Lignans from the Flower Buds of Magnolia liliflora Desr.

• Abstract
• Abbreviation
• Introduction
• Materials and Methods
• Instruments and chemicals
• Plant material
• Extraction and isolation
• Animals
• Dosage and treatment
• Comet assay
• Statistical analysis
• Supporting information
• Results and Discussion
• Acknowledgements
• References

Abstract

Six new lignans, 1–6, along with six known compounds were obtained from the flower buds of Magnolia liliflora Desr. The new lignans were elucidated as (1S*,2R*,5S*,6S*)-2-(3,5-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (1), (1R*,2R*,5R*,6S*)-2-(3,5-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (2), (1R*, 2R*,5R*,6S*)-2,6-bis (3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (3), (1R*,2S*,5R*,6R*)-2-(3,4-methylenedioxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (4), (7′S*,8R*,8′R*)-3,5′-dimethoxy-3′,4,9′-trihydroxy-7′, 9-epoxy-8,8′-lignan (5), and (7′R*,8′S*)-3,3′,4,5′-tetramethoxy-7-en-7′,9-epoxy-8,8′-lignan (6), by the analysis of 1D and 2D-NMR as well as HRESIMS data. The capacity of compound 1 to protect against damages to the DNA of rat lymphocyte cells induced by UV irradiation was assessed by the comet assay. It showed stronger antigenotoxicity than ascorbic acid from 6 × 10⁻³ mmol · L⁻¹ to 6 × 10⁻⁶ mmol · L⁻¹.

Abbreviation

SCGE: single-cell gel electrophoresis

Introduction

Many species of the genus Magnolia are used as traditional medicines in China to treat chill and low blood pressure as well as cure headaches and nasal sinusitis [1]. Previous studies reported that lignans were the main constituents of this family, especially furofurans and substituted tetrahydrofurans, showing anti-PAF [2] and cytotoxic activities [3], [4], [5]. Magnolia liliflora is commonly called “Xinyi” by the Shezu people of southeastern China [6]. In order to make modern use of medicinal plants used by local people in ethnical areas of China, the chemical constituents of dry buds of M. liliflora were studied and reported here for the first time. Six new lignans, 1–6 ([Fig. 1]), along with six known compounds were isolated. Their structures were elucidated by extensive spectroscopic methods including IR, HRESIMS, and 1D and 2D-NMR experiments. Furthermore, in this paper we report the antigenotoxicity effect of compound 1 on the UV-induced DNA damage in rat lymphocytes by comet assays.

Materials and Methods

Instruments and chemicals

Melting points were measured on an X-6 micro-melting point apparatus. Specific rotations were determined by using a Jasco-P-1020 polarimeter. CD data were collected with a JASCO J-810 spectrometer. UV spectra were recorded on a Shimadzu-UV-2450 UV/VIS spectrophotometer. IR spectra were measured on a Bruker-VERTEX 70 FT-IR spectrometer (KBr). 1D and 2D-NMR spectra were recorded on a Bruker-AV-500 spectrometer using TMS as the internal reference. ESIMS analyses were performed on an Agilent-1100-LC/MSD-Trap SL. HRESIMS spectra were measured on a LCT Premier XE TOF mass spectrometer. Column chromatography was performed using silica gel (Qingdao Marine Chemical Factory, 200–300 mesh) or Sephadex LH-20 (GE Healthcare). TLC and preparative TLC were performed using precoated silica gel GF₂₅₄ plates (Qingdao Marine Chemical Factory, 200 × 200 × 1 mm or 200 × 200 × 2 mm). Slide glass used was of type CATNO.701 (Xinkang Medical Apparatus Ltd.). Ultrapure water was obtained by using MiLLi-Q (Millipore Company). Sartorius-Sigma Laboratory Centrifuges offered the use of a Sigma Sartorius 1–15 k. Ultraviolet lamp model used was SB-100P/F (American Spectronics Company). Electrophoresis tank was of a model DYCP-34A (Beijing Liuyi Instrument Factory). Electrophoresis apparatus No. JY-CZ5 was manufactured by Beijing Junyi-Dongfang Electrophoresis Equipment Ltd. The image analysis system used was CASP1.22 (Comet Assay Software Project) attached to a fluorescence microscope OLYMPUS CX41 (Olympus Optical Company). Ascorbic acid was purchased from China National Medicines Corporation Ltd. Lymphocytes separation medium was obtained from Beijing Solarbio Science and Technology Ltd. All analytical reagents (ether, NaCl, KCl, NaOH, KH₂PO₄, and Na₂HPO₄ · 12H₂O) were bought from Beijing Chemical Works. DMSO and normal melting point agarose were obtained from Shanghai YiTo Bio-Instrument Company. Low melting point agarose was purchased from Promega Company. Amersco Company offered Na₂EDTA, Tris-base, sodium N-lauroylsarcosine, etidium bromide, and trypan.

