Two new 1,4-naphthoquinone derivatives from Impatiens balsamina L. flowers

Phytochemistry Letters 14 (2015) 8–11

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Two new 1,4-naphthoquinone derivatives from Impatiens balsamina L. flowers Qian Lia,b , Zhenghong Guoa,b , Kaibo Wanga,b , Xiaoshu Zhanga,b , Yuntian Loua,b , Yu-qing Zhaoa,b,* a

School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 June 2015 Received in revised form 28 July 2015 Accepted 18 August 2015 Available online xxx

Two new 1,4-naphthoquinone derivatives, balsaminone D (1), balsaminone E (2) along with two known compounds (3 and 4) were discovered from Impatiens balsamina L. flowers. Their structures were identified with spectroscopic methods including HR–EI–MS, 1D and 2D NMR, as well as the absolute configuration was determined by ECD calculation. In addition, new compounds 1 and 2 with IC50 value of 30.54 and 40.67 mg/mL exhibited better activities against activated t-HSC/Cl-6 cells than positive control Silymarin and Fufang Biejia Ruangan Pian, of which the IC50 value were 202.34 and 231.56 mg/mL, respectively. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Impatiens balsamina L. 1,4-Naphthoquinone Anti-hepatic fibrosis activity Electronic circular dichroism (ECD)

1. Introduction Impatiens balsamina (Garden Balsam or Rose Balsam) is an annual herb that has been used in Asian traditional medicine (Fransworth and Bunyapraphatsara, 1992). The aerial parts of I. balsamina are traditionally used for the treatment of articular rheumatism, bruises and beriberi (Ishiguro et al., 1992). Flowers are used to treat lumbago, neuralgia, burns and scalds (Ghani, 2003). Pharmacological active compounds derived from this plant include naphthoquinones, coumarins, phenolic acids, flavonoids, anthocyanidins, steroids and peptides (Bohm and Towers, 1962; Thevissen et al., 2005; Yang et al., 2001). Hepatic fibrosis has been a major world health problem and a major cause of mortality and morbidity worldwide (Friedman, 2000). A hallmark of pathology of the hepatic fibrosis is the activation of HSCs, which leads to the overexpression of extracellular matrix (ECM), the formation of scar tissue and the deterioration of hepatic function (McGuire et al., 1992). Currently, it is generally believed that downregulating activated HSCs or promoting the apoptosis of activated HSCs are potential therapeutic strategies for hepatic fibrosis (Wu et al., 2011). Silymarin is a

* Corresponding author at: School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China. Fax: +86 24 23986521. E-mail address: [email protected] (Y.-q. Zhao).

hepatoprotective agent with anti-inflammatory and anti-carcinogenic effects (Crocenzi and Roma, 2006), and Fufang Biejia Ruangan Pian (FBRP) showed definite clinical effect on hepatic fibrosis, (Jia and Wang, 2001) thus Silymarin and FBRP were used as positive control. In our continuing effort to discover more structurally diverse and biologically interesting natural products from I. balsamina, two new and two known 1,4-naphthoquinone derivatives were isolated from its flowers. Their structures were elucidated as 9S-(1methoxycarbonyl-4,5-dihydroxyl)phenyl-furanonaphthoquinone (1), 2-methoxy-10 -hydroxy-30 -methoxy[3,20 -binaphthalene]-1,4dione (2) balsaminone B (3) (Hui et al., 2012) and 2-methoxyl1,4-naphthoquinone (4) (Sultana et al., 2009) (Fig. 1). Herein, we presented the isolation, structural elucidation and their antihepatic fibrosis activity of the two new compounds. 2. Results and discussion Compound 1 was obtained as red violet powder (MeOH), had a molecular formula of C20H14O7 as determined by the positive HR– ESI–MS ion at m/z 367.0812 [M + H]+ (calcd for C20H15O7, 367.0812). The 13C NMR spectrum of 1 displayed signals for all 20 carbons in the molecule: fourteen aromatic carbons, three carbonyl, one methoxyl, one methylene and one methyne carbon. 1H NMR spectrum showed signals assignable a pair of 1,2-disubstituted benzene at dH 8.06 (1H, dd, J = 1.02, 7.62), 7.96 (1H, dd, J = 1.32, 7.50), 7.86 (1H, td, J = 1.02, 7.62), 7.81 (1H, td, J = 1.32, 7.50), a pair of

http://dx.doi.org/10.1016/j.phytol.2015.08.011 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Q. Li et al. / Phytochemistry Letters 14 (2015) 8–11

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Fig. 1. Structures of compounds 1–4.

