Five New Diarylbutyrolactones and Sesquilignans from Saussurea medusa and Their Inhibitory Effects on LPS-induced NO Production

• Abstract
• Introduction
• Results and Discussion
• Material and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• ECD calculations for 1 – 4
• Determination of NO production and cell viability assay
• Molecular docking study
• Measurement of iNOS expression
• Contributorsʼ Statement
• References

Abstract

Five new diarylbutyrolactones and sesquilignans (1a/1b – 4), including one pair of enantiomers (1a/1b), together with 10 known analogues (5 – 14), were isolated from the whole plants of Saussurea medusa. Compound 1 was found to possess an unusual 7,8′-diarylbutyrolactone lignan structure. Separation by chiral HPLC analysis led to the isolation of one pair of enantiomers, (+)-1a and (−)-1b. The structures of the new compounds were elucidated by extensive spectroscopic data. All compounds, except compounds 5, 7 and 9, were isolated from S. medusa for the first time. Moreover, compounds 1 –  4, 8 and 10 – 14 had never been obtained from the genus Saussurea previously. Compounds (+)- 1a, 2, 5, 7, and 9 – 11 were found to inhibit the lipopolysaccharide (LPS)-induced release of NO by RAW264.7 cells with IC₅₀ values ranging from 10.1 ± 1.8 to 41.7 ± 2.1 µM. Molecular docking and iNOS expression experiments were performed to examine the interactions between the active compounds and the iNOS enzyme.

Introduction

Saussurea medusa Maxim. is a rare subnival plant known as “snow lotus” that belongs to the genus Saussurea of the family Asteraceae [1]. The plant is found predominantly in the Qinghai-Tibet Plateau at heights of 3500 – 4500 m [2]. S. medusa is an important traditional Chinese medicinal herb used to treat anthrax, stroke, rheumatoid arthritis, placental retention and mountain sickness [3]. In a previous study, we found that an ethanol extract of S. medusa possessed potential anti-inflammatory properties [4]. The aim of the present study was to identify and characterize the anti-inflammatory compounds of S. medusa.

Herein, we report on the isolation and characterization of five new diarylbutyrolactones and sesquilignans, together with 10 known analogues from the whole plants of S. medusa. Extensive spectroscopic data and time-dependent density functional theory-based electronic circular dichroism (TDDFT-ECD) calculations [5] led to the identification of their chemical structures. The anti-inflammatory activities of the compounds were preliminary assessed in vitro by examining their abilities to inhibit the LPS-induced NO production in RAW264.7 macrophage-like cells. The interactions between the bioactive compounds and iNOS were further explored using molecular docking and iNOS expression experiments.

Results and Discussion

The ethyl acetate fraction from the whole plants of S. medusa was subjected to repeated chromatographic separations to afford five new lignans (1a/1b – 4), namely medusarins A−D (1a/1b – 4), see [Fig. 1].

Medusarin A (1) was obtained as a colorless gum. Its molecular formula was determined to be C₂₀H₂₀O₈ based on the sodium adduct [M + Na]⁺ at m/z 411.1056 in HRESIMS corresponding to 11 indices of hydrogen deficiency (IHDs). The IR spectrum of 1 displayed characteristic absorption bands of hydroxy (3359 cm⁻¹), carbonyl (1741 cm⁻¹) and C=C bond (1645 cm⁻¹) groups. The ¹H NMR spectroscopic data ([Table 1]) in conjunction with HSQC data revealed the presence of two aromatic rings, including an ABX coupling system at δ _H 7.08 (1H, d, J = 1.8 Hz, H-2′), 6.83 (1H, d, J = 8.3 Hz, H-5′) and 7.02 (1H, dd, J = 8.3, 1.8 Hz, H-6′), assignable to a 1,3,4-trisubstituted benzene ring. Two equivalent aromatic protons at δ _H 6.58 (2H, s, H-2, 6) indicated the existence of a 1,3,4,5-tetrasubstituted aromatic ring. In addition, an oxygenated methylene at δ _H 3.93 (1H, dd, J = 15.1, 8.1, H-9a) and 3.62 (1H, dd, J = 15.1, 7.0, H-9b), one allylic hydrogen signal at δ _H 7.49 (1H, s, H-7′), two methines at δ _H 3.66 (1H, dd, J = 8.1, 7.0, H-8) including one oxygenated at δ _H 5.60 (1H, brs, H-7), and two methoxy groups at δ _H 3.82 (6H, s, H-3, 5) were also observed. The ¹³C NMR and DEPT spectra revealed 20 carbon signals, consisting of 12 aromatic carbons, a double bond, one oxygenated methylene carbon, two methoxy groups, two methine carbons (one oxygenated) and a lactone carbonyl group signal. Two aromatic rings (A and B), a lactone carbonyl and a double bond group accounted for 10 out of 11 IHDs. The remaining IHD in the molecule implied the existence of the butyrolactone ring C in compound 1.

The aforementioned evidence indicated that compound 1 was similar to impecylenolide [6], a lignan previously isolated from Imperata cylindrica, except for the presence of a methoxy group at C-5 and the replacement of a methoxy group by a hydroxy group at C-3′ in 1. This was confirmed by analysis of the 2D NMR and was also consistent with its molecular formula.