Plant material

The dry buds of M. liliflora were bought from Biological Products Co. Ltd., Ningbo Dekang, Zhejiang Province, P. R. China, in December 2008. The plant was identified by Associate Professor Lin Yang (College of Life and Environmental Sciences, Minzu University of China). A voucher specimen (No. 20081201) was deposited in the herbarium of the College of Life and Environmental Sciences, Minzu University of China.

Extraction and isolation

The dry buds of M. liliflora (3 kg) were pulverized and extracted 3 times (each for 7 days) with MeOH (total amount 10 L) at room temperature. After filtration, the extracts were combined and evaporated under vacuum. The residue (220 g) was chromatographed on silica gel (1500 g, column: 80 × 7.0 cm) with a petroleum ether-acetone gradient (stepwise, 20 : 1 to 0 : 1, 20 L), and finally MeOH. Each elution was examined by TLC and combined to afford 10 fractions (Frs.1–10). Fr.3 (3.5 g) was chromatographed on silica gel (350 g, column: 80 × 5 cm) with a mixture of petroleum ether-acetone (10 : 1, 3 L) to afford compound 1 (526.3 mg). Fr.5 (1.3 g) was separated over silica gel (200 g, column: 70 × 4 cm) with a mixture of petroleum ether-acetone (8 : 1, 2 L) to afford compound 7 (17.5 mg).

Fr.6 (3.7 g) was divided into three subfractions (Fr.6–1 to Fr.6–3). Fr.6–1 (1.5 g) was separated over silica gel (200 g, column: 70 × 4 cm) with a mixture of petroleum ether-acetone (8 : 1, 2 L) to afford compound 9 (195.0 mg). Fr.6–2 (1.78 g) was separated over silica gel (200 g, column: 70 × 4 cm) with a mixture of petroleum ether-acetone (5 : 1, 2 L), further purified by gel permeation chromatography on Sephadex LH-20 (column: 110 × 4 cm) in methanol (500 mL) and then put on 1 preparative TLC plate (200 × 200 × 1 mm, petroleum ether-acetone 2 : 1, R_f = 0.65) to afford compound 8 (32.1 mg). Fr.6–3 (0.37 g) was separated over silica gel (60 g, column: 70 × 2 cm) with a mixture of petroleum ether-acetone (3 : 1, 1 L), further purified by gel permeation chromatography on Sephadex LH-20 (column: 110 × 4 cm) in methanol (500 mL) and then put on a preparative TLC plate (200 × 200 × 1 mm GF₂₅₄, R_f = 0.55) to afford compound 12 (71.3 mg).

Fr.7 (5.1 g) was separated over silica gel (350 g, column: 80 × 5 cm) with a mixture of chloroform-acetone (15 : 1, 3 L) to yield four subfractions (Fr.7–1 to Fr.7–4). Fr.7–1 (1.38 g) was separated on 6 preparative TLC plates (200 × 200 × 2 mm GF₂₅₄) and chromatographed by chloroform-acetone 5 : 1 to get crude 6 and 10 (6: R_f = 0.80, 10: R_f = 0.50), respectively. All crude fractions containing 6 were combined and further purified on Sephadex LH-20 (column: 120 × 4 cm) in methanol (500 mL) to get compound 6 (5.8 mg). All crude fractions containing 10 were combined and further purified on Sephadex LH-20 (column: 120 × 4 cm) in methanol (500 mL) to get compound 10 (39.3 mg). Compound 2 (11.5 mg) was obtained from Fr.7–3 (1.2 g) by 5 preparative TLC plates (200 × 200 × 2 mm, chloroform-acetone 5 : 1, R_f = 0.65) and further purified on Sephadex LH-20 (column: 120 × 4 cm) in methanol (500 mL). Compound 5 (12.7 mg) was obtained from Fr.7–4 (1.1 g) by 5 preparative TLC plates (200 × 200 × 2 mm, chloroform-acetone 5 : 1, R_f = 0.55) and further purified on Sephadex LH-20 (column: 120 × 4 cm) in methanol (500 mL).

Fr.8 (6.3 g) was chromatographed on a silica gel (350 g, column: 80 × 5 cm) with a mixture of chloroform-methanol (20 : 1, 3 L) to yield three subfractions (Fr.8–1 to Fr.8–3). Compound 3 (5.7 mg) was obtained from Fr.8–1 (2.4 g) by 10 preparative TLC plates (200 × 200 × 2 mm, chloroform-methanol 10 : 1, R_f = 0.80) and further purified on Sephadex LH-20 (column: 120 × 4 cm) in methanol (500 mL). Compound 11 (3.4 mg) was obtained from Fr.8–2 (2.2 g) by 10 preparative TLC plates (200 × 200 × 2 mm, chloroform-methanol 10 : 1, R_f = 0.55) and further purified on Sephadex LH-20 (column: 120 × 4 cm) in methanol (500 mL). Compound 4 (6.3 mg) was obtained from Fr.8–3 (1.1 g) by 5 preparative TLC plates (200 × 200 × 2 mm, chloroform-methanol 10 : 1, R_f = 0.70) and further purified on Sephadex LH-20 (column: 120 × 4 cm) in methanol (500 mL).

