fig. S5.10). Houpulignan F (6), a white amorphous powder, possessed the same molecular formula C55H54O10 as 4 and 5 according to a sodium adduct ion peak at m/z 897.3612 (calcd for C55H54O10Na, 897.3609) in the HRESIMS. The analysis of NMR data indicated that the structure of 6 was similar to that of 4 and 5 except that C-9 of subunit A was directly linked to C-2 of subunit E in 6 instead of via an ether bond as in 4 and 5. This observation was confirmed based on the HMBC correlations from H-9 (subunit A, δH 3.04, 3.20), H-6 (subunit E, δH 6.50), H-7 (subunit E, δH 3.21) to C-1 (subunit E, δC 130.9), C-2 (subunit E, δC 126.6). The threo configuration between two chiral centers at C-7 and C-8 of subunit A was determined by its large coupling constant (J = 9.6 Hz). The theoretically calculated ECD of (7R,8S)-6 matched well with the experimental ECD of 6 indicating the R,S configurations of C-7 and C-8 of subunit A, respectively (Fig. 3). Therefore, the structure of 6 was elucidated as shown in Fig. 1. Obovatol trimeric neolignans are unprecedented natural compounds. To the best of our knowledge, only one trimeric neolignan derived from two units of obovatol and one unit of magnolol has been isolated from the bark of M. obovata [15]. In this study, six trimeric neolignans are formed from three units of obovatol, a biphenyl ether neolignan. Compounds 1 and 2 are found to possess a rare 1,4-benzodioxepane moiety, whereas 3 contains a benzodihydropyran unit in the molecular structure.

Previous reports demonstrated that obovatol exhibited the anti-inflammatory activities [17,19]. Therefore, the isolated compounds were evaluated for inhibitory effect on NO production in LPS-stimulated RAW 264.7 macrophage cells with N-monomethyl-L-arginine as a positive control (IC50 35.1 ± 0.36 μM). The results showed that, of the isolates, compound 3 was found to exhibit the strongest inhibition (IC50 4.05 ± 0.31 µM). Compounds 1 and 2 displayed the significant inhibition of NO production with IC50 values of 8.01 ± 0.67 and 20.21 ± 2.20 µM, respectively. Three remaining trineolignans (4–6) were inactive (IC50 > 50 μM) (Table 2). The cytotoxicity of isolates was also evaluated using the MTT assay, no cytotoxic activity against RAW 264.7 macrophages cells was found at 50 μM. These findings suggest that the ring formation of subunits A and E might be responsible for the NO inhibitory activity. 4. Conclusion Phytochemical investigations of M. officinalis var. biloba leaves led to the isolation of six novel obovatol trimeric neolignans (1–6). All the isolates were tested for inhibitory activity against LPS-induced NO production in RAW 264.7 macrophages. Compounds 1–3 were the potent inhibitors with IC50 values of 8.01, 20.21, and 4.05 µM, respectively. This study contributes to enrich the structural diversity of natural neolignans and provides information for further pharmacological studies. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research was supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63), the Drug Innovation Major Project (2018ZX09711-001-007), and the 111 Project from Ministry of Education of China and the State Administration of Foreign Export Affairs of China (B18056).

Appendix A. Supplementary material Supplementary data connected to this article can be found online at.

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[16]. H. Nguyen Ngoc, M. Alilou, M. Stonig, D.T. Nghiem, L.T. Kim, J.M. Gostner, H. Stuppner, M. Ganzera, J. Nat. Prod. 82 (2019) 2941−2952. [17] M.S. Choi, S.H. Lee, H.S. Cho, Y. Kim, Y.P. Yun, H.Y. Jung, J.K. Jung, B.C. Lee, H.B. Pyo, J.T. Hong, Eur. J. Pharmacol. 556 (2007) 181–189. [18] W. Schuehly, W. Voith, H. Teppner, O. Kunert, J. Nat. Prod. 73 (2010) 1381−1384. [19] H. Matsuda, T. Kageura, M. Oda, T. Morikawa, Y. Sakamoto, M. Yoshikawa, Chem. Pharm. Bull. 49 (2001) 716–720.