The (E)-configuration of the C7′-C8′ double bond in 1 was deduced from the ROESY correlations ([Fig. 2]) between H-2′/H-6′ and H₂-9. This was also supported by a more de-shielded signal for H-7′ (7.49 ppm), which was in agreement with the reported chemical shifts (7.20 – 7.69 ppm) for the (E)-configuration [7], [8]. The ROESY correlation of H-7/H₂-9 indicated the trans orientation of H-7 and H-8, which was supported by a small coupling constant (J _7,8 = 0) [6]. Thus, the relative configuration of 1 was determined as 7S*,8R*.

An ECD spectrum was recorded to establish the absolute configuration of 1, but surprisingly, there was no obvious Cotton effect (CE), which suggested the racemic nature of 1. This prediction was confirmed by the presence of two peaks in chiral HPLC analysis. Compounds (+)-1a and (−)-1b were successfully separated in a ratio of approximately 1 : 1 (Fig. 46S, Supporting Information), showing typical antipodal ECD curves ([Fig. 3]) and specific rotations of opposite sign. By comparing their calculated ECD and experimental ECD ([Fig. 3]), the calculated ECD curve of (7S,8R)-form matched well with the experimental ECD spectrum of (+)-1a, which allowed the assignment of the absolute configuration of (+)-1a as 7S,8R. Thus, the almost mirror-image ECD curve of (−)-1b was assigned to the 7R,8S configuration.

Medusarin B (2) possessed a molecular formula of C₂₁H₂₄O₇ as deduced by (+)-HRESIMS at m/z 411.1424 [M + Na]⁺. The ¹H and ¹³C NMR spectra ([Table 1]) showed the existence of two benzene rings (one 1,3,4-trisubstituted, the other 1,2,4,5-tetrasubstituted), three methylenes (one oxygenated), two methines, three methoxy groups and a lactone carbonyl group. The ¹H and ¹³C NMR spectral features indicated that compound 2 was very similar to arctigenin [9], a compound (5) also isolated from this plant during this study. The difference was the existence of a hydroxy group at C-2 in compound 2. The HMBC correlations between H-6/H₂-7 and C-2, in combination with the different pattern of proton peaks in the aromatic region, supported this deduction, which was also in accordance with its molecular formula.

According to Corrie et al. [10], the relative configuration of the 8,8′-diarylbutyrolactone lignan can be determined by NMR comparison of the methylene protons at C-9. Equivalent chemical shifts of H₂-9 correspond to the cis-configuration, while different chemical shifts correspond to the trans-configuration. Thus, the configuration at C-8 and C-8′ was assigned as trans on the basis of the unequal chemical shifts observed for H₂-9 [δ _H 4.14 (1H, dd, J = 9.0, 6.7 Hz, H-9a) and 3.93 (1H, dd, J = 9.0, 7.0 Hz, H-9b)]. This deduction was confirmed by comparing the ¹H and ¹³C NMR data with those of arctigenin (5), an analog with the same trans-configuration. ECD calculations were used to determine the absolute configuration of 2, and the calculated ECD curve of the (8R,8′R)-form matched well with the experimental ECD spectrum of 2 ([Fig. 3]), indicating an 8R,8′R configuration for 2.

Medusarin C (3) possessed a molecular formula of C₃₁H₃₆O₁₀ based on the sodium adduct at m/z 591.2208 [M + Na]⁺ in (+)-HRESIMS. The ¹H NMR spectrum data ([Table 2]) of compound 3 combined with HSQC revealed three sets of ABX systems. An arylglyceryloxy moiety was revealed by signals of a vicinal coupling system attributed to two oxygenated methines at δ _H 4.94 (1H, d, J = 4.7 Hz, H-7″) and 4.13 (1H, m, H-8″) and an oxygenated methylene at δ _H 3.89 (1H, dd, J = 12.2, 4.0 Hz, H-9″a) and 3.66 (1H, dd, J = 12.2, 3.4 Hz, H-9″b). The ¹³C NMR and DEPT spectra showed 31 carbon signals assignable to 18 aromatic carbon signals, four methylene carbons (two oxygenated), four methine carbons (two oxygenated), four methoxy groups and a lactone carbonyl group. These data suggested that compound 3 was a sesquilignan, and its structure made up of two parts (Fig. 47S, Supporting Information).

The structure of 3 was established by further examination of the 2D NMR spectra. First, five spin-coupling units were identified via the ¹H−¹H COSY spectrum as show in Fig. 47S, Supporting Information. The connection of the five structural units with other functional groups was then made using the HMBC spectrum (Fig. 47S, Supporting Information). In the HMBC spectrum of 3, the long-range correlations from H-7″/C-1″, C-2″, C-6″; H-2″/C-4″, C-6″ confirmed that part I was a 3,4-disubstituted phenylglyceryl unit, and the HMBC correlations of 3″-OMe identified a methoxy group at C-3″. The HMBC correlations of H₂-7′/C-1′, C-2′, C-6′, C-9′; H-2′/C-4′, C-6′; 3′-OMe/C-3′; H₂-7/C-1, C-2, C-6; H-2/C-4, C-6; 3-OMe/C-3; 4-OMe/C-4; H₂-9/C-9′ indicated that part II was arctigenin (5), which was confirmed by comparing their 1D NMR data. Parts I and II were linked by the formation of an ether bond between C-8″ and C-4′, although a correlation from H-8″ to C-4′ was not observed in the HMBC spectrum of 3. NOE enhancements of H-2″, H-6″ and H-5′, observed after irradiation of H-8″ in a NOE difference experiment (Fig. 25S, Supporting Information), indicated a connection between C-8″ and C-4′ in 3. This deduction was also verified by the obvious downfield chemical shift of C-8″ (δ _C 87.4) compared to a typical hydroxylated carbon. Thus, the planar structure of 3 was established.

