Antioxidant and Anti-Osteoporosis Activities of Chemical Constituents of the Stems of Zanthoxylum piperitum

Two new lignans, zanthoxyloside C (1) and zanthoxyloside D (2), together with nine known compounds comprising lignans (3–5), flavonoids (6–8), and phenolics (9–11), were isolated from the methanol extract of the stems of Zanthoxylum piperitum. All isolates were evaluated for their antioxidant and anti-osteoporotic activities using oxygen radical absorbance capacity (ORAC), cupric reducing antioxidant capacity (CUPRAC), and tartrate-resistant acid phosphatase (TRAP) assays. Compounds 7–10 showed peroxyl radical-scavenging capacities and 4, 6–7, and 9 showed reducing capacities. Moreover, compounds 3, 6–9, and 11 significantly suppressed TRAP activities. These results indicated that the stems of Z. piperitum could be an excellent source for natural antioxidant and anti-osteoporosis.


Introduction
Osteoporosis, one of the metabolic diseases of the bones, occurs when the balance between bone resorption and bone formation is lost.To maintain bone mass and skeletal homeostasis, the dynamic process of resorption and formation continues in the bone tissues.Two types of bone cells, osteoclasts and osteoblasts perform specific functions in bone remodeling.Osteoclasts absorb bone, while osteoblasts synthesize and fill bone matrix; bone mass depends on the reciprocal function of these cells.A typical adult always maintains a balance between the amount of bone resorption and bone formation.However, once osteoporosis develops, due to aging, hormone abnormality, or lack of exercise, one's quality of life degrades as a result of severe pain and limited mobility [1][2][3][4][5].
Recently, there has been a growing interest in the relationship between osteoporosis and oxidative stress.Clinical studies have shown that there is a significant correlation between increased oxidative stress and decreased bone mineral density.The antioxidant levels in the blood of osteoporotic women turned out to be low, but their bone mineral density increased by taking antioxidant vitamins.It has been revealed in in vitro studies, as well, that reactive oxygen species (ROS) increase the activity of osteoclasts and depress the metabolism of osteoblasts.The inhibition of the metabolism of osteoblasts owing to oxidative stress can be mitigated by the medication of antioxidants [6][7][8].Therefore, if natural products without side effects on the human body were able to indirectly increase the intravital antioxidant defense system and directly eliminate excessive ROS, natural antioxidants could be applied as a functional material to prevent diseases caused by oxidative stress.
natural antioxidants could be applied as a functional material to prevent diseases caused by oxidative stress.
Zanthoxylum piperitum DC, widely distributed in South-East Asia, is an aromatic shrub belonging to the Rutaceae family.The fruits of Z. piperitum have been used as traditional herbal medicine as well as as a condiment.Most previous studies of Z. piperitum have focused on the fruits and leaves [9][10][11][12].Therefore, there is a lack of information on chemical constituents of Z. piperitum stems and their biological activities.As a part of our ongoing research into the bioactivity of natural products, eleven secondary metabolites were isolated from stems of Z. piperitum.Moreover, antioxidant and anti-osteoporosis activities of these compounds were evaluated.
Compound 2 was obtained as a white amorphous powder.The HR-ESI-MS spectrum of compound 2 contained quasi-molecular ion peaks at m/z 543.1873 [M + Na] + (Cald for C 26 H 32 NaO 11 , 543.1837), indicating its molecular formula to be C 26 H 32 O 11 .The 1 H-NMR spectrum of 2 showed two 1,3,4-trisubstituted benzene ring spin systems (δ H 6.71 (1H, dd, J = 2.1, 8.2 Hz, H-6), 6.83 (1H, d, J = 2.1 Hz, H-2), and 6.66 (1H, d, J = 8.2 Hz, H-5)) and (δ H 6.59 (1H, dd, J = 8.2, 1.3 Hz, H-2 ) , 6.63 (1H, d, J = 8.2 Hz, H-3 ) and 6.64 (1H, dd, J = 8.2, 1.3 Hz, H-6 )).The signal of a methylene dioxy proton at δ H 5.80 (2H, s) and a methoxy group at δ H 3.76 (3H, s) were also observed.The signal of one anomeric proton at δ H 4.20 (1H, d, J = 8.2 Hz) indicated the presence of one sugar unit in the structure of 2. The 13 C-NMR (Table 1) and DEPT-135 spectroscopic data of 2 also indicated signals of 26 carbons, which was in agreement with the structure of lignan glycoside as compound 1.However, the downfield shift of C-7 (δ C 84.3) and C-9 (δ C 73.7) suggested that compound 2 belonged the tetrahydrofuran lignans.The HMBC spectrum revealed significant correlations between the proton signal at δ H 6.65 (H-6 ) and the carbon signals at δ C 33.9 (C-7 ), 122.7 (C-2 ), 136.1 (C-1 ), 147.3 (C-4 ); between the proton signal at δ Correlations between the proton signal at δ H 6.83 (H-2) and the carbon signals at δ C 84.3 (C-7), 119.9 (C-6), 135.6 (C-1), 147.1 (C-4), 149.2 (C-3); and between the methoxy proton signal at δ H 3.76 and the carbon signal at δ C 149.2 (C-3) in HMBC led to the establishment of a hydroxyl and methoxy groups at C-4 and C-3, respectively.Correlations between the proton signals at δ H 6.83 (H-2), 6.70 (H-6) and the carbon signal at δ C 84.3 (C-7) revealed a second benzene ring moiety connected to the tetrahydrofuran ring at