(1S*,2R*,5S*,6S*)-2-(3,5-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (1): colorless crystal; m. p. 133–134 °C; [α]_D ²⁰ + 11.8 (c 0.12, CHCl₃); UV (CHCl₃) λ _max (log ε) = 257 (2.91), 296 (3.86) nm; IR (KBr) ν _max = 2856, 1590, 1514, 1244, 1029 cm⁻¹; CD (MeOH): 204 (Δε + 3.36), 211 (Δε − 4.57), 237 (Δε + 10.35), 290 (Δε − 2.76) nm; HRESIMS m/z: 371.1484 [M + H]⁺ (calcd. for C₂₁H₂₃O₆, calcd. mass for 371.1495); ¹H-NMR and ¹³C-NMR: [Table 1].

(1R*,2R*,5R*,6S*)-2-(3,5-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (2): colorless oil; [α]_D ²⁰ − 30 (c 0.15, CHCl₃); UV (MeOH) λ _max (log ε) = 239 (2.89), 297 (3.92) nm; IR (KBr) ν _max = 2863, 1579, 1456, 1221, 1027 cm⁻¹; CD (MeOH): 203 (Δε + 5.83), 211 (Δε − 3.12); HRESIMS m/z: 371.1487 [M + H]⁺ (calcd. for C₂₁H₂₃O₆, calcd. mass for 371.1495); ¹H-NMR and ¹³C-NMR: [Table 1].

(1R*,2R*,5R*,6S*)-2,6-bis(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (3): white amorphous solid; [α]_D ²⁰ − 43.3 (c 0.03, MeOH); UV (MeOH) λ _max (log ε) = 239 (3.40), 280 (3.97) nm; IR (KBr) ν _max = 2858, 1583, 1421, 1347, 1012 cm⁻¹; CD (MeOH): 201 (Δε + 4.92), 205 (Δε − 7.12); HRESIMS m/z: 387.1813 [M + H]⁺ (calcd. for C₂₂H₂₇O₆, calcd. mass for 387.1808); ¹H-NMR and ¹³C-NMR: [Table 1].

(1R*,2S*,5R*,6R*)-2-(3,4-methylenedioxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (4): white amorphous solid; [α]_D ²⁰ − 16.4 (c 0.06, MeOH); UV (MeOH) λ _max (log ε) = 231 (3.41), 284 (3.93) nm; IR (KBr) ν _max = 2854, 1586, 1427, 1335, 1021 cm⁻¹; CD (MeOH): 205 (Δε + 14.34), 298 (Δε − 1.02); HRESIMS m/z: 387.1797 [M + H]⁺ (calcd. for C₂₂H₂₇O₆, calcd. mass for 387.1808); ¹H-NMR and ¹³C-NMR: [Table 1].

(7′S*,8R*,8′R*)-3,5′-dimethoxy-3′,4,9′-trihydroxy-7′,9-epoxy-8,8′-lignan (5): purple oil; [α]_D ²⁰ − 6.2 (c 0.07, acetone); UV (MeOH) λ _max (log ε) = 239 (2.90), 298 (3.90) nm; IR (KBr) ν _max = 3215, 2878, 1613, 1317, 1211 cm⁻¹; HRESIMS m/z: 721.3218 [2 M + H]⁺ (calcd. for C₄₀H₄₉O₁₂, calcd. mass for 721.3224); ¹H-NMR and ¹³C-NMR: [Table 2].

(7′R*,8′S*)-3,3′,4,5′-tetramethoxy-7-en-7′,9-epoxy-8,8′-lignan (6): colorless oil; [α]_D ²⁰ − 26 (c 0.02, MeOH); UV (MeOH) λ _max (log ε) = 239 (2.96), 299 (4.03) nm; IR (KBr) ν _max = 2866, 1435, 1250, 1078 cm⁻¹; CD (MeOH): 250 (Δε + 1.71), 274 (Δε + 3.74), 320 (Δε − 1.97); HRESIMS m/z: 371.1805 [M + H]⁺ (calcd. for C₂₂H₂₇O₅, calcd. mass for 371.1858); ¹H-NMR and ¹³C-NMR: [Table 2].