The relative configuration in part II was assigned as trans on the basis of observed unequal chemical shifts of H₂-9 [δ _H 4.15 (1H, dd, J = 9.0, 8.0 Hz, H-9a) and 3.88 (1H, dd, J = 9.0, 7.0 Hz, H-9b)]. The absolute configuration of 8R,8′R was assigned based upon biogenetic considerations and also by comparison of its ¹H and ¹³C NMR spectral data with those of arctigenin (5). The 7″,8″-erythro configuration was deduced from the observed small coupling constant (J _7″,8″ = 4.7 Hz) [11]. The 7″S configuration was defined by a positive CE at 345 nm (the E band) in the Rh₂(OCOCF₃)₄-induced ECD spectrum of 3 ([Fig. 4]) [12], [13]. Therefore, the absolute configuration of 3 was 8R,8′R,7″S,8″R and this conclusion was further supported by the calculated ECD spectrum of (8R,8′R,7″S,8″R)-3, which exhibited a pattern similar to the experimental one ([Fig. 3]).

Medusarin D (4) was found to have a molecular formula of C₃₁H₃₆O₁₀ established by the observation of a (+)-HRESIMS ion at m/z 591.2211 [M + Na]⁺. The IR and the NMR data ([Table 2]) of 4 highly resembled those of 3, suggesting that they were isomers of each other. The main difference between 3 and 4 was the coupling constant of H-7″ and H-8″ (J _7″,8″ = 7.9 Hz), which indicated a 7″,8″-threo configuration of 4. The 7″R configuration was defined by a negative CE at 342 nm (the E band) in the Rh₂(OCOCF₃)₄-induced ECD spectrum of 4 ([Fig. 4]). Therefore, the absolute configuration of 4 was 8R,8′R,7″R,8″R, which was further verified by the ECD calculations.

Along with the new lignans, the 10 previously reported lignans, namely arctigenin (5) [9], (−)-traxillagenin (6) [14], (−)-matairesinol (7) [15], (+)-matairesinol (8) [16], (−)-7(S)-hydroxyarctigenin (9) [9], (+)-7(R)-hydroxyarctigenin (10) [9], phenaxolactone 1 (11) [17], acutissimalignan B (12) [18], (+)-7,8-didehydroarctigenin (13) [19] and arctignan A (14) [20], were also obtained and identified on the basis of spectroscopic analysis and comparison with literature data.

All the isolates were screened for their inhibitory effects on NO production in LPS-stimulated RAW264.7 macrophage-like cells ([Table 3]). Compounds 2, 5 and 11 exhibited marked inhibition with IC₅₀ values of 13.2 ± 1.3, 10.1 ± 1.8 and 10.3 ± 1.9 µM, respectively. These values were comparable to that of the positive control quercetin (IC₅₀ = 15.9 ± 1.2 µM). Compounds (+)-1a, 7, 9 and 10 displayed moderate inhibitory activities with IC₅₀ values ranging from 16.2 ± 2.0 to 41.7 ± 2.1 µM. Arctigenin (5), the major constituent in S. medusa, significantly inhibited the production of NO in LPS-stimulated RAW264.7 cells and might contribute to the reported anti-inflammatory effects of S. medusa extracts [4].

Some preliminary structure-activity relationships could be drawn. The phenolic hydroxy group (especially the 4′-OH group) was found to be essential for the observed inhibitory effects. Absence of the 4′-OH group resulted in a loss of activity as those sesquilignans that lacked this (compounds 3, 4 and 14) displayed poor inhibition of iNOS in LPS-induced RAW264.7 cells. Secondly, the C-8′ chiral environment was also deemed to be essential, as the introduction of a C7′-C8′ double bond led to the loss of activity (compounds 12 and 13). Also, compound 7 exhibited good activity because of its stereoselectivity. Compound 6 was inactive, likely because of the additional 3-OMe group on aromatic ring B. However, the presence of a 2-OH group instead (compound 2) enabled inhibition. Furthermore, it is interesting to note that compound (+)-1a showed inhibitory effects, while its enantiomer (−)-1b was inactive.

In order to explore the mechanisms by which these compounds inhibit NO production, molecular docking and iNOS expression studies ([Fig. 5]) were conducted. The active compounds (+)-1a, 2, 5, 7 and 9 – 11 and the positive control quercetin were selected for molecular docking studies to investigate their interactions with the iNOS enzyme. The docking results are presented in [Table 4]. With the exception of compound 9, the active compounds exhibited excellent docking scores (< − 7.0 kcal/mol) with iNOS. Of particular interest was the fact that compound 5 showed the lowest docking score with the iNOS enzyme, consistent with its strong inhibitory effect.

