8-O-4’-Neolignans from Flower Buds of Magnolia fargesii and their Biological Activities

The genus of Magnolia (Magnoliaceae) has traditionally been used as herb medicine in China for a long time. Especially, Xinyi (dried flower buds of Magnolia fargesii), has been used for the treatment of inflammatory-related diseases such as nasal congestion, empyema, sinusitis, and allergic rhinitis. Previous phytochemical investigations have reported that this species contains several secondary metabolites such as lignans, neolignans, sesquiterpenes, and essential oils, which show various biological activities. To search for more new bioactive compounds from this plant, we reexamined the flower buds of M. fargesii, which led to the isolation of three new 8-O-4’-neolignans, named fargesiphenols A-C (1-3), along with six known compounds (4-9). In addition, the anti-HIV-1 activities and their cytotoxicities were evaluated. Their structure elucidation and biological activities are described in this paper.


Introduction
The genus of Magnolia (Magnoliaceae) has traditionally been used as herb medicine in China for a long time.Especially, Xinyi (dried flower buds of Magnolia fargesii), has been used for the treatment of inflammatory-related diseases such as nasal congestion, empyema, sinusitis, and allergic rhinitis. 1,2Previous phytochemical investigations have reported that this species contains several secondary metabolites such as lignans, [3][4][5] neolignans, 6,7 sesquiterpenes, 2,8 and essential oils, 9 which show various biological activities.
To search for more new bioactive compounds from this plant, we reexamined the flower buds of M. fargesii, which led to the isolation of three new 8-O-4'-neolignans, named fargesiphenols A-C (1-3), along with six known compounds (4-9).In addition, the anti-HIV-1 activities and their cytotoxicities were evaluated.Their structure elucidation and biological activities are described in this paper.
Compound (1608, 1520, 1478 cm -1 ) could also be observed in its IR spectrum.The UV spectrum of 1 showed absorption maxima at 285 and 210 nm, which confirmed the existence of the aromatic functions.The 1 H-  147.4), suggested that 1 is a 8-O-4'neolignan possessing three methoxyl groups and a phenolic hydroxyl group. 14hree methoxyl groups located at C-3, C-4, C-3' were assigned from the HMBC correlations of the methoxyl proton signals (d H 3.77, 3.88, 3.74) with C-3 (d C 153.9), C-4 (d C 136.7), and C-3' (d C 151.9), respectively.The presence of a phenolic hydroxyl group at C-5 was supported by the HMBC correlations of the phenolic hydroxyl proton signal (d H 11.25) with C-4 (d C 136.7), C-5 (d C 151.9), and C-6 (d C 109.0), respectively.Thus, the plain structure of 1 was established.
The configuration of the two chiral carbons (C-7 and C-8) was considered to be threo according to the coupling constant (J 7.2 Hz) between H-7 and H-8, 15 which is distinct from that of the erythro diastereoisomers. 16,17The absolute configurations at C-7 and C-8 of 1 could be established on the basis of ROESY and CD spectroscopic evidence. 18The CD spectrum of 1 gave a positive cotton effect at 237 nm, and clear ROESY correlations observed between H-7 and H-8, H-7 and H-2, H-7 and H 3 -9 (Figure 3) indicated the 7S, 8S-configuration of 1, which was named fargesiphenol A.
Compounds 2 and 3 (fargesiphenols B and C) were all obtained as yellow gums.HRESIMS analysis of 2 demonstrated that it has the molecular formula C 22 H 28 O 7 (m/z 427.1730, for [M+Na] + , calc.427.1733).The NMR spectra of 2, when compared to those of 1, displayed an additional methoxy group (d H 3.85; d C 55.6), which was located at C-5' from the HMBC cross-peak between this carbon (d C 152.3) and the methoxyl protons (d C 3.85).
The NMR spectra of 3, when compared to those of O OH MeO HO OH  13 C NMR data with those of 1 (Table 1), the relative configurations of 2 and 3 were established as being the same as that of 1.The configuration of the two chiral carbons (C-7 and C-8) for 2 and 3 was considered to be threo according to the coupling constant (J 7.2 Hz) between H-7 and H-8, and a positive cotton effect at 237 nm for 2, and 238 nm for 3 in their CD spectrum indicated a 7S,8S-configuration. 15 Since some neolignans are reported to possess anti-HIV activities, 19,20 these have been tested for compounds 1-9.The cytotoxicity assay against C8166 cells (CC 50 ), and anti-HIV-1 activity were evaluated by the inhibition assay for the cytopathic effects of HIV-1 (EC 50 ), using azidothymidine (AZT) as a positive control (EC 50 = 0.034 µg mL -1 and CC 50 = 200 µg mL -1 ). 21The results (Table 2) revealed significant activity for compounds 2, 6, 7, and 8, with therapeutic index (TI) values above 30.Compounds 1, 4, and 9 showed moderate activity with TI values above 10.

Plant material
The flower buds of M. fargesii, indigenous to Nanzhao country, Henang province, were purchased from Kunming Herb Medicine Market in September 2010.A voucher specimen (YNNI-10-9-28) has been deposited in our laboratory.

Anti-HIV-1 assay
The cytotoxicity assay against C8166 cells (CC 50 ) was assessed using the MTT method, and anti-HIV-1 activity was evaluated by the inhibition assay for the cytopathic effects of HIV-1 (EC 50 ). 21

Cytotoxicity assay
The cytotoxicity tests for these compounds were performed against HL-60, Hep-G2, KB, and MDA-MB-231 tumor cell lines by the MTT-assay using camptothecin as positive control. 24

Table 3 .
Compound 2 revealed high cytotoxic activity to HL-60 and MDA-MB-231 cells, whereas compound 8 showed high cytotoxic activity to KB cells, both with IC 50 values close to those of positive control.The other compounds displayed moderate or weak cytotoxic activity.

Table 2 .
Anti-HIV activities of the compounds 1-9