Lignans from the Twigs of Litsea cubeba and Their Bioactivities

Litsea cubeba, an important medicinal plant, is widely used as a traditional Chinese medicine and spice. Using cytotoxicity-guided fractionation, nine new lignans 1–9 and ten known analogues 10–19 were obtained from the EtOH extract of the twigs of L. cubeba. Their structures were assigned by extensive 1D- and 2D-NMR experiments, and the absolute configurations were resolved by specific rotation and a combination of experimental and theoretically calculated electronic circular dichroism (ECD) spectra. In the cytotoxicity assay, 7′,9-epoxylignans with feruloyl or cinnamoyl groups (compounds 7–9, 13 and 14) were selectively cytotoxic against NCI-H1650 cell line, while the dibenzylbutyrolactone lignans 17–19 exerted cytotoxicities against HCT-116 and A2780 cell lines. The results highlighted the structure-activity relationship importance of a feruloyl or a cinnamoyl moiety at C-9′ or/and C-7 ketone in 7′,9-epoxylignans. Furthermore, compound 11 was moderate active toward protein tyrosine phosphatase 1B (PTP1B) with an IC50 value of 13.5 μM, and compounds 4–6, 11 and 12 displayed inhibitory activity against LPS-induced NO production in RAW264.7 macrophages, with IC50 values of 46.8, 50.1, 58.6, 47.5, and 66.5 μM, respectively.