To further explore the underlying mechanisms, we investigated the effect of selected compounds on iNOS expression. As reported in the literature, arctigenin (5) inhibits iNOS expression in LPS-induced RAW264.7 cells [21], [22]. In this study, compounds 2 and 11 were selected to investigate their inhibitory effects on iNOS expression. As shown in [Fig. 6], iNOS expression was significantly increased after LPS stimulation and both compounds 2 and 11 showed a dose-dependent reduction in the expression of iNOS in LPS-treated RAW264.7 cells. The results suggest that compounds 2 and 11 inhibit the production of NO by reducing iNOS expression.

In conclusion, five new diarylbutyrolactones and sesquilignans, together with ten known analogues, were separated from the whole plants of S. medusa. Among them, compounds 1, 2 and 5−13 were diarylbutyrolactone lignans, with compound 1 featuring an unusual 7,8′-diarylbutyrolactone lignan. Compounds 3, 4 and 14 were found to be sesquilignans. Overall, these findings not only provide more data on the chemical diversity of lignans present in S. medusa, but also indicate that diarylbutyrolactone lignans, such as arctigenin, may serve as potential lead compounds for further anti-inflammatory drug development. This should stimulate further studies on the anti-inflammatory activities of the constituents of S. medusa.

Material and Methods

General experimental procedures

Optical rotations (Na lamp, 589 nm) were measured on a Rudolph Autopol VI automatic polarimeter at room temperature. UV spectra were determined on a Shimadzu UV-2550 UV-visible spectrophotometer. ECD spectra were acquired on a JASCO J-815 spectrometer using a 0.1 cm path length sample cell and a JASCO LC-J1500 consisting of a MD-4014 photodiode array detector, an AS-4050 HPLC auto sampler, a PU-4185 binary and a CO-4060 column oven. IR spectra were recorded on a Thermo IS5 spectrometer with KBr panels. NMR experiments were performed on a Bruker Avance III 600 MHz spectrometer (Bruker Biospin AG) using TMS as the internal standard. (±)-ESIMS and (±)-HRESIMS data were obtained on a Bruker Daltonics Esquire 3000 Plus LC-MS instrument and a Waters Q-TOF Ultima mass spectrometer, respectively. Column chromatography (CC) was performed using silica gel (200 – 300 and 300 – 400 mesh, Qingdao Haiyang Chemical Co. Ltd.), Sephadex LH-20 (GE Healthcare), MCI gel (CHP20P, 75 – 150 µm, Mitsubishi Chemical Industries, Ltd.) and C18 reversed-phase silica gel (150 – 200 mesh, Merck). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.) were used for TLC detection. Semipreparative HPLC was carried out on a Waters 2695 instrument equipped with a Waters 2489 detector (210 and 254 nm) using a Waters X-Bridge Prep C18 column (250 × 10 mm, S-5 µm) or a YMC-Pack ODS-A column (250 × 10 mm, S-5 µm). A Daicel Chiralpak IG (250 × 4.6 mm, S-5 µm) column was used for chiral HPLC separation. Rh₂(OCOCF₃)₄ was purchased from Sigma-Aldrich. All solvents except HPLC solvents were purchased from Shanghai Chemical Reagents Co. Ltd. and were of analytical grade. Solvents used for HPLC were of HPLC grade and were obtained from J & K Scientific Ltd.

Plant material

The whole plants of S. medusa were collected from Yeniu Ditch (altitude 4100 m), Qilian County, Xining City, Qinghai Province in August 2018, and authenticated by Professor Lijuan Mei from Northwest Institute of Plateau Biology. The specimen was deposited in the Key Laboratory of Tibetan Medicine of the Chinese Academy of Sciences (access number: 0 341 202).

Extraction and isolation

The air-dried and powdered whole herbs of S. medusa (15.0 kg) were soaked overnight with 95% ethanol and then extracted with 95% ethanol (3 times, 75 L and 12 h) to obtain the crude extract (800 g). The extract was suspended in water (4 L) and successively partitioned with petroleum ether (5 × 4 L), EtOAc (5 × 4 L) and n-butanol (5 × 4 L). The EtOAc-soluble fraction (90 g) was subjected to column chromatography on MCI gel (5 × 40 cm, 100 – 200 mesh) eluted with MeOH-H₂O (10% to 100%) to give fractions F1−F7 based on TLC analysis. F5 (26.4 g) was separated by a silica gel column eluted with a gradient of CH₂Cl₂/MeOH (400 : 1 to 10 : 1) to yield fractions F5a−F5g. F5d (0.98 g) was fractioned via Sephadex LH-20 (MeOH) (3 × 150 cm), followed by RP semi-preparative HPLC (41% MeOH in H₂O) to yield 2 (19 mg, t _R = 41 min). Fraction F5f (1.6 g) was separated over a Sephadex LH-20 column (3 × 150 cm) eluted with MeOH to afford subfractions F5f1−F5f7. Fraction F5f2 (343 mg) was subjected to a silica gel column eluted with CH₂Cl₂/MeOH (400 : 1 to 1 : 1) in gradient to give subfractions F5f21−F5f24. F5f23 (61 mg) was then purified by semi-preparative HPLC with 44% MeOH in H₂O as the mobile phase to afford 3 (12 mg, t _R = 43 min) and 4 (8 mg, t _R = 46 min). Fraction F4 (15.8 g) was subjected to a silica gel column eluted with CH₂Cl₂/MeOH (400 : 1 to 1 : 1) in gradient to give subfractions F4a−F4k. Separation of F4k (1.0 g) with Sephadex LH-20 (MeOH) (3 × 150 cm) yielded subfractions F4k1−F4k3. Fraction F4k2 (243 mg) was subjected to a silica gel column eluted with n-hexane/isopropanol (80 : 1 to 1 : 1) in gradient to give subfractions F4k21−F4k23. F4k22 (97 mg) was then purified by RP semi-preparative HPLC (32% MeOH in H₂O) to yield 1 (15 mg, t _R = 21 min). The isolation procedure of the known compounds is described in the Experimental Section, Supporting Information.