1,2,4,5-tetrasubstituted benzene moiety at dH 7.92 (1H, s), 6.67 (1H, s). A methylene at 2.98 (1H, dd, J = 8.94, 15.45), 2.80 (1H, dd, J = 5.04, 15.48), a methyne 5.76 (1H, dd, J = 5.04, 8.82) and a methoxy group at 3.60 (3H, s) were also observed. The 1H spectral data of 1 (Table 1) were similar to those of 2,3-dihydro-2-phenylnaphtho [2,3-b]furan-4,9-diones, except for the 1,2,4,5-tetrasubstituted benzene moiety (Kobayashi et al., 1993). The HMBC correlations from 30 -OH (dH 9.60) to C-30 (dC 145.36), 40 -OH (dH 9.39) to C-40 (dC 147.48) and methoxy group (dH 3.60) to carbonyl (dC 169.71) were observed, indicating the moiety was methyl-3,4-dihydroxybenzoate (Kim et al., 2013). The HMBC correlations (Fig. 2) provided additional support for the assigned structure. Correlations between the signals of H-5 at dH 8.06 with dC 130.74 (C-4a), 133.58 (C-6), 183.26 (C-4), H-8 at dH 7.96 with dC 132.13 (C-8a), 134.26 (C-7), 178.17 (C-1), methyne proton at dH 5.76 to 150.80 (C-2), 39.26 (C-10) reinforced the structure of furanonaphthoquinone. Correlations from the methyne proton at dH 5.76 with signals at dC 112.02 (C-50 ), 115.73 (C-60 ), 124.03 (C-10 ) were observed, established the presence of linkage between C-9 and C-60 atoms.

Fig. 2. Key HMBC correlations of 1,2 and NOESY correlations of 1.

Significant NOE correlations (Fig. 2) among H-9/H-10, and H-10/ H-50 suggested that the atoms were co-facial. ECD spectrum was calculated using the GAUSSIAN09 program to provide the evidence of the absolute configuration (Fig. 3). In the 200–400 nm region, compared to the experimental positive and negative cotton effects at 206 nm and 286 nm, respectively, the predicted ECD curve of 9S showed the same pattern with the effects at 207 (+1) and 291 (+5). Therefore, the absolute configuration of C-9 was assigned as 9S. The structure of 1 was established as 9S-(1-methoxycarbonyl-4,5dihydroxyl)phenyl-furanonaphthoquinone, and named as balsaminone D. Compound 2 was isolated as violet powder (MeOH). It gave a molecular formula C22H16O5 on the basis of the positive HR-ESI-MS at m/z 383.0911 [M + Na]+ (calcd for C22H16O5Na 383.0890). The IR spectrum suggested the presence of hydroxyl and para-quinone carbonyl showing absorbance at 3395 cm1 (broad) and 1611 cm1, respectively. 1H NMR spectrum showed signals assignable to two

Table 1 NMR data for compounds 1 and 2 (TMS as the internal standard, d in ppm, J in Hz). 1 (DMSO-d6) Position 1 2 3 4 4a 5 6 7 8 8a 9 10 10 20 30 40 50 60 10 CO OCH3 30 OH 40 OH a b

600 MHz for 1H NMR. 150 MHz for 13C NMR.

2 (DMSO-d6)

dH a

8.06 (1H, dd, J = 1.02, 7.62) 7.86 (1H, td, J = 1.32, 7.50) 7.81 (1H, dd, J = 8.94, 15.45) 7.96 (1H, td, J = 1.32, 7.50) 5.76 (1H, dd, J = 5.04, 8.82) 2.98 (1H, dd, J = 8.94, 15.45) 2.80 (1H, dd, J = 5.04, 15.48) 7.92 (1H, s)

6.67 (1H, s)

3.59 (3H, s) 9.60 (1H, s) 9.39 (1H, s)

dC b

Position

178.67 150.80 119.39 183.26 130.74 126.13 133.58 134.26 125.26 132.13 74.66 39.26

1 2 3 4 4a 5 6 7 8 8a 10 20

124.03 114.73 145.46 147.48 112.02 115.73 169.71 51.59

30 40 4a0 50 60 70 80 8a0 2-OCH3 3'-OCH3

dHa

7.98 (1H, m) 7.87 (1H, m) 7.87 (1H, m) 8.09 (1H, m)

6.91 (1H, s) 7.46 (1H, d, J = 8.46) 7.24 (1H, td, J = 1.26, 6.72) 7.33 (1H, td, J = 1.26, 6.72) 8.10 (1H, m) 3.61 (3H, s) 3.73 (3H, s)

dC b 183.95 158.64 133.61 180.80 131.81 125.98 133.40 134.26 125.98 131.44 156.69 104.48 155.18 96.61 120.29 124.40 121.72 126.63 122.44 127.01 59.66 55.82