Table 1 1H (600 MHz) and 13C NMR (150 MHz) spectroscopic data for compounds 1–6. Position Subunit A 1 2 3 4 5 6 7 8 9 4-OH 7-OCH3 Subunit B 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ Subunit C 1 2 3 4 5 6 7 8 9 4-OH 5-OH Subunit D 1′

1a δH 5.98 br s

6.36 br s 4.86 d (9.6) 3.72 m 3.92 dd (12.0, 9.0) 4.21 dd (12.0, 3.2) 5.31 s

7.10 d (8.4) 6.63 d (8.4) 6.63 d (8.4) 7.10 d (8.4) 3.35 d (6.6) 5.95 m 5.09 m

6.57 s 2.96 dd (15.6, 6.0) 3.16 dd (15.6, 6.0) 5.75 m 4.89 m 5.09 m 4.56 s 5.35 s

2a δC 132.5 109.6 142.4 134.1 144.0 109.8 84.5 49.7 74.3

135.1 130.2 117.7 155.2 117.7 130.2 39.6 137.1 116.0 131.9 122.0 140.7 134.5 144.3 114.3 38.1 136.7 116.5

134.7

δH 6.27 br s

6.82 br s 5.12 d (9.6) 3.80 m 3.82 dd (12.0, 9.0) 4.06 dd (12.0, 3.2) 5.33 s

7.13 d (8.4) 6.71 d (8.4) 6.71 d (8.4) 7.13 d (8.4) 3.36 d (6.6) 5.94 m 5.06 m

6.53 s 2.98 dd (15.6, 6.0) 3.17 dd (15.6, 6.0) 5.72 m 4.88 m 5.06 m 4.45 s 5.27 s

3a δC 133.2 109.2 143.4 133.9 144.6 109.6 85.2 49.8 74.2

135.7 130.1 118.4 154.8 118.4 130.1 39.6 137.3 116.1 132.0 121.9 140.8 134.3 144.3 114.2 37.9 137.0 116.6

135.4

δH 6.40 br s

6.81 br s 5.39 d (9.6) 3.41 m 2.67 dd (16.6, 5.4) 2.80 dd (16.6, 12.0) 5.34 s

7.06 d (8.4) 6.72 d (8.4) 6.72 d (8.4) 7.06 d (8.4) 3.31 d (6.6) 5.94 m 5.08 m

6.58 s 3.03 dd (15.6, 6.0) 3.19 dd (15.6, 6.0) 5.76 m 4.86 m 4.99 m 4.55 s 5.29 s

4a δC 131.9 109.8 143.3 134.4 144.7 109.9 80.0 41.2 29.2

134.6 130.3 118.1 154.7 118.1 130.3 39.4 137.3 116.1 131.7 124.6 140.6 134.7 144.1 114.1 37.9 137.0 116.4

135.5

δH 6.06 d (1.8)

6.70 d (1.8) 4.41 d (9.6) 3.59 m 4.08 dd (9.6, 6.6) 4.48 dd (9.6, 6.6) 5.40 s 3.11 s 7.12 d (8.4) 6.66 d (8.4) 6.66 d (8.4) 7.12 d (8.4) 3.36 d (6.6) 5.95 m 5.06 m

6.54 s 2.79 dd (15.6, 6.0) 2.97 dd (15.6, 6.0) 5.61 m 4.86 m 4.94 m 4.63 s 5.33 s

5a δC 132.5 109.9 143.4 134.0 144.8 108.8 84.4 46.7 73.2 56.9 135.5 130.1 118.3 154.7 118.3 130.1 39.6 137.8 115.9 132.7 122.5 140.6 134.7 144.1 113.8 37.5 136.8 116.5

134.7

δH 5.96 d (1.8)