Animals

Male Kunming mice (18–22 g) obtained from the Department of Health Science, Peking University, were housed under standard laboratory conditions at constant temperature (22 ± 2 °C temperature, 50 ± 10 % relative humidity, 12 h:12 h light/dark cycles). Commercial food pellets and tap water were freely available. All animal use procedures were in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People's Republic of China, 1988.

Dosage and treatment

The capacity of compound 1 to protect against DNA damage of rat lymphocyte cells induced by UV irradiation was assessed by the comet assay [7]. Peripheral blood mononuclear cells (PMNC) were isolated from 1 mL samples of the rat's heart blood, collected into tubes and mixed with 30 µL heparin. The blood was transferred to tubes and mixed with an equal volume of phosphate buffered saline (PBS) (pH = 7.4) to dilute. The diluted blood (0.6 mL) was aspirated and carefully added into a tube within an equal volume of lymphocytes separation medium. The mixture was separated into layers after centrifugation at 3000 RPS for 15 min, with the lymphocytes being found in the middle layer. This layer was then aspirated and transferred into a clean tube where PBS (pH = 7.4) was added and diluted to 1.2 mL, followed by new centrifugation at 3000 RPS for 10 min. The lymphocyte layer was extracted into a clean tube and then PBS was added to afford a final concentration of 106–107 cells/mL. The cell viability was counted and checked by trypan blue staining [8]. Ascorbic acid was used as a positive control [9]. DMSO was used as a solvent for delivering compound 1 and ascorbic acid. The viability of the lymphocytes always exceeded 90 % when the concentration of DMSO was less than 20 %. Cell suspension was exposed to the fluorescent sunlamp that emitted UV irradiation with a wavelength of 365 nm. A UV radiometer was used to determine the irradiance and was fixed at 4.2 mM/cm² [10]. The viability of the lymphocytes was found to surpass 90 % when the cell suspension was exposed to UV irradiation for less than 50 min. Compound 1 and the ascorbic acid used were also exposed to UV irradiation for 50 min as described previously, then dissolved in DMSO in a series of concentrations and added to the lymphocytes. After the cells were exposed to UV irradiation for 50 min, it was seen that the lymphocytes viability surpassed 90 % if the concentration of compound 1 was lower than 0.1 mM. A blank used as a control was made up of lymphocytes only exposed to UV irradiation.

Comet assay

Comet assays were performed under alkaline conditions according to the method previously reported [11]. Briefly, 100 µL of cell suspension were mixed with 200 µL of 0.75 % low melting point agarose and layered on a precoated slide with a thin layer of 0.75 % normal melting point agarose [12]. The slides were then placed into a lysis solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris-base, 35 mM sodium N-lauroylsarcosine, 0.2 M NaOH, and 20 % DMSO, pH = 10) at 4 °C for 2 h. Then the slides were washed with ultrapure water three times and placed into a horizontal gel electrophoresis tank filled with fresh alkaline buffer (300 mM NaOH and 1 mM Na₂EDTA, pH = 13) to unwind DNA for 40 min at 4 °C. Following this procedure, electrophoresis was performed for 20 min under 27 V. The slides were finally washed with neutralizing buffer (400 mM Tris-base, pH = 7.5) and then stained with etidium bromide [13]. Valuable data were acquired using a CASP1.22 image analysis system attached to an OLYMPUS CX41 fluorescence microscope and selected randomly for each condition. The cell parameters studied automatically generated the tail length, tail moment, and olive tail (tail length × percentage migrated DNA). Images from 100 cells were analyzed.

Statistical analysis

The data obtained was statistically analyzed using one-way ANOVA. Values were expressed as means ± standard error (SE). The results of the comet assay data were tested with the SPSS analysis system at p values less than 0.05. In all cases, the data indicated that compound 1 and ascorbic acid showed statistically significant effects on DNA as compared to the control blank.

Supporting information

The ¹H, ¹³C-NMR, and HRESIMS spectra of compounds 1 (Fig. 1S–7S), 2 (Fig. 8S–10S), 3 (Fig. 11S–13S), 4 (Fig. 14S–16S), 5 (Fig. 17S–19S), and 6 (Fig. 20S–22S) are available as Supporting Information.

Results and Discussion

The MeOH extract prepared from the flower buds of M. liliflora was subjected to column chromatography (CC) over silica gel and Sephadex LH-20 to afford compounds 1–12, which included six new lignans (1–6; [Fig. 1]), as well as six previously known and identified compounds isolated from other plants. Using their MS and NMR data as well as their observed optical activities as a comparison with data from published literature, the known compounds were identified as magnolin (7) [14], (+)-2α- (3′,4′-dimethoxyphenyl)-6α- (3′′-hydroxy-4′′,5′′-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (8) [15], lirioresinol-B dimethyl ether (9) [14], (−)-pinoresinol monomethyl ether (10) [16], 3′-O-demethylep ipinoresionl (11) [17], and (+)-pinoresinol (12) [18]. All compounds were isolated and identified from M. liliflora for the first time.

