Honokiol Dimers and Magnolol Derivatives with New Carbon Skeletons from the Roots of Magnolia officinalis and Their Inhibitory Effects on Superoxide Anion Generation and Elastase Release

Two honokiol dimers, houpulins A and B (1 and 2), and two magnolol derivatives, houpulins C and D (3 and 4), were isolated and characterized from an ethanol extract obtained from the roots of Magnolia officinalis. The chemical structures were determined based on spectroscopic and physicochemical analyses, which included 1D and 2D NMR, as well as mass spectrometry data. These four oligomers possess new carbon skeletons postulated to be biosynthesized from the coupling of three or four C6-C3 subunits. In addition, the new oligomers were evaluated for inhibition of superoxide anion generation and elastase release, and houpulin B (2) was identified as a new anti-inflammatory lead compound.

in d values (ppm) with tetramethylsilane as an internal standard. Mass spectra were measured on Bruker APEXII spectrometer with ESI ionization (positive-ion mode). Column chromatography was performed in silica gel (70-230 mesh, 230-400 mesh), and PTLC was executed on Merck pre-coated Si gel 60 F 254 plates, using UV light to visualize the spots.

Plant material
The root of M. officinalis was provided by Chuang Song-Zong Pharmaceutical Factory and authenticated by Prof. C. S. Kuo, Department of Life Science, National Cheng Kung University. A voucher specimen (2010000013) has been deposited in the Herbarium of National Cheng Kung University, Tainan, Taiwan.

Extraction and isolation
Dried and powdered roots of M. officinalis (5.0 kg) were refluxed with ethanol (6620 L) and filtered. The filtrate was concentrated to afford the crude extract (2.0 kg). The crude extract was suspended in water and partitioned with dichloromethane. The organic layers were combined and concentrated to yield a dichloromethane extract (550.0 g). The dichloromethane extract was further partitioned with 5% HCl aqueous solution to afford dichloromethane solubles (440.0 g) and 5% HCl aqueous extract (90.0 g). The dichloromethane solubles were subjected to open column chromatography over silica gel by eluting with a mixture of n-hexane and EtOAc (19:1) and stepwise gradient of EtOAc to obtain 10 fractions. Fraction 6 was further purified by column chromatography over silica gel with a mixed eluent of n-hexane and acetone (4:1) followed by repeated column and thin-layer chromatography to afford compounds 1 (17.4 mg) and 2 (14.7 mg). Similarly, repeated column chromatography, preparative thin-layer chromatography, and HPLC of fraction 7 yielded 3 (2.2 mg) and 4 (5.8 mg). Houpulin

Preparation of human neutrophils
Blood was taken from healthy human donors (20-30 years old) by venipuncture, using a protocol approved by the Institutional Review Board at Chang Gung Memorial Hospital. All donors gave written consent. The Medical Ethics Committee of Chang Gung Memorial Hospital approved this consent procedure. Neutrophils were isolated with a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes. Purified neutrophils that contained .98% viable cells, as determined by the trypan blue exclusion method, were re-suspended in calcium (Ca 2+ )-free HBSS buffer at pH 7.4, and were maintained at 4uC before use.

Measurement of O 2 N 2 generation
The O 2 N 2 generation assay was based on the SOD-inhibitable reduction of ferricytochrome c. In brief, after supplementation with 0.5 mg/mL ferricytochrome c and 1 mM Ca 2+ , neutrophils (6610 5 cells/mL) were equilibrated at 37uC for 2 min and incubated with test compound or an equal volume of vehicle (0.1% DMSO) for 5 min. Cells were activated with 100 nM FMLP during the preincubation of 1 mg/mL cytochalasin B (FMLP/ cytochalasin B) for 3 min. Changes in the absorbance with a reduction in ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioner spectrophotometer with constant stirring (Hitachi U-3010, Tokyo, Japan).

Measurement of elastase release
Degranulation of azurophilic granules was determined by elastase release as described previously. Experiments were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 mM), neutrophils (6610 5 /ml) were equilibrated at 37uC for 2 min and incubated with test compound or an equal volume of vehicle (0.1% DMSO) for 5 min. Cells were activated by 100 nM FMLP and 0.5 mg/mL cytochalasin B, and changes in absorbance at 405 nm were continuously monitored to assay elastase release. The results were expressed as the percent of elastase release in the FMLP/ cytochalasin B-activated, drug-free control system.