6.73 d (1.8) 4.30 d (9.6) 3.34 m 4.13 dd (9.6, 6.6) 4.28 dd (9.6, 6.6) 5.36 s 3.12 7.12 d (8.4) 6.65 d (8.4) 6.65 d (8.4) 7.12 d (8.4) 3.36 d (6.6) 5.90 m 5.05 m

6.50 s 2.59 dd (15.6, 6.0) 2.76 dd (15.6, 6.0) 5.43 m 4.66 m 4.70 m 4.50 s 5.27 s

6b δC 132.6 109.8 143.5 133.8 144.8 108.7 86.0 46.8 76.6 57.0 135.7 130.0 118.5 154.5 118.5 130.0 39.6 137.7 116.1 132.4 123.2 140.5 134.5 144.1 113.7

δH 6.01 br s

6.65 br s 4.15 d (9.6) 3.40 m 3.04 d (9.6) 3.20 d (9.6) 2.87 s 7.15 d (8.4) 6.59 d (8.4) 6.59 d (8.4) 7.15 d (8.4) 3.35 d (6.6) 5.96 m 5.07 m

136.1

6.36 s 2.60 dd (15.6, 6.0) 2.70 dd (15.6, 6.0) 5.20 m

116.2

4.75 m

37.2

134.7

δC 134.2 112.2 143.8 136.4 147.1 110.0 85.2 49.7 28.1 56.4 135.0 130.5 118.2 157.0 118.2 130.5 39.9 139.0 115.7 131.8 125.5 142.1 137.4 145.0 112.7 37.4 138.8 115.5

133.2

2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ Subunit E 1 2 3 4 5 6 7 8 9 5-OH Subunit F 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ a

7.04 d (8.4) 6.58 d (8.4) 6.58 d (8.4) 7.04 d (8.4) 3.37 d (6.6) 5.95 m 5.09 m 6.61 br s

6.56 br s 3.37 d (6.6) 5.95 m 5.09 m

6.86 d (8.4) 6.63 d (8.4) 6.63 d (8.4) 6.86 d (8.4) 3.27 d (6.6) 5.95 m 5.09 m

Data were recorded in CDCl3 were recorded in acetone-d6

b Data

130.0 115.0 154.7 115.0 130.0 39.4 138.0 115.7 133.7 116.9 148.7 140.9 154.0 117.1 39.6 137.3 116.2 135.8 129.2 116.6 157.4 116.6 129.2 39.4 137.5 116.3

7.00 d (8.4) 6.56 d (8.4) 6.56 d (8.4) 7.00 d (8.4) 3.33 d (6.6) 5.93 m 5.07 m 6.48 d (1.8)

6.64 d (1.8) 3.20 d (6.6) 5.94 m 5.06 m

6.96 d (8.4) 6.70 d (8.4) 6.70 d (8.4) 6.96 d (8.4) 3.31 d (6.6) 5.93 m 5.07 m

130.4 115.1 154.7 115.1 130.4 39.6 137.3 115.8 134.8 116.4 148.0 142.8 152.3 118.2 39.5 136.6 116.2 135.4 129.6 117.2 156.7 117.2 129.6 39.6 137.8 116.0

7.13 d (8.4) 6.72 d (8.4) 6.72 d (8.4) 7.13 d (8.4) 3.36 d (6.6) 5.94 m 5.07 m

130.1 115.1 154.9 115.1 130.1 39.6 137.7 116.0

6.34 s 2.90 d (6.6) 5.52 m 4.74 m

128.9 117.2 143.6 135.3 140.7 113.9 36.5 136.1 115.5

7.08 d (8.4) 6.88 d (8.4) 6.88 d (8.4) 7.08 d (8.4) 3.32 d (6.6) 5.94 m 5.07 m

134.2 129.7 117.2 156.4 117.2 129.7 39.6 137.8 115.8

6.95 d (8.4) 6.65 d (8.4) 6.65 d (8.4) 6.95 d (8.4) 3.28 d (6.6) 5.95 m 5.06 m 6.42 d (1.8)