C-7.The glucosyl linkage was confirmed by the HMBC correlation between an anomeric proton at δ H 4.20 (H-1") with δ C 68.53 (C-8).The coupling constant of the anomeric proton was 8.2 Hz in doublet multiplicity in the 1 H-NMR spectrum, which confirmed the β-configuration of glucopyranoside.Finally, the absolute configurations were determined by examinations of the CD spectrum and NOE correlation.The NOE correlations, including proton H-8 (δ H 2.41) with both protons H-2 (δ H 6.83) and H-6 (δ H 6.71), proton H-7 (δ H 4.73) with H-9 (δ H 3.50), proton H-9 (δ H 3.50) with H-7 (δ H 2.91), were clearly observed in the NOESY spectrum of 2, which confirmed their close proximity, as shown in Figure 3.In addition, the CD spectrum of 2 showed the opposite trend of Cotton effects (positive effects at 241 nm (+0.24) and 289 nm (+0.13)) in comparison with those of (+)-(7S,8R,8 R)-lariciresinol (negative effects at 244 nm (−0.42) and 290 nm (−0.26)) _ENREF_4 [25], which indicated 7R,8S,8 S configurations of compound 2. Thus, the structure of compound 2 was determined to be (7R,8S,8 S)-3-methoxy-4,9-dihydroxy-3 ,4 -methylendioxy-7,9 -epoxylignan 9-O-β-D-glucopyranoside, and named zanthoxyloside D (see Supplementary Materials).The antioxidant activities of the isolated compounds 1-11 were evaluated with respect to their peroxyl radical-scavenging and reducing capacity.Table 2 shows the scavenging activities of compounds 1-11 on peroxyl radicals, which were generated from 2,2ʹ-azobis(2-amidinopropane) dihydrochloride (AAPH) in the oxygen radical absorbance capacity (ORAC) assay.All isolated compounds showed significant peroxyl radical-scavenging activities, with values of 5.91 ± 0.11 to 26.91 ± 1.05 μM at a concentration of 10 μM.The ability of compounds 1-11 to stimulate the reduction of copper ions (Cu 2+ to Cu + ) by donating electrons was investigated to determine whether their peroxyl radical-scavenging capacities, with the donation of hydrogen atoms, could be related to their reduction capacities.As shown in Table 2, compounds 1-9 showed significant reducing capacities, with values of 9.60 ± 0.26 to 33.04 ± 0.17 μM at a concentration of 10 μM.The rest of the compounds (10 and 11) showed weak activities.These results suggest that the peroxyl radical-scavenging and reducing capacity of all the tested compounds, due to transfer of hydrogen atoms and single electron, may be relevant to the hydroxyl groups of the benzene rings [26][27][28].The antioxidant activities of the isolated compounds 1-11 were evaluated with respect to their peroxyl radical-scavenging and reducing capacity.Table 2 shows the scavenging activities of compounds 1-11 on peroxyl radicals, which were generated from 2,2ʹ-azobis(2-amidinopropane) dihydrochloride (AAPH) in the oxygen radical absorbance capacity (ORAC) assay.All isolated compounds showed significant peroxyl radical-scavenging activities, with values of 5.91 ± 0.11 to 26.91 ± 1.05 μM at a concentration of 10 μM.The ability of compounds 1-11 to stimulate the reduction of copper ions (Cu 2+ to Cu + ) by donating electrons was investigated to determine whether their peroxyl radical-scavenging capacities, with the donation of hydrogen atoms, could be related to their reduction capacities.As shown in Table 2, compounds 1-9 showed significant reducing capacities, with values of 9.60 ± 0.26 to 33.04 ± 0.17 μM at a concentration of 10 μM.The rest of the compounds (10 and 11) showed weak activities.These results suggest that the peroxyl radical-scavenging and reducing capacity of all the tested compounds, due to transfer of hydrogen atoms and single electron, may be relevant to the hydroxyl groups of the benzene rings [26][27][28].The antioxidant activities of the isolated compounds 1-11 were evaluated with respect to their peroxyl radical-scavenging and reducing capacity.Table 2 shows the scavenging activities of compounds 1-11 on peroxyl radicals, which were generated from 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH) in the oxygen radical absorbance capacity (ORAC) assay.All isolated compounds showed significant peroxyl radical-scavenging activities, with values of 5.91 ± 0.11 to 26.91 ± 1.05 µM at a concentration of 10 µM.The ability of compounds 1-11 to stimulate the reduction of copper ions (Cu 2+ to Cu + ) by donating electrons was investigated to determine whether their peroxyl radical-scavenging capacities, with the donation of hydrogen atoms, could be related to their reduction capacities.As shown in Table 2, compounds 1-9 showed significant reducing capacities, with values of 9.60 ± 0.26 to 33.04 ± 0.17 µM at a concentration of 10 µM.The rest of the compounds (10 and 11) showed weak activities.These results suggest that the peroxyl radical-scavenging and reducing capacity of all the tested compounds, due to transfer of hydrogen atoms and single electron, may be relevant to the hydroxyl groups of the benzene rings [26][27][28].The anti-osteoporotic activities were investigated using TRAP assay on RAW 264.7 cells.The inhibitory effects of isolated compounds were tested based on the suppression of excessive bone resorption by osteoclasts.As shown in Table 3, compounds 3, 6-9, and 11 showed significant inhibitory activities, with values of 77.73 to 92.42% relative to the RANKL-treated control (100%).