Medusarin A (1): colorless gum; [α]_D ²⁵ + 0.7 (c 0.57 in MeOH); ¹H and ¹³C NMR (CD₃OD) data, see [Table 1]; IR (KBr) ν _max 3359, 2922, 2851, 1741, 1645, 1468, 1384, 1260, 1041 cm⁻¹; UV (MeOH) λ _max (log ε) 237 (3.25), 340 (3.37) nm; (+)-ESIMS m/z 799.1 [2 M + Na]⁺; (−)-ESIMS m/z 387.4 [M – H]⁻; (+)-HRESIMS m/z 411.1056 [M + Na]⁺ (calcd for C₂₀H₂₀NaO₈, 411.1050, Δ − 1.39 ppm).

1a: colorless gum; [α]_D ²⁵ + 83.8 (c 0.1 in MeOH); ECD (MeOH) λ (Δε) 209 (− 14.95), 239 (− 7.47), 305 (+ 7.90), 336 (+ 9.07) nm;

1b: colorless gum; [α]_D ²⁵ − 87.2 (c 0.1 in MeOH); ECD (MeOH) λ (Δε) 209 (+ 16.50), 239 (+ 9.68), 305 (− 9.38), 336 (− 10.42) nm;

Medusarin B (2): white amorphous solid; [α]_D ²⁵ + 5.2 (c 0.23 in MeOH); ¹H and ¹³C NMR (CDCl₃) data, see [Table 1]; IR (KBr) ν _max 3422, 2933, 1751, 1612, 1518, 1452, 1384, 1204, 1117, 1031 cm⁻¹; UV (MeOH) λ _max (log ε) 230 (3.44), 286 (3.18); ECD (MeOH) λ (Δε) 211 (− 8.59), 233 (− 6.56), 290 (+ 0.85) nm; (+)-ESIMS m/z 406.4 [M + NH₄]⁺; (−)-ESIMS m/z 387.4 [M – H]⁻; (+)-HRESIMS m/z 411.1424 [M + Na]⁺ (calcd for C₂₁H₂₄NaO₇, 411.1414, Δ − 2.44 ppm).

Medusarin C (3): light yellow amorphous solid; [α]_D ²⁵ − 11.8 (c 0.22 in MeOH); ¹H and ¹³C NMR (CDCl₃) data, see [Table 2]; IR (KBr) ν _max 3447, 2936, 1763, 1591, 1514, 1463, 1421, 1265, 1235, 1123, 1028 cm⁻¹; UV (MeOH) λ _max (log ε) 230 (3.68), 280 (3.27); ECD (MeOH) λ (Δε) 238 (− 7.78), 282 (− 2.17) nm; (+)-ESIMS m/z 591.6 [M + Na]⁺; (−)-ESIMS m/z 567.3 [M – H]⁻; (+)-HRESIMS m/z 591.2208 [M + Na]⁺ (calcd for C₃₁H₃₆NaO₁₀, 591.2201, Δ − 1.28 ppm).

Medusarin D (4): light yellow amorphous solid; [α]_D ²⁵ − 26.7 (c 0.31 in MeOH); ¹H and ¹³C NMR (CDCl₃) data, see [Table 2]; IR (KBr) ν _max 3471, 2936, 1763, 1605, 1515, 1464, 1266, 1156, 1028 cm⁻¹; UV (MeOH) λ _max (log ε) 230 (3.61), 278 (3.23); ECD (MeOH) λ (Δε) 211 (+ 4.97), 236 (− 8.28) nm; (+)-ESIMS m/z 591.5 [M + Na]⁺; (−)-ESIMS m/z 567.3 [M – H]⁻; (+)-HRESIMS m/z 591.2211 [M + Na]⁺ (calcd for C₃₁H₃₆NaO₁₀, 591.2201, Δ − 1.78 ppm).

ECD calculations for 1 – 4

The absolute configurations of 1 – 4 were determined by TDDFT-ECD calculations. For calculation details see the Experimental Section, Supporting Information.

Determination of NO production and cell viability assay

Measurements of NO production in an activated macrophage-like cell line were performed as described previously [23]. Briefly, RAW264.7 cells (1 × 10⁵ cells/well) were cultured in 96-well plates with a DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS), 1 mM pyruvate, 2.0 mM glutamine, 100.0 U/mL of penicillin and 10.0 µg/mL of streptomycin at 37 °C in a humidified atmosphere with 5% CO₂. The cells were treated with 1.0 µg/mL of LPS and with the test compounds for 24 h. Absorbance was measured at 540 nm after incubating the culture media (100 µL/each well) with Griess reagent (100 µL) (Sigma-Aldrich) at room temperature. The concentration of NO was calculated using a NaNO₂ solution standard. Cell viability was measured using the MTT-based colorimetric assay (for experimental details see the Experimental Section, Supporting Information).