Structure Elucidation
The EtOAc extract of the twigs of L. cubeba was subjected to column chromatography on silica gel to give 13 fractions (F 1 -F 13 ). Cytotoxicity assays found that F 9 displayed potent activities against HCT-116, NCI-H1650, and A270 cell lines. Fractionation of F 9 by Sephadex LH-20, RP-18, preparative TLC, and preparative HPLC led to the discovery of nine new lignans 1-9 and the ten known ones 10-19.
Compound 1 was obtained as a white amorphous powder. The presence of amide (1643 cm −1 ), aromatic ring (1611, 1516, and 1459 cm −1 ), and hydroxy (3372 cm −1 ) functionalities were evident in its IR spectrum. Its molecular formula of C 30 H 33 NO 9 with fifteen degrees of unsaturation was established by HREIMS based on the [M + H] + ion at m/z 552.2234 (calcd. 552.2228) and 13 C-NMR spectrum.
Further analysis of 2D-NMR data permitted the tyramine and 3-methoxytyramine moieties to be located at C-9 and C-9 in 3, the reverse of 2, via the amide bonds (Figure 2), respectively. Analysis of the 1D NOE difference spectrum of 3 and its optical rotation indicated that 3 had the same absolute configuration as 2. Therefore, the structure of 3 was confirmed as (−)- Compound 4 was obtained as a yellow solid and its molecular formula was deduced as C 31 H 36 O 10 from HRESIMS. The IR spectrum exhibited absorption bands at 3391, 1608, and 1516 cm −1 due to the aromatic and hydroxy groups. The NMR data of 4 showed signals similar with secoisolariciresinol ( Table 1; Table 2) [21,22]. However, both the H 2 -9 and C-9 were shifted downfield when compared with secoisolariciresinol. Besides, the 1 H-and 13 C-NMR signals attributed to a trans-cinnamyloxy unit were present (Table 1; Table 2). These were consistent with the substitution of the trans-cinnamyloxy at C-9, which was verified by the key HMBC correlation from H 2 -9 to C-9". The positive optical rotation of 4 supported the same (8S,8 S) configuration as that of the known compound (+)-(8S,8 S)-9,9 -di-O-(E)-feruloylsecoisolariciresinol (11), which has been also isolated from this plant [12]. The (8S,8 S) configuration was confirmed by the evidence that compound 4 showed optical rotation opposite to that of (−)-1-O-feruloylsecoisolariciresinol [21]. Thus, the structure of 4 was defined as (+)-(8S,8 S)-9-O-(E)-cinnamoylsecoisolariciresinol.
The molecular formula of compound 5 was C 32 H 38 O 11 from the HRESIMS data. Analysis of the 1D-and 2D-NMR data revealed that its planar structure was completely identical to the known lignan, (−)-(8R,8 R)-9-O-(E)-feruloyl-5,5 -dimethoxysecoisolariciresinol, but their specific rotation was inverse [23]. Taking into account that 4 was the 5-methoxy analogue of 5 and they displayed similar specific rotation, it is proposed that they both have the ( The molecular formula of compound 8 was C 33 H 38 O 12 as indicated by the HRESIMS. The NMR spectra of 8 and (+)-9 -O-trans-feruloyl-5,5 -dimethoxylariciresinol were closely comparable [24], except for the replacement of (E)-feruloyl group by the (E)-cinnamoyl group. The structure of 8 was confirmed by the 2D-NMR HSQC, COSY, HMBC, and NOESY data. Also, the NOESY correlations of H-7 /H 2 -9 and H 2 -7/H 2 -9 revealed that compounds 7 and 8 have the same relative configuration. Therefore, on the basis of the positive optical rotation of 8 and biosynthetic considerations, the structure of 8 was deduced as (+)-(8R,7 S,8 R)-9 -O-(E)-cinnamoyl-5,5 -dimethoxylariciresinol.
Compound 9 was shown to have the molecular formula of C 33 H 38 O 12 , as established by the HRESIMS. The 1 H-and 13 C-NMR spectra of 9 closely resembled those of 7, the only discernable difference being the presence of a new methoxy moiety and lack of a ketone moiety in 9, suggesting that compound 9 contains a methoxy moiety rather than a ketone moiety at C-7. This was confirmed from the COSY correlation of H-7/H-8 and HMBC correlation of OMe/C-7. In the NOESY spectrum of 9, the NOE correlations of H-7/H 2 -9 and H-7 /H 2 -9 also verified that H-7 was oriented opposite to H-8 and H-8 . Thus, the structure of 9 was defined as 9 -O-(E)-feruloyl-5,7,5 -trimethoxy-lariciresinol.
Compound 7, an amorphous powder, was determined to have the molecular formula of C32H34O12 by HRESIMS. The NMR spectra of 7 were similar to the co-occurring (+)-9′-O-trans-feruloyl-5,5′-dimethoxylariciresinol (13) [24], with the only difference being the replacement of the CH2 group by a ketone. These data demonstrated the presence of a ketone moiety at C-7 in 7. This inference was confirmed by the HMBC cross-peak of H-2(6)/C-7, H2-9/C-7, and H-  The molecular formula of compound 8 was C33H38O12 as indicated by the HRESIMS. The NMR spectra of 8 and (+)-9′-O-trans-feruloyl-5,5′-dimethoxylariciresinol were closely comparable [24], except for the replacement of (E)-feruloyl group by the (E)-cinnamoyl group. The structure of 8 was

Cytotoxic Activity
The task of IC 50 assessment for all isolates against human colon cancer (HCT-116), human non-small-cell lung carcinoma (NCI-H1650), and human ovarian cancer (A2780) cell lines began immediately following the purification and characterization of each lignan.
Of the compounds, only 7 ,9-epoxylignans with feruloyl or cinnamoyl group (compounds 7-9, 13 and 14) were selectively cytotoxic against NCI-H1650 cell line, with IC 50 values of less than 20 µM. These results suggested the presence of a feruloyl or a cinnamoyl moiety at C-9 in 7 ,9-epoxylignans is essential for cytotoxicity against NCI-H1650 cell line. It is noteworthy that compound 7 displayed 4-6 folds more active than 8, 9, 13, and 14, indicating that the presence of the C-7 ketone could enhance the bioactivity. In addition, the dibenzylbutyrolactone lignans (17)(18)(19) exerted cytotoxicities against HCT-116 and A2780 cell lines, with IC 50 values ranging from 0.28 to 18.47 µM (Table 3), but less potent than the positive control taxol (IC 50 = 0.005 and 0.02 µM, respectively). Interestingly, the addition of the double bond at C-7−C-8 on 19 resulted in 4-40 folds less active than 17 and 18. This implied that the C-7−C-8 double bond could reduce the cytotoxicity, especially against the A2780 cell line.