Plant Material
Dried stems of Z. piperitum were purchased at Daekwang Farm, Busan, Korea in November 2012 and were taxonomically identified by one of the authors (Prof.Young Ho Kim).A voucher specimen (CNU12107) was deposited at the Herbarium of College of Pharmacy, Chungnam National University, Daejeon, Korea.

Extraction and Isolation
Dried stems of Z. piperitum DC. (3.0 kg) were extracted with methanol at room temperature three times.After removal of the solvent under reduced pressure, the crude extract (120.0 g) was dissolved in 4.0 L of H 2 O to form a suspension that was successively partitioned with n-hexane, CH 2 Cl 2 , EtOAc, and BuOH to give n-hexane (45.0 g), CH 2 Cl 2 (29.0 g), EtOAc (2.5 g), and BuOH (28.0 g) extracts, respectively.

Acid Hydrolysis and Sugar Identification
Compounds 1 and 2 (2 mg each) were heated in 3 mL 10% HCl (dioxane-H 2 O, 1:1) at 90 • C for 3 h.The residue was partitioned between EtOAc and H 2 O to give aglycone and sugar, respectively.The aqueous layer was evaporated until dry to yield a residue; this was dissolved in anhydrous pyridine (200 µL) and then mixed with a pyridine solution of 0.1 M L-cysteine methyl ester hydrochloride (200 µL).After warming to 60 • C for 1 h, trimethylsilylimidazole solution was added, and the reaction solution was warmed at 60 • C for 1 h.The mixture was evaporated in vacuo to yield a dried product, which was partitioned between n-hexane and H 2 O.The n-hexane layer was filtered and analyzed by gas chromatography.Retention times of the persilylated monosaccharide derivatives were as follows: D-glucose (t R , 14.11 min) was confirmed by comparison with those of authentic standards (Sigma-Aldrich, St. Louis, MO, USA).( 1 H-NMR (CD 3 OD, 600 MHz) and 13 C-NMR data (CD 3 OD, 150 MHz), see

