Four New Lignans from Kadsura Interior and Their Bioactivity

A phytochemical investigation of the stems of Kadsura interior has led to an isolation of four new lignans, named kadsutherin E–H (1–4), together with two known lignans (5–6). The structures of the four new compounds were established on the basis of comprehensive spectroscopic analyses. Compounds 1–6 exhibited inhibition against adenosine diphosphate (ADP) induced platelet aggregation. Among the isolated compounds, kadsutherin F (2) showed the strongest anti-platelet aggregation activity with an inhibition of 49.47%.


Four New Identified Compounds (1-4)
Kadsutherin E (1), which was obtained as a white powder, had the molecular formula C28H28O8, as it was revealed by its high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z 515.1691 [M + Na] + ). The UV spectrum of 1 showed a maximum absorption at 221 nm, along with the 1 H-and 13 C-NMR spectra (Table 1), which indicated that 1 was a dibenzocyclooctene lignan [11,12].

Four New Identified Compounds (1-4)
Kadsutherin E (1), which was obtained as a white powder, had the molecular formula C 28 H 28 O 8 , as it was revealed by its high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z 515.1691 [M + Na] + ). The UV spectrum of 1 showed a maximum absorption at 221 nm, along with the 1 H-and 13 C-NMR spectra (Table 1), which indicated that 1 was a dibenzocyclooctene lignan [11,12].
Kadsutherin F (2), which was obtained as a white powder, had the molecular formula C 28 H 28 O 8 , as revealed by its HRESIMS (m/z 521.1791 [M + Na] + ). The UV spectrum of 2 showed a maximum absorption at 221 nm. The 1 H-NMR and 13 C-NMR spectra (Tables 1 and 2) indicated that 2 was also a dibenzocyclooctene lignan. The characteristic proton signals at δ H 4.46, 5.57(2H, d, J = 8.4 Hz) and a quaternary carbon signal at δ C 83.7 indicated that 2 possessed a spiroenone ring, similar to kadsutherin D [14]. The 1 H-NMR spectrum (Table 1) exhibited two aromatic singlets at δ H 6.31 (H-4) and δ H 6.39 (H-11), two methoxy groups at δ H 3.69, 3.85 (3H each), one methylenedioxy (-OCH 2 O-) group at δ H 5.99 (2H, s). In the cyclooctadiene ring, two doublet methyl groups at δ H 1.02 and 1.04 (each 3H, d, J = 7.2 Hz) were located at C-7and C-8, respectively. Furthemore, the characteristic signals of an angeloxy group (δ H 5.87, 1.77, 1.66 for H-3 , H-4 and H-5 ) were found in the 1 H-and 13 C-NMR spectra (Tables 1 and 2). The HMBC correlations of H-11 with C-12 (δ C 150.6) and C-13 (δ C 129.8), H-11 with C-9 (δ C 79.2), and two H-atoms (δ H 5.97, 5.98) of the methylenedioxy moiety with C-12 and C-13, indicated that the methylenedioxy moiety was located at C-12 and C-13 ( Figure 2). Moreover, two methoxy groups (1-OCH 3 , 2-OCH 3 ) were deduced from the HMBC correlations of 1-OCH 3 (δ H 3.85, 3H, s) with C-1(δ C 168.     The CD spectrum of 2 exhibited a positive Cotton effect around 276 nm and a negative Cotton effect around 216 nm, which was contrary to the CD spectrum of the 1, which suggested that 2 possessed an R-biphenyl conformation [14]. A twist-boat conformation of the cycloctadiene ring was deduced from the NOE correlations of H-9 with H-11, H-9 with CH 3 -18, H-6a (δ H 4.13, 1H, d, J = 10.8 Hz) with H-4, and CH 3 -18 with H-4 ( Figure 2). According to the above data, the structure of 2 was elucidated as kadsutherin F. Kadsutherin G (3), which was obtained as a white powder, had the molecular formula C 29 H 28 O 9 , as it was revealed by its HRESIMS (m/z 543.1647 [M + Na] + ). The 1 H-NMR spectrum of 3 was similar to that of 2, but the prominent difference in the 1 H-NMR spectrum (Table 1) was the presence of a benzoyl group in 3, which was substituted the angeloxy group in 2. The data of the 13 C-NMR spectrum of 3 (Table 2) also confirmed this deduction. In the HMBC spectrum of 3, H-9 was correlated with the carbonyl carbon (δ C 168.5, C=O) of the benzoyl group and δ C 122.0 (C-10), 156.4 (C-15), 101.7 (C-11), 39.8 (C-8), and 44.0 (C-7), which clearly indicated that the benzoyl group was located at C-9.
The CD spectrum of 3 exhibited a positive Cotton effect around 311 nm and a negative Cotton effect around 224 nm, which suggested that 3 possessed an R-biphenyl conformation. A twist-boat conformation of 3 was deduced from the NOE correlations of CH 3 -17 with CH 3 -18, H-9b (δ H 5.72, 1H, d, J = 7.2 Hz) with CH 3 -18, and H-6a (δ H 4.26, 1H, d, J = 10.2 Hz) with CH 3 -17 ( Figure 2). On the basis of the above data, the structure of 3 was educidated as kadsutherin G.
Kadsutherin H (4), which was obtained as a white powder, had the molecular formula C 24 H 26 O 9 , as it was revealed by its HRESIMS (m/z 481.1478 [M + Na] + ). The 1 H-NMR spectrum of 4 was also similar to that of 2, but the prominent difference in the 1 H-NMR spectrum (Table 1) was the presence of an acetoxy group in 4, which was substituted the angeloxy group in 2. The data of the 13 C-NMR spectrum of 4 ( Table 2) also confirmed this deduction. In the HMBC spectrum of 4, H-9 was correlated with the carbonyl carbon (δ C 171.9, C=O) of the acetoxy group and δ C 122.2 (C-10), 146.6(C-15), 101.3 (C-11), 43.3 (C-8), and 40.4 (C-7), which clearly indicated that the acetoxy group was located at C-9.
The CD spectrum of 4 exhibited a positive Cotton effect around 245 nm and a negative Cotton effect around 223 nm, which suggested that 4 possessed an R-biphenyl conformation. Compound 4 had a twist-boat conformation, which was deduced from the NOE correlations of H-9 with H-11, H-9 with CH 3 -18, H-6a (δ H 4.11, 1H, d, J = 10.2 Hz) with H-4, and CH 3 -18 with H-4 ( Figure 2). Thus, 4 was elucidated as kadsutherin G.
A comparison of the NMR data with the reported values led to the identification of the structures of the known compounds 5-6 as acetoxyl oxokadsurane (5) [15] and heteroclitin D (6) [16].