Inhibitory Activity of Protein Tyrosine Phosphatase 1B
The isolates were also evaluated for inhibitory activities against protein tyrosine phosphatase 1B (PTP1B). Only compound 11 was moderate active toward PTP1B with an IC 50 value of 13.5 µM. The positive control oleanolic acid gave an IC 50 value of 3.82 µM.

Anti-Inflammatory Activity
The inhibitory activity of compounds 1-19 against LPS-induced NO production in RAW264.7 macrophages was examined in this study. As a result, compounds 4-6, 11 and 12 displayed inhibitions against LPS-induced NO production in RAW264.7 macrophages, with IC 50 values of 46.8, 50.1, 58.6, 47.5, and 66.5 µM, respectively. Dexamethasone was used as positive control with an IC 50 value of 9.5 µM.

General Experimental Procedures
Optical rotations were measured on an Autopol III automatic polarimeter (Rudolph Research, Hackettstown, NJ, USA). UV spectra were measured on a Cary 300 spectrometer (Agilent, Melbourne, Australia). ECD spectra were recorded on a J-815 spectrometer (JASCO, Tokyo, Japan). IR spectra were acquired on an Impact 400 FT-IR Spectrophotometer (Nicolet, Madison, WI, USA). Standard pulse sequences were used for all NMR experiments, which were run on either a Bruker spectrometer (600 MHz for 1 H or 150 MHz for 13 C, Karlsruhe, Germany) or a Varian INOVA spectrometer (500 MHz for 1 H or 125 MHz for 13 C, Palo Alto, CA, USA) equipped with an inverse detection probe. Residual solvent shifts for acetone-d 6 were referenced to δ H 2.05, δ C 206.7 and 29.9, respectively. Accurate mass measurements were obtained on a Q-Trap LC/MS/MS (Turbo ionspray source) spectrometer (Sciex, Toronto, ON, Canada). Column chromatography (CC) was run using silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). HPLC separation was done on Waters HPLC components (Milford, MA, USA) comprising of a Waters 600 pump, a Waters 600 controller, a Waters 2487 dual λ absorbance, with GRACE preparative (250 × 19 mm) Rp C 18 (5 µm) columns.

Plant Material
The twigs of Litsea cubeba were collected in Zhaotong, Yunnan Province, People's Republic of China, in May 2013, and identified by Prof. Gan-Peng Li at Yunnan Minzu University. A herbarium specimen was deposited in at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Beijing 100050, People's Republic of China (herbarium No. 2013-05-10).

Extraction and Isolation
The air-dried twigs of L. cubeba (12 kg) were ground and extracted using 30.0 L of 95% EtOH under ambient temperature for 3 × 48 h. The EtOH extract was concentrated in vacuo and the residue was suspended in H 2 O, then partitioned with EtOAc, to afford EtOAc and H 2 O soluble extracts.

Cytotoxicity Assay
The cytotoxic activity was determined against human colon cancer (HCT-116), human non-small-cell lung carcinoma (NCI-H1650), and human ovarian cancer (A2780) cell lines which were bought from the Cell Bank of Shanghai Institute of Cell Biology (Chinese Academy of Sciences) and originally obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in RPMI 1640 (GIBCO, New York, NY, USA) supplemented with 10% fetal calf serum (Life Technologies, Carlsbad, CA, USA), penicillin G (100 U/mL), and streptomycin (100 µg/mL) at 37 • C in a 5% CO 2 and seeded in 96-well plates (CLS3635, Corning ® , Sigma, Santa Clara, CA, USA) at a cell density of 3000 per well over night, and then were treated with various diluted concentrations (each concentration was arranged triple) of compounds 1-19, which were prepared with DMSO (Sigma) to 100 µM stock solution and stored in −20 • C in advance. After 24 h of treatment, 10 µL of MTT (5 mg/mL in PBS) was then added directly to all wells and the plates were placed in the dark at 37 • C for 3 h incubation. Cell viability was measured by observing absorbance at 570 nm on a SpectraMax 190 microplate reader (Molecular Devices, Silicon Valley, CA, USA). IC 50 values were calculated using Microsoft Excel software (version 2010, Redmond, WA, USA). Taxol was used as a positive control.