Oxygen Radical Absorbance Capacity (ORAC) Assay
ORAC assay was carried out using a Tecan GENios multifunctional plate reader (Salzburg, Austria) with fluorescent filters (excitation wavelength: 485 nm, emission filter: 535 nm).In the final assay mixture, fluorescein (40 nM) was used as a target of free radical attack with AAPH (20 mM) as a peroxyl radical generator in the peroxyl radical-scavenging capacity assay.The analyzer was programmed to record fluorescein fluorescence every 2 min after AAPH had been added.All fluorescence measurements were expressed relative to the initial reading.Final values were calculated based on the difference in the area under the fluorescence decay curve between the blank and test sample.All data are expressed as net protection area (net area).Trolox (1 µM) was used as the positive control to scavenge peroxyl radicals [29].
3.8.Tartrate-Resistant Acid Phosphatase (TRAP) Assay TRAP Staining.RAW 264.7 cells (macrophages (pre-osteoclasts) from BALB/c mouse) were seeded in 12-well plates (3 × 10 4 cells/well) containing DMEM medium plus 10% FBS, and the medium was replaced with test samples in differentiation medium containing 50 ng/mL RANKL.The differentiation medium was changed every 2 days.After 5 days, the medium was removed, and the cell monolayer was gently washed twice using ice-cold PBS.The cells were fixed in 3.5% formaldehyde for 10 min and ethanol-acetone (1:1) for 1 min.Subsequently, the dried cells were incubated in 50 mM citrate buffer (pH 4.5) containing 10 mM sodium tartrate and 6 mM PNPP.After 1 h incubation, the reaction mixtures were transferred to new well plates containing an equal volume of 0.1 N NaOH.Absorbance was measured at 405 nm using an enzyme-linked immunoassay reader, and TRAP activity was expressed as the percent of the untreated control [31].

Statistical Analysis
All data represent the mean ± S.D. of at least three independent experiments performed in triplicates.Statistical significance is determined by one-way ANOVA followed by Dunnett's multiple comparison test, p < 0.05, using the SPSS 21 (IBM Crop., Armonk, NY, USA) program.

Conclusions
This study confirmed that the phenolic constituents of Z. piperitum stems have potentialities for antioxidant and anti-osteoporosis activities.When comparing the results of two activities, there was no significant correlation between antioxidant and anti-osteoporotic activities.Therefore, further study may be required to determine whether the significant anti-osteoporotic activities of compounds 3, 6-9, and 11 are indirectly related to the antioxidant activity.

Supplementary Materials:
The following are available online.1D/2D-NMR, CD, and HR-ESI-MS spectra of compounds 1 and 2.
* Overlapped signals; assignments were done by HMQC, HMBC, and NOESY experiments.a Measured at 600 MHz.b Measured at 150 MHz.

Table 2 .
The antioxidant activities of compounds isolated from the stems of Z. piperitum.

(10 μM) Peroxyl Radical-scavenging Capacity (TE, μM) a
All data are expressed as the mean ± standard deviation of three individual experiments.a Values are expressed as μM of Trolox equivalents (TE), one ORAC unit is equivalent to the net protection area provided by 1 μM of Trolox.

Table 2 .
The antioxidant activities of compounds isolated from the stems of Z. piperitum.
All data are expressed as the mean ± standard deviation of three individual experiments.a Values are expressed as μM of Trolox equivalents (TE), one ORAC unit is equivalent to the net protection area provided by 1 μM of Trolox.

Table 2 .
The antioxidant activities of compounds isolated from the stems of Z. piperitum.
All data are expressed as the mean ± standard deviation of three individual experiments.a Values are expressed as µM of Trolox equivalents (TE), one ORAC unit is equivalent to the net protection area provided by 1 µM of Trolox.

Table 3 .
Inhibitory effects of the isolated compounds on RANKL-induced osteoclast differentiation.a Inhibition of osteoclast differentiation was reflected in the reduction of TRAP activity.TRAP-positive multinucleated osteoclasts (control, obtained from RANKL-induced RAW 264.7 cells) served as a positive control, while untreated cells (untreated control, without RANKL induction) served as a negative control.Values are expressed as a percentage of the control (mean ± standard deviation, n = 3).