Compound 1 was obtained as a colorless crystal. The molecular formula was determined to be C₂₁H₂₂O₆ by the pseudomolecular ion peak [M + H]⁺ at m/z 371.1484 in HRESIMS, in accordance with the data in ¹³C and DEPT spectra ([Table 1]). The ¹H-NMR spectrum displayed a methylene dioxy (δ _H 5.97) and an ABX system [δ _H 6.79 (d, J = 7.9 Hz), δ _H 6.84 (dd, J = 7.9, 1.2 Hz), δ _H 6.90 (brs, J = 1.2 Hz)], as well as three single signals [δ _H 6.95, δ _H 6.88 (2H, s)] indicating 1,3,4-trisubstituted and 1,3,5-trisubsitituted aromatic rings. Using the ¹H-¹H COSY spectrum, correlation sequences of H-2/H-1/H-5 (H-8)/H-6 (H-4) supported the structure determination as 3,7-dioxabicyclo[3.3.0]octane. Assignments of ¹H and ¹³C-NMR data were based on the HMQC experiment ([Table 1]). The location of two phenyl groups on the furan were confirmed by cross peaks in HMBC between H-6 and C-1′, C-2′, and C-6′, respectively, as well as cross peaks between H-2 and C-1′′, C-2′′, and C-6′′, respectively ([Fig. 2]). Thus, the planar structure of compound 1 was elucidated as 2-(3,5-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, a specific type of lignan structure called furofuran.

In order to minimize ring tension, furofurans are biogenetically cis-blend [19]. It was reported by Kakisawa [20] that only one signal of H-1 and H-5, as well as only one signal of H-2 and H-6 could be observed in ¹H-NMR if the two phenyl groups were in the same side of the furofuran plane. On the contrary, if the two phenyl groups were in the opposite direction, four signals of H-1, H-5, H-2, and H-6 would appear respectively in ¹H-NMR. Thus, ¹H-NMR of compound 1 ([Table 1]) indicated the opposite stereo-relationship of the two phenyl groups ([Fig. 1]). Moreover, in 1D-NOE spectra, when H-5 was irradiated, the enhancement of H-2, as well as H-2′ and H-6′, was observed, showing that H-2 and the 1,3,4-trisubstitued benzene were in the cis-orientation of H-5 ([Fig. 3]). In this fashion, the relative stereochemistry of this compound was confirmed.

In addition to the same molecular formula, compound 1 showed almost the same NMR spectra with (+)-fargesin [21], except for the replacement of a 3,4-dimethoxyphenyl in (+)-fargesin by a 3,5-dimethoxyphenyl in 1. Moreover, considering the same specific rotation of these compounds, the stereochemistry of 1 should be consistent with that of (+)-fargesin. As a result, compound 1 was deduced as (1S*,2R*,5S*,6S*)-2-(3,5-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane.

Compound 2 was isolated as colorless oil. The molecular formula was assigned as C₂₁H₂₂O₆ by the pseudomolecular ion peak [M+H]⁺ at m/z 371.1487 in HRESIMS, which is the same as for 1. Meanwhile, the ¹H-NMR and ¹³C-NMR data implied a furofuran structure with the same planar configuration of 1 ([Table 1]), which was confirmed by HMBC. The four signals of H-1 and H-5, H-2 and H-6 in ¹H-NMR also indicated the two phenyl groups were on different sides of the furofuran. However, compound 2 showed to be levorotatory in the specific rotation spectrum. Thus it was deduced as (1R*,2R*,5R*,6S*)-2- (3,5-dimethoxyphenyl)-6- (3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane.

Compound 3 was obtained as a white amorphous solid. Its molecular formula was identified as C₂₂H₂₆O₆ by the pseudomolecular ion peak [M + H]⁺ at m/z 387.1813 in HRESIMS. There were 4 × CH₃, 2 × CH₂, 10 × CH, and 6 × C in the ¹³C-NMR and DEPT data, indicating it was also a furofuran ([Table 1]). The fragment of 3,7-dioxabicyclo[3.3.0]octane was confirmed by the correlation sequence H-2/H-1/(H-8)H-5/H-6 (H-8) in the ¹H-¹H COSY spectrum. In accordance with four methoxys [δ _H 3.85, δ _H 3.84, δ _H 3.82 (6H, s)] in the ¹H-NMR, the six singlets [δ _H 7.06, δ _H 7.00, δ _H 6.97, δ _H 6.95, δ _H 6.93, δ _H 6.92] showed two 3,5-dimethoxyphenyl groups. The assignment of ¹H and ¹³C-NMR data was confirmed by HMQC and HMBC spectra ([Table 1]). As there were four peaks of H-1 and H-5, H-2 and H-6 ([Table 1]) in ¹H-NMR, the two phenyl groups should be in the opposite direction in space. In 1D-NOE spectrum, the irradiation of H-1 led to the enhancement of H-6, H-2′′ and H-6′′, which were attributed to the cis-orientation between H-1 and H-6, as well as trans-orientation between H-1 and H-2 ([Fig. 3]). Moreover, the specific rotation of compound 3 was also levorotatory, suggesting that the stereochemistry of 3 was the same as the one of 2. Eventually, it was assigned as (1R*,2R*,5R*,6S*)-2,6-bis (3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane.

