Japonicasins A and B, two new isoprenylated flavanones from Sophora japonica

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Lai-Bin Zhang a, Jie-Li Lv a,⁎, Hong-Li Chen b a School of Pharmacy, Xinxiang Medical University, Xinxiang 453003, People's Republic of China b School of Life Science and Technology, Xinxiang Medical University, Xinxiang 453003, People's Republic of China a r t i c l e i n f o a b s t r a c t Article history: Received 20 January 2013 Accepted in revised form 17 March 2013 Available online 2 April 2013 Two new flavanones with a C15 isoprenoid group, japonicasins A and B (1 and 2), were isolated from the leaves of Sophora japonica. This is the first report on the presence of the (2E,7E)-6-isopropyl-3,9-dimethyldeca-2,7,9-trien-1-yl group (C15 isoprenoid group) in isoprenylated flavonoids. Their structures were determined by spectroscopic methods, including UV, IR, 1D and 2D NMR, HRESIMS, and CD experiments. In addition, the antioxidant activities of compounds 1 and 2 were determined through DPPH radical scavenging assays. They exhibited potential antioxidant activities, with IC50 values of 35.1 ± 0.8 μM and 88.7 ± 1.1 μM for compounds 1 and 2, respectively. © 2013 Elsevier B.V. All rights reserved. Keywords: Leguminosae Sophora japonica Isoprenylated flavanone Antioxidant activity 1. Introduction The genus Sophora (Leguminosae) includes about 70 species worldwide and occurs mainly in the tropical and temperate zones. There are 21 species, 14 varieties and 2 forms grown in China [1]. Sophora japonica L. is widely cultivated in all parts of China, whose buds and fruits have been used as a hemostatic agent in traditional Chinese medicine [2]. Previous phytochemical studies indicated that flavoniods were the main chemical constituents of S. japonica, which have been isolated from its seeds, fruits, stem barks, woods, and leaves. In addition, some of these flavoniods showed the activities of anti-platelet aggregating, antioxidant and aldose reductase inhibitory [3–7]. In the present study, two new isoprenylated flavanones, japonicasins A and B (1 and 2), were isolated from an ethanol extract of the leaves of S. japonica. Both of them possess a (2E,7E)-6-isopropyl-3,9-dimethyldeca-2,7,9-trien-1-yl group (C15 isoprenoid group), which has not been reported on the presence in isoprenylated flavonoids. Herein, we reported the isolation and structural identification of two new compounds. Moreover, 1,1-diphenyl-2-picrylhydrazyl (DPPH) was used to assay their antioxidant activities. 2. Experimental 2.1. General experimental procedures Optical rotations were measured on a JASCO P-1020 polarimeter. UV spectra were recorded on a Hitachi U-2900 spectrophotometer. CD spectra were obtained on a JASCO J-810 spectrometers. IR spectra were measured on a Nicolet Avatar-360 spectrometer with KBr pellets. NMR spectra were obtained on Varian Mercury Plus 400 instruments. Chemical shifts were reported with TMS as internal standard or with respect to acetone-d6 (δH 2.04, δC 206.0 ppm). Mass spectra were recorded on Agilent LC/MSD and Agilent Q-TOF mass spectrometers. Column chromatography (CC) was performed on silica gel (200–300 mesh, Yantai Institute of Chemical Technology, Yantai, People's Republic of China) and Sephadex LH-20 gel (GE Healthcare Amersham Biosciences, Uppsala, Sweden). TLC analysis was run on precoated silica gel GF254 plates (10–40 μM, Yantai Institute of Chemical Technology). 1,1-Diphenyl-2-picrylhydrazyl Fitoterapia 87 (2013) 89–92 ⁎ Corresponding author. Tel./fax: +86 373 3831652/3029879. E-mail address: ruoxin0371@163.com (J.-L. Lv). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.03.019 Contents lists available at SciVerse ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote (DPPH) and α-tocopherol were purchased from Sigma Chemical Company. 2.2. Plant material The leaves of S. japonica were collected in July 2008 from Henan province, People's Republic of China, and identified by Prof. Sui-Qing Chen (School of Pharmacy, Henan University of Traditional Chinese Medicine). A voucher specimen (SJ200807) has been deposited at the Herbarium, School of Pharmacy, Xinxiang Medical University. 2.3. Extraction and isolation The air-dried, powdered leaves of S. japonica (9.5 kg) were extracted with 90% ethanol three times (3 × 100 L) at room temperature. The solvent was removed under reduced pressure to give a residue (1050 g), which was suspended in H2O and successively partitioned with petroleum ether, CHCl3 and EtOAc, respectively. The CHCl3 extract (110 g) was subjected to column chromatography (CC) on silica gel eluted with a gradient of petroleum ether–EtOAc (1:0, 10:1, 5:1, 2:1, 1:1, 1:2) to give fractions A–I. Fraction D was isolated by CC on silica gel eluted with a gradient of petroleum ether–EtOAc (40:1 → 1:1) to yield fractions D1–D5. Fraction D3 was purified by CC on Sephadex LH-20 eluted with MeOH to yield compound 2 (6 mg). Fraction E was separated by CC on silica gel eluted with a gradient of CH2Cl2–Me2CO (20:1 → 1:1) to afford fractions E1–E6. Compound 1 (8 mg) was afforded from fraction E4 by CC on Sephadex LH-20 eluted with MeOH. Japonicasin A (1), yellowish gum; [α]25D −33.5 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 227 (4.01), 297 (3.82) nm; CD (MeOH, nm) λmax (Δε) 291 (−1.77), 335 (+0.41); IR (KBr) νmax 3431, 2927, 1642, 1601, 1447, 1287, 1159, 1113, 821 cm−1; 1H NMR and 13C NMR spectroscopic data, see Table 1; ESIMS m/z 515 [M + Na]+; HRESIMS m/z 515.2412 [M + Na]+ (calcd for C30H36O6Na, 515.2409). Japonicasin B (2), yellowish gum; [α]25D −24.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 224 (4.07), 286 (3.84) nm; CD (MeOH, nm) λmax (Δε) 289 (−1.62), 331 (+0.37); IR (KBr) νmax 3426, 2925, 1637, 1603, 1451, 1283, 1156, 1117, 817 cm−1; 1H NMR and 13C NMR spectroscopic data, see Table 1 NMR spectroscopic data of compounds 1 and 2 (multi, J in Hz). position 1a 2a δH δC δH δC 2 5.36 (dd, 3.1, 12.8) 79.9 5.42 (dd, 3.1, 12.9) 79.9 3 2.71 (dd, 3.1, 17.1) 3.12 (dd, 12.8, 17.1) 43.7 2.72 (dd, 3.1, 16.8) 3.17 (dd, 12.9, 16.8) 43.6 4 197.3 197.3 5 162.3 162.3 6 109.0 109.0 7 164.7 164.8 8 6.04 (s) 95.2 6.05 (s) 95.2 9 161.9 162.0 10 103.1 103.1 1′ 131.7 130.9 2′ 6.86 (br s) 119.2 7.39 (d, 8.6) 129.0 3′ 146.3 6.89 (d, 8.6) 116.1 4′ 7.03 (br s) 114.7 158.6 5′ 145.9 6.89 (d, 8.6) 116.1 6′ 6.86 (br s) 115.9 7.39 (d, 8.6) 129.0 1″ 3.26 (br d, 7.3) 21.5 3.26 (br d, 7.1) 21.5 2″ 5.22 (br t, 7.3) 123.5 5.22 (br t, 7.1) 123.5 3″ 135.0 134.9 4″ 1.75 (br s) 16.1 1.75 (br s) 16.1 5″ 1.97 (m) 1.85 (overlap) 38.3 1.97 (m) 1.85 (overlap) 38.3 6″ 1.33 (m) 1.60 (overlap) 31.2 1.33 (m) 1.60 (m) 31.2 7″ 1.82 (overlap) 49.6 1.82 (overlap) 49.6 8″ 1.61 (overlap) 32.7 1.60 (m) 32.7 9″ 0.82 (d, 7.0)b 19.4b 0.82 (d, 7.0)b 19.4b 10″ 0.85 (d, 6.7)b 21.1b 0.85 (d, 7.1)b 21.1b 11″ 5.44 (dd, 9.8, 15.9) 133.5 5.44 (dd, 9.7, 15.8) 133.5 12″ 6.03 (d, 15.9) 134.9 6.03 (d, 15.8) 134.9 13″ 142.9 142.9 14″ 4.83 (br s) 4.81 (br s) 114.6 4.83 (br s) 4.81 (br s) 114.6 15″ 1.81 (br s) 18.9 1.81 (br s) 18.9 OH-5 12.48 (s) 12.49 (s) OH-7 –c 9.76 (br s) OH-3′ –c OH-4′ 8.58 (br s) OH-5′ –c a Data were measured at 400 MHz for 1H NMR and 100 MHz for 13C NMR in actone-d6. b The assignments are exchangeable. c Signal wasn't observed. 