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that the treatment of hepatic fibrosis could be an important addition to 1,4-naphthoquinone derivatives. 3. Experimental 3.1. General experimental procedures

Fig. 3. Comparison of the experimental and calculated ECD spectra of 1.

pairs of 1,2-disubstituted benzene at dH 8.09 (1H, m), 7.98 (1H, m), 7.87 (2H, m) and 8.10 (1H, m), 7.46 (1H, d, J = 8.46), 7.24 (1H, td, J = 1.26, 6.72), 7.33 (1H, td, J = 1.26, 6.72), one isolated proton at dH 6.91 (1H, s), and two methoxy group dH 3.73 (3H, s), 3.61 (3H, s). The 13C NMR (Table 1) and spectra of 2 exhibited 22 carbon signals including two carbonyl, eighteen aromatic carbons, two methoxy carbon signals. These data indicated that the molecular structure was close to that of compound 10 -hydroxy-40 -methoxy-[2,2’binaphthalene]-1,4-dione (Ogata et al., 2004), except for a methoxy attached to C-2 and a methoxy attached to C-30 instead of to C-40 . The HMBC correlations (Fig. 2) between H-5 at dH 8.09 with 133.40 (C-6), 180.80 (C-4), H-8 at dH 7.98 with dC 131.13 (C-8a), 133.71 (C-7), 183.95 (C-1), H-6 at dH 7.87 with dC 131.81 (C-4a), 125.98 (C-8) and a methoxy group dH 3.61 with dC 158.64 (C-2), reinforced the structure of 2-methoxyl-1,4 -naphthoquinone moiety. The correlations from a methoxy group dH 3.73 to dC 155.18 (C-30 ), from H-40 dH 6.91 to dC 104.48 (C-20 ), 155.18 (C-30 ) and 120.29 (C-4a0 ), in the HMBC spectrum helped further reinforced the structure of compound 2. The above evidences allowed compound 2 to be determined as 2-methoxy-10 -hydroxy-30 methoxy[3,20 -binaphthalene]-1,4-dione and given the name of balsaminone E. All the compounds were evaluated for their activity against activated t-HSC/Cl-6 cells (Table 2) with Silymarin (IC50 = 202.34 mg/mL) and FBRP (IC50 = 231.56 mg/mL) were used as positive control. New compounds 1 and 2 exhibited potent inhibitory activity with IC50 value of 30.54 and 40.67 mg/mL, respectively. On the basis of analysis of the structure-activity relationship of these compounds in the activated t-HSC/Cl-6 cells inhibiting assay, it may be suggested that the glycosylation of OH in the molecule was unfavorable to the inhibitory activity. This can be illustrated by, compounds 1, 2, 4 had more potent activity than that of compound 3 which contains a glycosyl attached to C-40 . The results revealed

Table 2 IC50 values of compounds 1–4 against activated t-HSC/Cl-6 cells, values are the mean of triplicate experiments. Compounds

IC50 (mg/mL)a

1 2 3 4 Silymarinb Fufang Biejia Ruangan Pianb

30.54 40.67 60.86 >100 202.34 231.56

a

IC50 value was calculated from the least-squares regression equations in the plot of the logarithm of three graded concentrations vs. % inhibition (mg/mL). b Positive control.