Compound 4 was obtained as a white amorphous solid. Its molecular formula was identified as C₂₂H₂₆O₆ by the pseudomolecular ion peak [M + H]⁺ at m/z 387.1797 in HRESIMS, which is the same as for compound 3. The ¹H-NMR and ¹³C-NMR spectra implied a furofuran ([Table 1]). In the ¹H-NMR spectrum, in addition to three aromatic singlets [δ _H 6.99, δ _H 6.94 (2H, s)], there was an ABX system [δ _H 6.90 (dd, J = 8.2, 1.3 Hz), δ _H 6.85 (d, J = 8.2 Hz), δ _H 6.94 (brs, J = 1.3 Hz)], indicating a 3,5-dimethoxyphenyl group and a 3,4-dimethoxyphenyl group, respectively. The cross peak in HMBC between H-6 and C-1′, C-2′, and C-6′, as well as cross peaks between H-2 and C-1′′, C-2′′ and H-6′′ confirmed the position of two phenyl groups on the furofuran. The enhancement of H-2′, H-6′, and H-2 observed in 1D-NOE spectra, while H-1 was irradiated, suggested that the phenyl group attached on C-6 was in the cis-orientation of H-1, and the phenyl group on C-2 was in the trans-orientation of H-1. In combination with the specific rotation data of compound 4, the compound was assigned as (1R*,2S*,5R*,6R*)-2- (3,4-methylenedioxyphenyl)-6- (3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane.

The molecular formula C₂₀H₂₄O₆ of compound 5 was determined by the pseudomolecular ion peak [2 M + H]⁺ at m/z 721.3218 in HRESIMS. Its IR spectrum exhibited a hydroxy group at 3215 cm⁻¹. In addition to ABX system signals [δ _H 6.66 (dd, J = 8.0, 1.6 Hz), δ _H 6.74 (d, J = 8.0 Hz), δ _H 6.84 (brs, J = 1.6 Hz)], three aromatic single peaks in ¹H-NMR ([Table 2]) showed a 3,4-disubstituted phenyl group and a 3,5-disubstituted phenyl group, respectively. Moreover, in the ¹H-¹H COSY spectrum, the sequence of H-9/H-8/H-8′/H-7 indicated a tetrahydrofuran ring, showing that the compound should be a kind of lignan called tetrahydrofuran. 2 × CH₃, 3 × CH₂, 9 × CH, and 6 × C were observed in the ¹³C-NMR and DEPT spectra, which was assigned to ¹H-NMR by the analysis of HMQC ([Table 2]). The positions of the benzyl and aryl groups was confirmed by the key correlations between C-1′ and H-2′, H-6′ and H-7′, and correlations between C-1 and H-7, H-2 and H-6 in HMBC, respectively ([Fig. 2]).

In the 1D-NOE spectra, the irradiation of H-7′ led to the enhancement of H-8 and H-8′ ([Fig. 3]), which showed that all of them were oriented in the same direction. Compound 5 was then identified as (7′S*,8R*,8′R*)-3,5′-dimethoxy-3′,4,9′-trihydroxy-7′,9-epoxy-8,8′-lignan.

Compound 6 was isolated as colorless oil. The molecular formula was deduced as C₂₂H₂₆O₅ by the pseudomolecular ion peak [M + H]⁺ at m/z 371.1805 in HRESIMS. The degree of unsaturation was found to be 10. Four methoxy [δ _H 3.85, δ _H 3.83 (6H, s), δ _H 3.82], and an ABX system [δ _H 6.96 (dd, J = 8.3, 1.2 Hz), δ _H 6.79 (d, J = 8.3 Hz), δ _H 6.95 (brs, J = 1.2 Hz)] as well as three singlets in aromatic fields [δ _H 7.07, δ _H 6.95, δ _H 6.85] in its ¹H-NMR spectrum indicated the existence of a 3,4-dimethoxyphenyl group and a 3,5-dimethoxyphenyl group, respectively. A trisubstituted double bond C-7 (δc 119.49) and C-8 (δc 144.30) could be found in the ¹³C-NMR and DEPT spectra ([Table 2]). In consideration of the observed degree of unsaturation, there should be a ring in the compound. In combination with the correlation sequence H-7′/H-8′/H-9′ in the ¹H-¹H COSY spectrum, the cross peak between C-8 and H-9 and H-8′ in HMBC supported the assignment of a tetrahydrofuran unit in 6. Furthermore, the benzyl and aryl groups were attached to the tetrahydrofuran by the key cross peaks in HMBC ([Fig. 2]). In the 1D-NOE spectra, the irradiation of H-7′ led to the enhancement of H-9′, showing that they were in the same direction ([Fig. 3]). Compound 6 then was identified as (7′R*,8′S*)-3,3′,4,5′-tetramethoxy-7-en-7′,9-epoxy-8,8′-lignan.