Molecular docking study

Chemical structures of active compounds were drawn using the ChemDraw program and converted to their three-dimensional (3D) coordinates in Chem3D. Each of them was subjected to energy minimization by the MM2 method and saved in “pdb” format. The 3D crystal structure of iNOS (PDB ID: 3E6T) was obtained from the RCSB Protein Data Bank (https://www.rcsb.org/pdb) [24] and handled in the Biovia Discovery Studio Visualizer 2020 program for checking any missing residue/atom and deleting co-crystallized molecules such as cofactors, inhibitors and water. The proteins and ligands were processed and converted to “pdbqt” format. A grid box with dimensions of 30, 30 and 30 points in x, y, and z directions, respectively, were built. Molecular docking was performed using AutoDock Vina with default parameters, and the binding sites were defined within 10 Å around the co-crystallized ligands. Each docking involved nine independent runs. The docked model with the lowest docking energy was selected to represent its most favorable binding pattern.

Measurement of iNOS expression

iNOS expression was measured according to a previous report [21]. Briefly, after the treatment with LPS (1.0 µg/mL) and target compounds for 24 h, cells were washed with PBS and suspended in a lysis buffer. Cell debris were removed by centrifugation. After the protein concentration for each aliquot was determined with BCA reagent, suspensions were boiled in an SDS-PAGE loading buffer. The proteins were subjected to gel electrophoresis and electrophoretically transferred onto PVDF membranes. The membranes were blocked with blocking solution at r. t. for 2 h. After washing, the membranes were incubated with a 1 : 1000 dilution of monoclonal anti-iNOS antibody and a 1 : 5000 dilution of β-actin antibody overnight at 4 °C. Blots were then washed thrice with TBST and incubated with a 1 : 3000 dilution of secondary antibody solution for 1 h at r. t. Blots were again washed thrice with TBST and then detected by using enhanced chemiluminescence reagent and exposed to photographic films. Images were collected and the related bands were quantitated by densitometric analysis.

Contributorsʼ Statement

Prof. Ruitao Yu, Yanduo Tao and Lijuan Mei were responsible for the experimental design; Ms. Jingya Cao was responsible for isolation and writing the article; Mr. Zhiyao Wang contributed to the spectrometric identification; Mr. Ye Zhao performed computational calculations; Ms. Qi Dong completed the biological experiments and data analysis; and Prof. Alan J. Stewart contributed to the revision of the article and the experimental analysis of the molecular docking. All the authors reviewed and validated the present manuscript prior to submission.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors are thankful to Prof. Jian Min Yue at Shanghai Institute of Materia Medica, Chinese Academy of Sciences for providing the necessary facilities for this research. This work was supported by the Natural Science Foundation of Qinghai Province (No. 2022-ZJ-930), the science and innovation platform for the development and construction of special projects of Key Laboratory of Tibetan Medicine Research of Qinghai Province (No. 2022-ZJ-Y03).