Anti-Platelet Effects of Compounds 1-6
The anti-platelet effects of the compounds from the stems of the K. interior plants were tested in vitro, using the turbidimetric method in washed rat platelets that were induced by ADP (100 µM.). The anti-platelet aggregation data are shown in Table 3. The clinically applied anti-platelet agent aspirin, was used as the positive control. From the results of our anti-platelet aggregation tests, kadsutherin E (1), kadsutherin F (2), kadsutherin G (3), kadsutherin H (4), acetoxyl oxokadsurane (5), and heteroclitin D (6) exhibited inhibition (with inhibition in the range of 11.77-49.47%) against the ADP induced platelet aggregation. Among these compounds, kadsutherin F (2) showed the strongest anti-platelet aggregation activity with an inhibition of 49.47 ± 2.93%. Table 3. Inhibitory effects of compounds on the aggregation of rat platelets induced by adenosine diphosphate (ADP) (100 µM, n = 9).

Plant Material
The

Extraction and Isolation
The dried and powdered stems (8 kg) of the K. interior plants were extracted three times with 90% EtOH. The extract was concentrated under a reduced pressure to dryness, which was then partitioned between EtOAc and H 2 O to provide the EtOAc-soluble fraction. The EtOAc fraction (243 g) was purified by CC on silica gel (200-300 mesh; petroleum ether/acetone gradient) to afford eight fractions, as follows: Fr 1 -Fr 8 . Fr 5 was applied to silica gel CC with PE/EtOAc and was then separated by Sephadex LH-20 CC with CHCl 3 /MeOH (3:2 v/v) and preparative HPLC with MeOH/H 2 O, so as to yield compounds 1 (26 mg), 2 (77 mg), 3 (15 mg), 4 (20 mg), 5 (6 mg), and 6 (120 mg).

Anti-Platelet Aggregation Assay
The platelet aggregation assay was carried out according to the methodology that was reported in previous studies [17]. The blood was collected by catheterization of the abdominal aorta in rats (mean weight: 232.9 ± 4.8 g), anticoagulated with acid citrate dextrose (ACD) (9:1, v/v) and centrifuged for 15 min at 100× g at room temperature, so as to obtain platelet rich plasma (PRP). The platelet numbers were counted by a Coulter counter and were adjusted to 5.0 × 10 5 platelets/µL. The platelet aggregation was measured at 37 • C using the turbidimetric method. The assays were performed at 37 • C in cuvettes using 300 µL of PRP under stirring, and the aggregation was triggered by the addition of adenosine diphosphate (ADP). All of the tested compounds (1mg/mL) were dissolved in 0.5% dimethyl sulfoxide (DMSO) [18] and were incubated with PRP for 2 min at 37 • C, and then, the platelet aggregation was triggered by adding ADP (10 µM). Aspirin was used as a positive control. The percentages of the inhibition were calculated as follows: Inhibition (%) = [OD(control) − OD(compound)]/OD(control) × 100%.
The results were expressed as the mean ± SD and all of the anti-platelet aggregation data were statistical analyzed by SPSS (version 19.0, Chicago, IL, USA).

Conclusions
Four new lignans, named kadsutherin E-H (1-4), together with two known lignans (5-6), were isolated from the stems of the K. interior plants. The structures of those compounds were established on the basis of spectroscopic data. The anti-platelet effects of the six compounds were evaluated by suppressing the ADP-induced platelet aggregation in washed rat platelets. The results of the anti-platelet aggregation experiments indicated that compounds 1-6 could inhibit ADP-induced platelet aggregation at 100 µM. Kadsutherin F (2) showed the strongest anti-platelet aggregation activity with an inhibition of 49.47%.