The comet assays are based on the quantification of denatured DNA fragments migrating out of the cell nucleus during electrophoresis. The resulting image obtained is a “comet” with a distinct head consisting of intact DNA and a tail that contains damaged or broken pieces of DNA [22] ([Fig. 4]).

When the concentration of compound 1 was lower than 6 × 10⁻³ mmol · L⁻¹, the migration of DNA from the nucleus of cells was less than that of the control blank, indicating that it was capable of protecting DNA from the damage induced by UV irradiation in cells. It was found that the protective effect of ascorbic acid upon DNA migration was dose-dependent. However, compound 1 showed a stronger protective effect as the concentration decreased from 6 × 10⁻³ mmol · L⁻¹ to 6 × 10⁻⁶ mmol · L⁻¹. The concentration which provided the strongest protection to DNA was observed at 6 × 10⁻⁵ mmol · L⁻¹ ([Table 3] and [Fig. 5]). The result indicates that compound 1 has antigenotoxic activity but probably due to a different mechanism than the one of ascorbic acid.

Acknowledgements

The project was supported by the 985 Project (MUC98504-14, MUC98507-08) Minzu University of China, Major Project for Young Teachers in Minzu University of China CUN10A, together with the “Programme of Introducing Talents of Discipline to Universities” (B08044), and the “Project for Scientific and Technical Achievements in Industrialization”, Beijing Education Commission.

The authors would like to acknowledge the work of Dr. Chunlin Long, Dr. Edward J. Kennelly, and Adam Negrin in reviewing and editing this manuscript in preparation for publication.

Conflict of Interest

All authors agreed to the publishing of the manuscript in this journal. Moreover, the sequence of authors was listed according to the contribution of each. There were no conflicts between any of these authors.

References

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  Search in Google Scholar
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• 20 Kakisawa H, Chen Y P, Hsij H Y. Lignans in flower buds of Magnolia fargesii.  Phytochemistry. 1972;  11 2289-2293
  Search in Google Scholar
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  Search in Google Scholar
• 22 Mustafayeva K, Di Giorgio C, Elias R, Kerimov Y, Ollivier E, De Méo M. DNA-damaging, mutagenic, and clastogenic activities of gentiopicroside isolated from Cephalaria kotschyi roots.  J Nat Prod. 2010;  73 99-103
  Search in Google Scholar