7 6 5 10 9 8 4 3 2 1 1' 1'' 2'' 3'' 5'' 4'' 6'' 7'' 10'' 8'' 9'' 6' 5' 4' 3' 2' 12'' 11'' 13'' 14'' 15'' 1 2 3 1 3 2 1 3 2 Fig. 1. Structures of compounds 1 and 2 from Sophora japonica. 90 L.-B. Zhang et al. / Fitoterapia 87 (2013) 89–92 Table 1; ESIMS m/z 499 [M + Na]+; HRESIMS m/z 499.2464 [M + Na]+ (calcd for C30H36O5Na, 499.2460). 2.4. DPPH radical scavenging assay The assay was carried out according to the method in the literature [8,9]. The reaction mixture containing 10 μL of sample solution (different concentrations in ethanol) and 190 μL of DPPH (300 μM) in ethanol was taken in a 96-well micro liter plate and incubated at 37 °C for 30 min. The absorbance was measured at 517 nm. Percent radical scavenging activity was determined by comparison with an ethanol containing control. Inhibition percentage (%IP) was derived from the equation: %IP ¼ A ½   ð Þ c−As =Ac 100 where A c and As are the absorbencies of the control and of the test sample, respectively. IC50 values represent the concentration of compounds to scavenge 50% of DPPH radicals and are expressed as means ± SD of three separate experiments. α-Tocopherol was used as a positive control. 3. Results and discussion Compound 1 was isolated as a yellowish gum. Its molecular formula C30H36O6 was determined by HRESIMS [M + Na]+ at m/z 515.2412 (calcd. 515.2409). The IR spectrum showed absorptions for OH (3431 cm−1), carbonyl (1642 cm−1) and aromatic (1601 and 1447 cm−1) groups. The UV spectrum resembled that of a flavanone [10]. The 1H NMR spectrum of 1 (Table 1) showed an ABX coupling system signals at δH 2.71 (1H, dd, J = 3.1, 17.1 Hz, H-3a), 3.12 (1H, dd, J = 12.8, 17.1 Hz, H-3b) and 5.36 (1H, dd, J = 3.1, 12.8 Hz, H-2), which can be typically assigned to H-2 and H2-3 of a flavanone skeleton. The 1H NMR spectrum of 1 also exhibited a hydrogen-bonded hydroxy proton signal at δH 12.48 (1H, s, OH-5) that can be assigned to the OH-5 group, as further confirmed on the basis of the correlations between δH 12.48 (OH-5) and δC 162.3 (C-5), 109.0 (C-6) and 103.1 (C-10) in the HMBC spectrum (Fig. 2). Meanwhile, the A ring displayed only one aromatic singlet at δH 6.04 (1H, s, H-8) that showed the HMBC correlations with four quaternary carbons as C-6 (δC 109.0), C-7 (δC 164.7), C-9 (δC 161.9), and C-10 (δC 103.1). These data suggested the presence of a 5,6,7-trisubtituted A ring. Additionally, ring B was assigned as a 1′,3′,5′-trisubstituted benzene ring on the basis of the 1H NMR signals of three meta-coupled aromatic protons at δH 6.86 (2H, br s, H-2′, H-6′) and 7.03 (1H, br s, H-4′) as well as HMBC correlations from H-2′ to C-2, C-1′ and C-4′, from H-4′ to C-2′, C-3′, C-5′, and C-6′, and from H-6′ to C-2, C-1′, C-4′, and C-5′. The chemical shifts for C-3′ (δC 146.3) and C-5′ (δC 145.9) were consistent with the substitution of hydroxyl groups at these positions. While ring B is symmetric in its substitution pattern, the nonequivalence in chemical shift of these two carbons as well as C-2′ (δC 119.2) and C-6′ (δC 115.9) indicates that there is restricted rotation of this ring. Similar NMR spectroscopic data have been reported for other flavanones with the same substituted B ring [11,12]. In the 13C NMR spectrum of 1 (Table 1), 30 carbon signals were observed. Among them, the signals at δC 79.9, 43.7 and 197.3 are characteristic for C-2, C-3 and C-4 of a flavanone skeleton. The above spectroscopic data, therefore, suggested that 1 is a flavanone containing a C15 side chain. The C15 side chain was deduced to be a 6-isopropyl-3,9-dimethyldeca- 2,7,9-trien-1-yl group (C15 isoprenoid group) according