• References

• 1 The angiosperm phylogeny group. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. Bot J Linn Soc 2016; 181: 1-20
  CrossrefPubMedSearch in Google Scholar
• 2 Li HH, Qiu J, Chen FD, Lv XF, Fu CX, Zhao DX, Hua XJ, Zhao Q. Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in Saussurea medusa . Mol Biol Rep 2012; 39: 2991-2999
  CrossrefPubMedSearch in Google Scholar
• 3 Northwest Institute of Plateau Biology. The Chinese Academy of Sciences. The Tibetan Medicine Glossary. Xining: Qinghai Peopleʼs Press; 1991: 222-223
  Search in Google Scholar
• 4 Yu RX, Jiang L, Mei LJ, Tao YD, Yu RT, Xia XC. Anti-inflammatory effects of alcohol extract from Saussurea medusa Maxim. against lipopolysaccharides-induced acute lung injury mice. Int J Clin Exp Medic Res 2019; 3: 112-118
  PubMedSearch in Google Scholar
• 5 Pescitelli G, Bruhn T. Good computational practice in the assignment of absolute configurations by TDDFT calculations of ECD spectra. Chirality 2016; 28: 466-474
  CrossrefPubMedSearch in Google Scholar
• 6 Liu X, Zhang BF, Yang L, Chou GX, Wang ZT. Four new compounds from Imperata cylindrica . J Nat Med 2013; 68: 295-301
  CrossrefPubMedSearch in Google Scholar
• 7 Mali RS, Babu KN. Efficient synthesis of α-benzylidene-γ-methyl-γ- butyrolactones. Helv Chim Acta 2002; 85: 3525-3531
  CrossrefPubMedSearch in Google Scholar
• 8 Datta A, Ila H, Junjappa H. Polarized ketene dithioacetals 63. Tetrahedron 1987; 43: 5367-5374
  CrossrefPubMedSearch in Google Scholar
• 9 Fischer J, Reynolds AJ, Sharp LA, Sherburn MS. Radical carboxyarylation approach to lignans. Total synthesis of (−)-arctigenin, (−)-matairesinol, and related natural products. Org Lett 2004; 6: 1345-1348
  CrossrefPubMedSearch in Google Scholar
• 10 Corrie J, Green GH, Ritchie E, Taylor WC. The chemical constituents of Australian Zanthoxylum species. V. The constituents of Z. pluviatile Hartley; the structures of two new lignans. Aust J Chem 1970; 23: 133-145
  CrossrefPubMedSearch in Google Scholar
• 11 Gan ML, Zhang YL, Sheng L, Liu MT, Song WX, Zi JC, Yang YC, Fan XN, Shi JG, Hu JF, Sun JD, Chen NH. Glycosides from the root of Iodes cirrhosa . J Nat Prod 2008; 71: 647-654
  CrossrefPubMedSearch in Google Scholar
• 12 Frelek J, Szczepek WJ. [Rh₂(OCOCF₃)₄] as an auxiliary chromophore in chiroptical studies on steroidal alcohols. Tetrahedron Asymmetry 1999; 10: 1507-1520
  CrossrefPubMedSearch in Google Scholar
• 13 Frelek J, Klimek A, Ruskowska P. Dinuclear transition metal complexes as auxiliary chromophores in chiroptical studies on bioactive compounds. Curr Org Chem 2003; 7: 1081-1104
  CrossrefPubMedSearch in Google Scholar
• 14 Jang YP, Kim SR, Kim YC. Neuroprotective dibenzylbutyrolactone lignans of Torreya nucifera . Planta Med 2001; 67: 470-472
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Tiwari AK, Srinivas PV, Kumar SP, Rao JM. Free radical scavenging active components from Cedrus deodara . J Agric Food Chem 2001; 49: 4642-4645
  CrossrefPubMedSearch in Google Scholar
• 16 Chang H, Wang YW, Gao X, Song ZH, Awale S, Han N, Liu ZH, Yin J. Lignans from the root of Wikstroemia indica and their cytotoxic activity against PANC-1 human pancreatic cancer cells. Fitoterapia 2017; 31: 1-27
  PubMedSearch in Google Scholar
• 17 Piccinelli AL, Mahmood N, Mora G, Poveda L, Simone FD, Rastrelli L. Anti-HIV activity of dibenzylbutyrolactone-type lignans from Phenax species endemic in Costa Rica. J Pharm Pharmacol 2005; 57: 1109-1115
  CrossrefPubMedSearch in Google Scholar
• 18 Tuchinda P, Kornsakulkarn J, Pohmakotr M, Kongsaeree P, Prabpai S, Yoosook C, Kasisit J, Napaswad C, Sophasan S, Reutrakul V. Dichapetalin-type triterpenoids and lignans from the aerial parts of Phyllanthus acutissima . J Nat Prod 2008; 71: 655-663
  CrossrefPubMedSearch in Google Scholar
• 19 Matsumoto T, Hosono-Nishiyama K, Yamada H. Antiproliferative and apoptotic effects of butyrolactone lignans from Arctium lappa on leukemic cells. Planta Med 2006; 72: 276-278
  Thieme ConnectPubMedSearch in Google Scholar
• 20 Umehara K, Sugawa A, Kuroyanagi M, Ueno A, Taki T. Studies on differentiation-inducers from Arctium fructus. Chem Pharm Bull 1993; 41: 1774-1779
  CrossrefPubMedSearch in Google Scholar
• 21 Zhao F, Wang L, Liu K. In vitro anti-inflammatory effects of arctigenin, a lignan from Arctium lappa L., through inhibition on iNOS pathway. J Ethnopharmacol 2009; 122: 457-462
  CrossrefPubMedSearch in Google Scholar
• 22 Kou XJ, Qi SM, Dai WX, Luo L, Yin ZM. Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway. Int Immunopharmacol 2011; 11: 1095-1102
  CrossrefPubMedSearch in Google Scholar
• 23 Cuong TD, Hung TM, Kim JC, Kim EH, Woo MH, Choi JS, Lee JH, Min BS. Phenolic compounds from Caesalpinia sappan heartwood and their anti-inflammatory activity. J Nat Prod 2012; 75: 2069-2075
  CrossrefPubMedSearch in Google Scholar
• 24 Zhang Y, Liu JZ, Wang MY, Sun CJ, Li XB. Five new compounds from Hosta plantaginea flowers and their anti-inflammatory activities. Bioorg Chem 2020; 95: 1-7
  CrossrefPubMedSearch in Google Scholar