Dr. Wen-Shu Wang

College of Life and Environmental Sciences
Minzu University of China

Zhongguancun South Avenue 27#

Beijing 100081

P. R. China

Phone: +86 10 68 93 22 42

Fax: +86 10 68 93 69 27

Email: wangws@muc.edu.cn

References

• 1 Liu Y H, Luo X R, Wu Y F. Flora of China. Vol. 30: Menispermaceae, Magnoliaceae. Beijing: Science Press; 1996: 140-141
  Search in Google Scholar
• 2 Jung K Y, Kim D S, Oh S R, Park S H, Lee I S, Lee J J, Shin D H, Lee H K. Magnone A and B, novel anti-PAF tetrahydrofuran lignans from the flower buds of Magnolia fargesii.  J Nat Prod. 1998;  61 808-811
  Search in Google Scholar
• 3 Ho K Y, Tsai C C, Chen C P, Huang J S, Lin C C. Antimicrobial activity of honokiol and magnolol isolated from Magnolia officinalis.  Phytother Res. 2001;  15 139-141
  Search in Google Scholar
• 4 Syu W J, Shen C C, Lu J J, Lee G H, Sun C M. Antimicrobial and cytotoxic activities of neolignans from Magnolia officinalis.  Chem Biodivers. 2004;  1 530-537
  Search in Google Scholar
• 5 Youn U J, Chen Q C, Jin W Y, Lee I S, Kim H J, Lee J P, Chang M J, Min B S, Bae K H. Cytotoxic lignans from the stem bark of Magnolia officinalis.  J Nat Prod. 2007;  70 1687-1689
  Search in Google Scholar
• 6 Li D Q. Pharmacopoeia of the P.R.C. Beijing: Chemical and Technologic Press; 2005: 126-127
  Search in Google Scholar
• 7 Tice R R, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu J C, Sasaki Y. The single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.  Env Mol Mutagen. 2000;  35 206-221
  Search in Google Scholar
• 8 Kimura T, Sonoda Y, Iwai N, Satoh M, Yamaguchi M, Izui T, Suda M, Sasaki K, Nakano T. Proliferation and cell death of embryonic primitive erythrocytes.  Exp Hematol. 2000;  28 635-641
  Search in Google Scholar
• 9 Yu P, Wang X L, Yan Q C. Study on protection of procyanidins against UV-induced oxidative damage of lens epitheliaI cells.  Int J Ophthalmol. 2010;  10 1477-1480
  Search in Google Scholar
• 10 Lyons N M, O'Brien N M. Modulatory effects of an algal extract containing astaxanthin on UVA-irradiated cells in culture.  J Dermatol Sci. 2002;  30 73-84
  Search in Google Scholar
• 11 Singh N P, McCoy M T, Tice R R, Schneider E L. A simple technique for quantitation of low levels of DNA damage in individual cells.  Exp Cell Res. 1988;  175 184-191
  Search in Google Scholar
• 12 Agner A R, Bazo A P, Ribeiro L R, Salvadori D M. DNA damage and aberrant crypt foci as putative biomarkers to evaluate the chemopreventive effect of annatto (Bixa orellana L.) in rat colon carcinogenesis.  Mutat Res. 2005;  582 146-154
  Search in Google Scholar
• 13 Méndez-Robles M D, Permady H H, Jaramillo-Flores M E, Lugo-Cervantes E C, Cardador-Martínez A, Canales-Aguirre A A, López-Dellamary F, Cerda-García-Rojas C M, Tamariz J. C-26 and C-30 apocarotenoids from seeds of Ditaxis heterantha with antioxidant activity and protection against DNA oxidative damage.  J Nat Prod. 2006;  69 1140-1144
  Search in Google Scholar
• 14 Chen C C, Huang Y L, Chen H T, Chen Y P, Hsu H Y. On the calcium-antagonistic principles of the flower buds of Magnolia fargesii.  Planta Med. 1988;  54 438-440
  Search in Google Scholar
• 15 Seo S M, Lee H J, Lee O K, Jo H J, Kang H Y, Choi D H, Paik K H, Khan M. Furofuran lignans from the bark of Magnolia kobus.  Chem Nat Comp. 2008;  44 419-423
  Search in Google Scholar
• 16 Miyazawa M, Kasahara H, Kameoka H. Biotransformation of lignans: metabolism of (+)-eudesmin and (+)-magnolin in Spodoptera litura.  Phytochemistry. 1995;  39 1027-1030
  Search in Google Scholar
• 17 Yang G Z, Hu Y, Yang B, Chen Y. Lignans from the bark of Zanthoxylum planispinum.  Helv Chim Acta. 2009;  92 1657-1664
  Search in Google Scholar
• 18 Xie L H, Akao T, Hamasaki K, Deyama T, Hattori M. Biotransformation of pinoresinol diglucoside to mammalian lignans by human intestinal microflora, and isolation of Enterococcus faecalis strain PDG-1 responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol.  Chem Pharm Bull. 2003;  51 508-515
  Search in Google Scholar
• 19 Xu R S, Ye Y, Zhao W M. Introduction to Natural Products Chemistry. Beijing: Science Press; 2006: 318-320
  Search in Google Scholar
• 20 Kakisawa H, Chen Y P, Hsij H Y. Lignans in flower buds of Magnolia fargesii.  Phytochemistry. 1972;  11 2289-2293
  Search in Google Scholar
• 21 Yoshida S, Yamanaka T, Miyake T, Moritani Y, Ohmizu H, Iwasaki T. Asymmetric syntheses of lignans utilizing novel diastereoselective Michael addition of cyanohydrin: syntheses of (+)-fargesin and (−)-picropodophyllone.  Tetrahedron. 1997;  53 9585-9598
  Search in Google Scholar
• 22 Mustafayeva K, Di Giorgio C, Elias R, Kerimov Y, Ollivier E, De Méo M. DNA-damaging, mutagenic, and clastogenic activities of gentiopicroside isolated from Cephalaria kotschyi roots.  J Nat Prod. 2010;  73 99-103
  Search in Google Scholar

Dr. Wen-Shu Wang

College of Life and Environmental Sciences
Minzu University of China

Zhongguancun South Avenue 27#

Beijing 100081

P. R. China

Phone: +86 10 68 93 22 42

Fax: +86 10 68 93 69 27

Email: wangws@muc.edu.cn

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