PTP1B Inhibition Assay
The recombinant GST-hPTP1B (gluthathione S-transferase-human protein tyrosine phosphatase 1B) bacteria pellets were purified by a GST bead column. The dephosphorylation of para-nitrophenyl phosphate (p-NPP) was catalyzed to para-nitrophenol by PTP1B. Enzyme activity involving an end-point assay, which intensified the yellow color, was measured at a wavelength of 405 nm. All compounds were dissolved in 100% dimethyl sulfoxide (DMSO), and reactions, including controls, were performed at a final concentration of 10% DMSO. Selected compounds were first evaluated for their ability to inhibit the PTPase reaction at a 10 µM concentration at 30 • C for 10 min, in a reaction system with 3 mM p-NPP in HEPES assay buffer (pH 7.0). The reaction was initiated by addition of the enzyme and quenched by addition of 1 M NaOH. The amount of the produced p-nitrophenol was determined at 405 nm using a microplate spectrophotometer (uQuant, Bio-Tek, Winooski, VT, USA). IC 50 values were evaluated using a sigmoidal dose-response (variable slope) curve-fitting program of GraphPad Prism 4.0 software (La Jolla, CA, USA). Oleanolic acid was used as a positive control.

Nitric Oxide (NO) Production in RAW264.7 Macrophages
The RAW 264.7 macrophages were cultured in The RPMI 1640 medium (Hyclone, Logan, UT, USA) containing 10% FBS. The compounds were dissolved in DMSO and further diluted in medium to produce different concentrations. The cell mixture and culture medium were dispensed into 96-well plates (2 × 105 cells/well) and maintained at 37 • C under 5% CO2. After preincubation for 24 h, serial dilutions of the test compounds were added into the cells, up to the maximum concentration 25 µM, then added with LPS to a concentration 1 µg/mL and continued to incubate for 18 h. The amount of NO was assessed by determined the nitrite concentration in the cultured RAW264.7 macrophage supernatants with Griess reagent. Aliqueots of supernatants (100 µL) were incubated, in sequence, with 50 µL 1% sulphanilamide and 50 µL 1% naphthylethylenediamine in 2.5% phosphoric acid solution. The sample absorbance was measured at 570 nm by a 2104 Envision Multilabel Plate Reader (PerkinElmer, Inc., Waltham, MA, USA). Dexamethasone was used as a positive control.

Conclusions
In summary, bioassay-guided isolation of cytotoxic fractionsof the twigs of L. cubebarevealed the presence of nine new lignans 1-9 and ten analogues 10-19. Initially, all of the isolated compounds were evaluated against HCT-116, NCI-H1650, and A2780 tumor cell lines. Of the compounds, only 7 ,9-epoxylignans with feruloyl or cinnamoyl group (7-9, 13 and 14) were selectively cytotoxic against NCI-H1650 cell line, with IC 50 values of less than 20 µM, whereas, the dibenzylbutyrolactone lignans 17-19 exerted cytotoxicity against HCT-116 and A2780 cell lines, with IC 50 values ranging from 0.28 to 18.47 µM. The results highlighted the structure-activity relationship importance of a feruloyl or a cinnamoyl moiety at C-9 or/and C-7 ketone in 7 ,9-epoxylignans. The isolates were also examined for inhibitory activities against PTP1B and LPS-induced NO production in RAW264.7 macrophages. As a result, compound 11 was moderate active toward PTP1B with an IC 50 value of 13.5 µM and compounds 4-6, 11 and 12 displayed inhibitions against LPS-induced NO production in RAW264.7 macrophages, with IC 50 values of 46.8, 50.1, 58.6, 47.5, and 66.5 µM, respectively. The present results provide additional phytochemical and bioactive information of this medicinal and spiced plant.
Supplementary Materials: The following are available online, IR, UV, HRMS, NMR and ECD spectra of compounds 1-9 as well as other supporting data.