6.26 d (1.8) 3.12 d (6.6) 5.80 m 4.98 m

7.10 d (8.4) 6.87 d (8.4) 6.87 d (8.4) 7.10 d (8.4) 3.34 d (6.6) 5.95 m 5.06 m

130.2 114.9 154.6 114.9 130.2 39.4 137.5 116.0 131.1 114.9 142.9 137.8 147.1 111.5 39.6 137.3 115.8 134.0 129.6 117.1 156.3 117.1 129.6 39.6 137.7 115.8

6.97 d (8.4) 6.57 d (8.4) 6.57 d (8.4) 6.97 d (8.4) 3.30 d (6.6) 5.90 m 5.05 m 6.22 d (1.8)

6.58 d (1.8) 3.22 d (6.6) 5.90 m 5.05 m 8.03 s 7.00 d (8.4) 6.67 d (8.4) 6.67 d (8.4) 7.00 d (8.4) 3.30 d (6.6) 5.90 m 5.05 m

130.4 115.0 154.5 115.0 130.4 39.5 137.4 116.0 135.2 112.1 149.3 135.5 151.5 111.5 40.1 137.2 116.0 134.1 129.6 117.3 156.0 117.3 129.6 39.6 137.7 116.0

6.83 d (8.4) 6.56 d (8.4) 6.56 d (8.4) 6.83 d (8.4) 3.25 d (6.6) 5.91 m 5.03 m

129.8 116.3 156.1 116.3 129.8 39.9 139.0 115.5

6.50 s 3.21 d (6.6) 5.74 m 4.92 m

130.9 126.6 142.1 137.2 145.0 113.9 37.2 139.4 115.2

7.03 d (8.4) 6.67 d (8.4) 6.67 d (8.4) 7.03 d (8.4) 3.33 d (6.6) 5.95 m 5.05 m

133.2 129.7 116.1 157.6 116.1 129.7 39.9 139.2 115.5

Table 2 NO inhibitory activity of compounds 1–6. Compounds 1 2 3 4 5 6 N-monomethyl-L-argininea

NO inhibitory effect (IC50) 8.01 ± 0.67 µM 20.21 ± 2.20 µM 4.05 ± 0.31 µM > 50 µM > 50 µM > 50 µM 35.1 ± 0.36 μM

a

Positive control.

b

RAW 264.7 cell viability treated with samples at 50 µM.

RAW 264.7 cell viabilityb (%) 98.84 ± 3.56 99.17 ± 4.23 101.28 ± 2.77 94.62 ± 4.10 102.55 ± 5.68 92.44 ± 4.00 95.47 ± 5.86

7' 1'

2'

D

3' OH HO

5

6

6' 9'

8' 7'

5'

3' OH HO

O

5

E

7 O 4

1 2

O

3 O

8

6 1

9

9

7

5

2 4' 3'

3

F

5'

2' 1'

6'

3'

8'

9'

9'

7'

1'

7'

1'

8'

6' 5'

F

2'

2 9

7

1

4

E

5 O 6 6 HO 1 5 HO 4 5'

B

8' 9'

7'

1'

O 9

A

6'

4'

OH

3 8

2

7 2

5

1

6

HO 4 5'

7 8 6'

9

8'

3'

7'

9'

1'

B

1

2

8

7

6

E O 3

7

1

A

8

7 9

2

6

9

5 O

4 OH 5'

2

3

6'

O

4'

3'

F

2'

1'

4'

7'

3'

8'

2'

9'

3

9' 8' 7'

C

1 7 OCH3

9 OH

5

7

1

8 6

6

2

4' 3 O

E

4

OH 6

HO

9

5

3

A

HO 4 5'

O 6'

3'

B

8'

2' 9'

7'

1'

2'

2' 9

HO 5'

F

4

OH 5

E

3

4' O 3'

6 1

2

7

5 OH

C

O 4' OCH3 5'

2

3

6'

O

8 8

5

OH 3'

D

7

7

HO 4 5'

2' 1'

A

7'

6'

8' 9' 9'

B

8'

4'

7'

1'

3 O

4' 3'

2'

3'

5

Fig. 1. Chemical structures of compounds 1–6.