to the HMBC correlations from H-2′′ to C-1′′, C-4′′ and C-5′′, from H3-4′′ to C-2′′, C-3′′ and C-5′′, from H-7′′ to C-5′′, C-6′′, C-9′′, and C-12′′, from H3-10′′ to C-7′′, C-8′′ and C-9′′, from H-11′′ to C-7′′ and C-13′′, and from H3-15′′ to C-12′′, C-13′′ and C-14′′. The C15 isoprenoid group was located at C-6 on the basis of the HMBC correlations from H2-1′′ to C-5, C-6 and C-7. The coupling constant of H-11′′/H-12′′ (J = 15.9 Hz) suggested the E-configuration of the double bond at C-11′′/C-12′′. In addition, the E-configuration of the double bond at C-2′′/C-3′′ was established by the ROESY correlations (Fig. 2) of H2-1′′/H3-4′′ and H-2′′/H2-5′′. The absolute configuration at C-2 was established as S based on a negative Cotton effect at 291 nm and a positive Cotton effect at 335 nm in the CD spectrum of 1 [13]. However, the absolute configuration at C-7′′ was uncertain, for compound 1 could be a mixture of diastereomers. Thus, the structure of compound 1 was elucidated as (2S)-5,7,3′,5′-tetrahydroxy-6-[(2E,7E)-6- isopropyl-3,9-dimethyldeca-2,7,9-trien-1-yl]flavanone, which was named japonicasin A (Fig. 1). The molecular formula of compound 2 was assigned as C30H36O5 by HRESIMS [M + Na]+ at m/z 499.2464 (calcd. 499.2460). The 1H and 13C NMR spectroscopic data of 2 was found to be similar to that of compound 1 except the chemical shifts in the B ring (Table 1). In the 1H NMR spectrum of 2, signals at δH 6.89 (2H, d, J = 8.6 Hz, H-3′, H-5′) and 7.39 (2H, d, J = 8.6 Hz, H-2′, H-6′) suggested the Fig. 2. Key HMBC and ROESY correlations of compound 1. L.-B. Zhang et al. / Fitoterapia 87 (2013) 89–92 91 presence of 4′-hydroxylated B ring. Similarly, the absolute configuration at C-2 was established as S based on a negative Cotton effect at 289 nm and a positive Cotton effect at 331 nm in the CD spectrum of 2 [13]. Thus, the structure of compound 2 was elucidated as (2S)-5,7,4′-trihydroxy-6-[(2E,7E)-6-isopropyl- 3,9-dimethyldeca-2,7,9-trien-1-yl] flavanone, which was named japonicasin B. Compounds 1 and 2 are two new isoprenylated flavanones with a C15 isoprenoid group [(2E,7E)-6-isopropyl- 3,9-dimethyldeca-2,7,9-trien-1-yl group]. To the best of our knowledge, this is the first time that the C15 isoprenoid group occurs in the side chain of flavonoids. The isolated compounds (1 and 2) were screened antioxidant activities using DPPH radical scavenging assay. Compound 1 showed significant antioxidant activity with the IC50 value of 35.1 ± 0.8 μM compared to the positive control, α-tocopherol (IC50 = 21.2 ± 0.9 μM). Similarly, compound 2 showed moderate antioxidant activity with the IC50 value of 88.7 ± 1.1 μM. The antioxidant activity of compound 1 was stronger than that of compound 2, which may be related to the presence of two hydroxyl groups in the B ring of compound 1 [14]. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (no. 81202916), the Project for Provincial-level Key Disciplines of Xinxiang Medical University (no. ZD200967), and by the High Educated Talents Fund of Xinxiang Medical University. References [1] Ma QY. Zhongguo zhi wu zhi, vol. 40. Beijing, China: Science Press; 199464–5. [2] Wang JH, Lou FC, Wang YL, Tang YP. A flavonol tetraglycoside from Sophora japonica seeds. Phytochemistry 2003;63:463–5. [3] Kim MJ, Yun-Choi HS. Anti-platelet effects of flavonoids and flavonoidglycosides from Sophora japonica. Arch Pharm Res 2008;31(7):886–90. [4] Park HY, Kim SH, Kim GB, Sim JY, Lim SS, Kim MJ, et al. A new isoflavone glycoside from the stem bark of Sophora japonica. Arch Pharm Res 2010;33(8):1165–8. 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