NMR spectra were recorded on Bruker AV-600 spectrometer with TMS as internal standard, J in Hz; IR spectra: Bruker IFS55 spectrophotometer; KBr pellets; cm1. HR–TOF–MS: BIC micro TOF-Q mass spectrometer; in m/z (rel. %). The circular dichroism (CD) spectra were measured on a Bio-Logic Science (MOS-450, France). Column chromatography (cc): silica gel (SiO2: 200– 300 mesh, Qingdao Marine Chemical Group, Co.); Sephadex LH20 (pharmacia, Co.). Prep. HPLC (Beijing CXTH3000 system): P3000 pump, UV3000 spectrophotometric detector at 210 nm, YMC C18 reversed-phase column (5 mm, 10  250 nm; flow rate 3.0 mL/min). The cell culture reagents were from Gibco/Invitrogen (Grand Island, NY, USA). Silymarin and FBRP were purchased from Furui Medical Technology Co., Ltd. (Nei Menggu, China). MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3.2. Plant material The flowers of I. balsamina were purchased from Nanjing Zelang Phar. Co. Ltd. (Nanjing, China) in November 2010. The voucher specimen of this herb (No. 20101002) was identified by Prof. Jincai Lu of Shenyang Pharmaceutical University. 3.3. Extraction and isolation The air-dried flowers of I. balsamina (10.0 kg) were extracted with 75% ethanol (3  120 L, 2 h each) under refluxing at 78  C. The extract was concentrated in vacuum and suspended in water (8 L), then partitioned with petroleum ether (3  5 L), dichloromethane (3  5 L), ethyl acetate (3  5 L) and n-butanol (3  5 L). The dichloromethane fraction (160.0 g) was subjected to a silica gel column chromatography (CC) (200–300 mesh, 1.6 kg), using a gradient of petroleum ether–acetone (PE–acetone) as eluant to provide nine fractions (Fr. 1–Fr. 9) based on TLC analysis (PE– acetone, 3:1, V/V). Fr. 4 (4 g) was subjected to repeated silica gel CC using PE ether–ethyl acetate (PE–EtOAc) elution (100:0 ! 100:100, V/V) to yield five fractions D1–D5. D5 was conducted by Sephadex LH-20CC with CH2Cl2–MeOH (1:1) as the eluting solvent to obtain compound 1 (5 mg). D2 was further purified using preparative HPLC (YMC, ODS, 75% MeOH), affording compounds 2 (3 mg). Fr. 5 (6.7 g) was chromatographed on a silica gel (200–300 mesh) column with a PE–acetone gradient, then recrystallized to yield compound 4 (560 mg). The n-butanol fraction (300 g) was subjected to a Diaion HP20 resin column eluted with EtOH–H2O in gradient. The 70% EtOH– H2O eluent (100 g) was then subjected to polyamide CC with MeOH–H2O (0:100 ! 100:0, V/V) as the eluent to obtain five fractions (A–E) based on TLC analysis (CH2Cl2–MeOH–EtOAc–H2O, 2:2:4:1, V/V/V/V). Fraction A was subjected to a silica gel CC, using a gradient of CH2Cl2–MeOH as eluant to provide nine fractions (A1– A9). A7 was subjected to a silica gel CC, using a gradient of CH2Cl2– MeOH–H2O (7:3:1, V/V/V) to yield compound 3 (36 mg). 3.3.1. Balsaminone D (1) Red violet powder (MeOH), HR–ESI–MS: (m/z 367.0812 [M + H]+, calcd 367.0812); 1H NMR (600 MHz, DMSO) and 13C NMR (150 MHz, DMSO) spectroscopic data, see Table 1.

Q. Li et al. / Phytochemistry Letters 14 (2015) 8–11

3.3.2. Balsaminone E (2) Violet powder (MeOH), IR (KBr) vmax 3395, 2920, 1611 cm1; HR–ESI–MS (m/z 383.0911 [M + Na]+, calcd 383.0890); 1H NMR (600 MHz, DMSO) and 13C NMR (150 MHz, DMSO) spectroscopic data, see Table 1. 3.4. ECD experiment and calculations The CONFLEX (Goto and Osawa, 1989) searches based on molecular mechanics with MMFF94S force fields were performed for 1, which gave 8 stable conformers. Selected conformers with the low energies were further optimized by the density functional theory method at the B3LYP/6-31G (d) level in Gaussian 09 program package, (Frisch et al., 2013) which were further checked by frequency calculation and resulted in no imaginary frequencies. The ECD of the conformers of 1 was then calculated by the TDDFT method at the B3LYP/6-31G (d) level with the CPCM model in methanol solution. The calculated ECD curve was generated using SpecDis 1.51 (Bruhn et al., 2013) with s = 0.16 ev according to Boltzmann weighting. 3.5. Cell culture The murine Hepatic Stellate Cells (t-HSC/Cl-6) cell line were routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented with 10% heat inactivated fetal bovine serum and antibiotics (100 U/mL penicillin and streptomycin) in a humidified atmosphere containing 5% CO2 at 37  C. 3.6. Cell viability assay t-HSC/Cl-6 cells were plated in 96-well plates (1 10,430 cells/ well) for overnight, then cells were treated by compounds at various concentrations with or without TNF-a together for another 48 h. 100 mL fresh medium with MTT reagent (5 mg/mL) was added to each well for 3 h of incubation at 37  C after removing all the left liquid in the wells. After the incubation period, the formazan crystal was dissolved with DMSO, and the reduction of cell viability was determined at 490 nm using a microplate reader (Lammer et al., 1998). Acknowledgements The research was supported by E&T modern center for Natural Products of Liaoning Province of China (No. 2008402021), Constraction of R&D institute of state original new drug at Benxi of Liaoning Province (2010ZX09401-304-105B). We are glad to acknowledge the Analytical Center of Shenyang Pharmaceutical University for NMR measurements.

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