Statistical analysis
Results were expressed as mean 6 S.E.M. Computation of 50% inhibitory concentration (IC 50 ) was computer-assisted (PHARM/ PCS v.4.2). Statistical comparisons were made between groups using Student's t test. Values of P less than 0.05 were considered to be statistically significant.  [17], [18], [19], the chemical structure of 1 should be an o,o-/o,p-linked tetramer containing four C 6 -C 3 subunits (moieties A-D shown in Figure 1). The connections of these moieties were further elucidated via 2D-correlational techniques, including HMBC and NOESY analyses. In the HMBC spectrum, 2   subunits A and B were linked through C-3/C-59 similarly to honokiol. In addition, the long range HMBC cross-peaks from  -69), and 151.7 (C-29); and from d 3.33 (H-7) to d 128.8 (C-6) and 131.2 (C-2) supported the presence of a C-3/C-59 linkage between subunits A and B, as also found in 1. However, a comparison of the proton and carbon NMR spectra of 2 with those of 1 suggested that the two honokiol moieties in 2 are connected symmetrically. Thus, the two honokiol fragments are connected between C-39 and C-30, rather than C-39 and C-50. Therefore, the structure of 2 was elucidated as shown in Figure 1, and the compound has been named houpulin B. Figure 2 depicts our proposed biogenetic pathway to compounds 1 and 2, with the compounds being derived by bimolecular coupling between two honokiol radical derivatives. Because the radical intermediate would be more stable if the radical was located at the ortho-position to the hydroxy group, the major honokiol radical intermediates are 5 and 6, and the resultant coupling products are compounds 1 and 2, which exhibit the new carbon skeletons characterized in the present work.

Characterization of new compounds
Compound 3 was purified as an optically active gum with a molecular formula of C 27 H 26 O 3 , which was deduced by HR-ESI-MS analysis. The IR absorption bands at 3375, 1612, and 1496 cm -1 indicated the presence of hydroxy and phenyl groups in 3. The 1 H NMR spectrum of 3 exhibited ABX-type aromatic  [20], [21] in the CD spectrum. Therefore, the absolute configuration at C-70 was assigned as S. The structure of 3 was elucidated as shown in Figure 1, and the compound given the name houpulin C.
Compound 4 was obtained as optically active syrup with a molecular formula of C 36 H 34 O 4 , which was determined by a pseudomolecular ion peak at m/z 553.2352 in the HR-ESI-MS analysis. The IR spectrum displayed absorption bands at 3356, 1608, and 1496 cm 21 , which are consistent with the presence of hydroxy and benzyl functionalities, respectively. The 1 H NMR spectrum of 4 exhibited three sets of ABX-type aromatic signals, three sets of allyl groups, one set of meta-coupled protons, and five aliphatic protons, which were very similar to those of compound 3. From the MS and NMR ( 13 C, DEPT135, and HSQC) data, one additional C 6 -C 3 subunit was present in compound 4. This postulation was further corroborated by HMBC analysis. In the HMBC spectrum of 4, 2 J, 3 [20], [21] observed in the CD spectrum. Consequently, the structure of 4 ( Figure 1) was established unambiguously, and the compound has been named houpulin D.

Biological results
Compounds 1-4 were evaluated for inhibition of superoxide anion generation and elastase release by human neutrophils in response to FMLP/cytochalasin B [22], and the data are shown in Table 2. Compounds 1, 2, and 4 inhibited superoxide anion generation and elastase release in FMLP/cytochalasin B activated human neutrophils in a concentration-dependent manner. Although compound 3 significantly inhibited elastase release with an IC 50 value of 3.4060.53 mM, it also induced superoxide generation by human neutrophils. Among the tested compounds, compound 2 demonstrated the most significant inhibition towards superoxide anion generation and elastase release with IC 50 values of 2.8560.16 and 2.0060.50 mM, respectively, compared with the reference compound sorafenib (IC 50 of 3.2360.42 and 2.0160.13 mM for inhibition of superoxide anion generation and elastase release, respectively).
The anti-neutrophilic effect of compound 2 was further evaluated in preliminary mechanistic studies. Compound 2 did not alter activation of ERK, p38 MAPK, JNK, or Akt ( Figure 3). Notably, compound 2 failed to alter the peak [Ca 2+ ] i values in FMLP-induced cells, but the time it took for [Ca 2+ ] i to return to half of the peak value (t 1/2 ) was significantly shortened by compound 2 (Figure 4). Many neutrophil functions, such as respiratory burst and degranulation, are regulated by calcium signals; thus, calcium clearance mechanisms are increasingly viewed as novel targets for pharmacological control of neutrophilic inflammation [23]. Compound 2 merits further investigation and development as an anti-inflammatory clinical trial candidate.