6

1 2

9

8

9

6

HO

4

3

6 1

9

8

8 2

1

6' 1'

8'

7 9

7'

5'

O

5

8

6'

F

3'

OH 4

9'

1'

2'

4'

4

HO

2

E

7 O 5

8

5'

3

1

9

3

6'

D 4' O

C

6

2

6' 5'

5'

2'

8'

D

3'

O 3

3'

8

2'

4'

4

3 O

9'

9'

HO 5

O

O

2

1 7' 1'

6'

4'

8

1

F

3' 9

1 2 7

2' 3' OH 4

1'

2'

4'

B

8'

1' 8'

7'

3

A

HO 4 5'

6'

7'

2'

6

HO

6' 5'

8'

7'

1'

4' O

C

8

9'

9'

D

4

5 6

9

4'

B

1'

2'

8

A

HO 4 5'

6'

2 7

HO

7'

O

1 8

8'

8'

3

C

6 9

9'

4'

4

5

9'

7

6

1

2

OH 5

C 3 O

OCH3

4 OH 3' 4' 5'

D 6'

2' 8' 1'

7'

9'

8'

7'

OH 4

HO 6

1'

D

D

2

9

1 5

HO

A

8

E

7 O 4

2 O

HO 4

9'

1'

A

1'

7'

8'

7'

9'

8'

B

1'

8'

F

O 3

9

2

8 7

E 5 O

1

OH

C

HO

8 1

5

A

HO 4

2

2 7

9

1 7 OCH3

OH

7

6

8'

1'

B

E

4

7'

7'

8' 9'

9

8

7

3

O 4' OCH3

D

2

HO 4

9'

1'

B

7'

COSY

4'

O

8'

1' 7'

O 8'

2

9' 9'

8'

1'

4'

7'

6

5 HMBC

B

ROESY

Fig. 2. COSY and key HMBC and ROESY correlations of compounds 1–6.

OH

1

C 2 7

3 O

OCH3

A

HO 4

6

7

9

1 5

4 OH

9

1 7

8 HO

C

A

4'

2

OH

1 2

6

E

3

4'

9

6

7

1

5

F

O

9

HO

HO 4

1'

9

8

8

7' 8'

O

5 OH

7'

4

4' O

2

1

8

6

O 9'

1'

4'

OH 9'

F

9

B

1'

3

1'

O 3 4

OH

4'

OH

8'

7'

8'

4'

O

4

9'

D

4'

3

F

2

1'

1'

O 2

O 8' 9'

9'

7'

6

E

HO 4

9

1

9'

A

8

4'

7'

9'

7

1

7 1

5

8

7 8 9 2

7

HO

2 1

2

O

F

7'

E

7 O 5

2

1

9

9

4' 3 O

C

6

O

4

HO 4

4'

4'

B

1 5

O

8

OH 4

HO

4'

9

7

HO

7

F

2

1

9

2

O 8'

1

9

D

1'

4' 3 O

C

6

O5

8

HO

OH 4

1' 8'

7'

8'

7'

9'

1'

1 7

9

8'

7'

4' 3 O

C

9'

9'

9'

5 4 OH 4'

D

8'

1' 7'

9'

Fig. 3. Comparison of calculated and experimental ECD spectra of compounds 1, 3, 4, 6

Highlights

 Six new obovatol trimeric neolignans, houpulignans A–F (1–6) were isolated from the leaves of Magnolia officinalis var. biloba.  Compounds 1 and 2 are the first examples of neolignans formed from three obovatol units bearing a rare 1,4-benzodioxepane moiety.  The active compounds were found to be 1–3 which inhibited NO production with IC50 values of 8.01, 20.21, and 4.05 µM, respectively.

Graphical Abstract

Conflict of interest The authors declare no conflict of interest.