Correspondence

Publication History

Received: 04 June 2022

Accepted after revision: 06 October 2022

Accepted Manuscript online:
06 October 2022

Article published online:
10 January 2023

© 2022. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 The angiosperm phylogeny group. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. Bot J Linn Soc 2016; 181: 1-20
  CrossrefPubMedSearch in Google Scholar
• 2 Li HH, Qiu J, Chen FD, Lv XF, Fu CX, Zhao DX, Hua XJ, Zhao Q. Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in Saussurea medusa . Mol Biol Rep 2012; 39: 2991-2999
  CrossrefPubMedSearch in Google Scholar
• 3 Northwest Institute of Plateau Biology. The Chinese Academy of Sciences. The Tibetan Medicine Glossary. Xining: Qinghai Peopleʼs Press; 1991: 222-223
  Search in Google Scholar
• 4 Yu RX, Jiang L, Mei LJ, Tao YD, Yu RT, Xia XC. Anti-inflammatory effects of alcohol extract from Saussurea medusa Maxim. against lipopolysaccharides-induced acute lung injury mice. Int J Clin Exp Medic Res 2019; 3: 112-118
  PubMedSearch in Google Scholar
• 5 Pescitelli G, Bruhn T. Good computational practice in the assignment of absolute configurations by TDDFT calculations of ECD spectra. Chirality 2016; 28: 466-474
  CrossrefPubMedSearch in Google Scholar
• 6 Liu X, Zhang BF, Yang L, Chou GX, Wang ZT. Four new compounds from Imperata cylindrica . J Nat Med 2013; 68: 295-301
  CrossrefPubMedSearch in Google Scholar
• 7 Mali RS, Babu KN. Efficient synthesis of α-benzylidene-γ-methyl-γ- butyrolactones. Helv Chim Acta 2002; 85: 3525-3531
  CrossrefPubMedSearch in Google Scholar
• 8 Datta A, Ila H, Junjappa H. Polarized ketene dithioacetals 63. Tetrahedron 1987; 43: 5367-5374
  CrossrefPubMedSearch in Google Scholar
• 9 Fischer J, Reynolds AJ, Sharp LA, Sherburn MS. Radical carboxyarylation approach to lignans. Total synthesis of (−)-arctigenin, (−)-matairesinol, and related natural products. Org Lett 2004; 6: 1345-1348
  CrossrefPubMedSearch in Google Scholar
• 10 Corrie J, Green GH, Ritchie E, Taylor WC. The chemical constituents of Australian Zanthoxylum species. V. The constituents of Z. pluviatile Hartley; the structures of two new lignans. Aust J Chem 1970; 23: 133-145
  CrossrefPubMedSearch in Google Scholar
• 11 Gan ML, Zhang YL, Sheng L, Liu MT, Song WX, Zi JC, Yang YC, Fan XN, Shi JG, Hu JF, Sun JD, Chen NH. Glycosides from the root of Iodes cirrhosa . J Nat Prod 2008; 71: 647-654
  CrossrefPubMedSearch in Google Scholar
• 12 Frelek J, Szczepek WJ. [Rh₂(OCOCF₃)₄] as an auxiliary chromophore in chiroptical studies on steroidal alcohols. Tetrahedron Asymmetry 1999; 10: 1507-1520
  CrossrefPubMedSearch in Google Scholar
• 13 Frelek J, Klimek A, Ruskowska P. Dinuclear transition metal complexes as auxiliary chromophores in chiroptical studies on bioactive compounds. Curr Org Chem 2003; 7: 1081-1104
  CrossrefPubMedSearch in Google Scholar
• 14 Jang YP, Kim SR, Kim YC. Neuroprotective dibenzylbutyrolactone lignans of Torreya nucifera . Planta Med 2001; 67: 470-472
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Tiwari AK, Srinivas PV, Kumar SP, Rao JM. Free radical scavenging active components from Cedrus deodara . J Agric Food Chem 2001; 49: 4642-4645
  CrossrefPubMedSearch in Google Scholar
• 16 Chang H, Wang YW, Gao X, Song ZH, Awale S, Han N, Liu ZH, Yin J. Lignans from the root of Wikstroemia indica and their cytotoxic activity against PANC-1 human pancreatic cancer cells. Fitoterapia 2017; 31: 1-27
  PubMedSearch in Google Scholar
• 17 Piccinelli AL, Mahmood N, Mora G, Poveda L, Simone FD, Rastrelli L. Anti-HIV activity of dibenzylbutyrolactone-type lignans from Phenax species endemic in Costa Rica. J Pharm Pharmacol 2005; 57: 1109-1115
  CrossrefPubMedSearch in Google Scholar
• 18 Tuchinda P, Kornsakulkarn J, Pohmakotr M, Kongsaeree P, Prabpai S, Yoosook C, Kasisit J, Napaswad C, Sophasan S, Reutrakul V. Dichapetalin-type triterpenoids and lignans from the aerial parts of Phyllanthus acutissima . J Nat Prod 2008; 71: 655-663
  CrossrefPubMedSearch in Google Scholar
• 19 Matsumoto T, Hosono-Nishiyama K, Yamada H. Antiproliferative and apoptotic effects of butyrolactone lignans from Arctium lappa on leukemic cells. Planta Med 2006; 72: 276-278
  Thieme ConnectPubMedSearch in Google Scholar
• 20 Umehara K, Sugawa A, Kuroyanagi M, Ueno A, Taki T. Studies on differentiation-inducers from Arctium fructus. Chem Pharm Bull 1993; 41: 1774-1779
  CrossrefPubMedSearch in Google Scholar
• 21 Zhao F, Wang L, Liu K. In vitro anti-inflammatory effects of arctigenin, a lignan from Arctium lappa L., through inhibition on iNOS pathway. J Ethnopharmacol 2009; 122: 457-462
  CrossrefPubMedSearch in Google Scholar
• 22 Kou XJ, Qi SM, Dai WX, Luo L, Yin ZM. Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway. Int Immunopharmacol 2011; 11: 1095-1102
  CrossrefPubMedSearch in Google Scholar
• 23 Cuong TD, Hung TM, Kim JC, Kim EH, Woo MH, Choi JS, Lee JH, Min BS. Phenolic compounds from Caesalpinia sappan heartwood and their anti-inflammatory activity. J Nat Prod 2012; 75: 2069-2075
  CrossrefPubMedSearch in Google Scholar
• 24 Zhang Y, Liu JZ, Wang MY, Sun CJ, Li XB. Five new compounds from Hosta plantaginea flowers and their anti-inflammatory activities. Bioorg Chem 2020; 95: 1-7
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material