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Flavonoids from Podocarpus macrophyllus and their cardioprotective activities a
b
c
Yun Qiao , Wei-Wei Sun , Jian-Feng Wang & Ji-Dong Zhang
a
a
Department of Traditional Chinese Medicine, Qilu Hospital, Shandong University, Jinan, 250012, China b
Basic Medical College of Shandong University of Traditional Chinese Medicine, Changqing University Science & Technology Park, Jinan, 250355, China c
Department of Pain Managent, Qilu Hospital, Shandong University, Jinan, 250012, China Published online: 09 Dec 2013.
To cite this article: Yun Qiao, Wei-Wei Sun, Jian-Feng Wang & Ji-Dong Zhang (2014) Flavonoids from Podocarpus macrophyllus and their cardioprotective activities, Journal of Asian Natural Products Research, 16:2, 222-229, DOI: 10.1080/10286020.2013.861821 To link to this article: http://dx.doi.org/10.1080/10286020.2013.861821
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 2, 222–229, http://dx.doi.org/10.1080/10286020.2013.861821
Flavonoids from Podocarpus macrophyllus and their cardioprotective activities Yun Qiaoa, Wei-Wei Sunb*, Jian-Feng Wangc and Ji-Dong Zhanga* a
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Department of Traditional Chinese Medicine, Qilu Hospital, Shandong University, Jinan 250012, China; bBasic Medical College of Shandong University of Traditional Chinese Medicine, Changqing University Science & Technology Park, Jinan 250355, China; cDepartment of Pain Managent, Qilu Hospital, Shandong University, Jinan 250012, China (Received 18 July 2013; final version received 30 October 2013) One new 8-aryl flavone, podocarflavone A (1), together with 15 previously reported flavonoids were isolated from the twigs and leaves of Podocarpus macrophyllus. Their structures were established on the basis of extensive spectroscopic analysis and by the comparison with spectroscopic data reported in the literature. Antioxidant capacities of the isolated substances were determined using the 1,1-diphenyl-2-picrylhydrazyl, ferrous ions, and 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical in vitro assays, and their cytoprotective activities were also tested on H2O2-induced apoptosis in H9c2 cardiomyocytes. The results showed that those flavonoids exhibited significant cardioprotective effects by decreasing the H2O2-induced death of H9c2 cell, and the levels of lactate dehydrogenase and creatine kinase, and by inhibiting the elevated intracellular concentration of reactive oxygen species. Keywords: Podocarpus macrophyllus; flavonoids; cardioprotective effect; H2O2induced apoptosis
1. Introduction Podocarpus macrophyllus var. maki is a small- to medium-size evergreen tree of the family Podocarpaceae distributed throughout Australia to the tropical and subtropical areas of eastern Asia [1]. A number of flavonoids, phenolics, sesquiterpenoids, norditerpene dilactones, triterpenoids, and steroids have been isolated from this plant [2 – 4]. Because of their antioxidant, antitumor, insecticidal, antifeedant, allelopathic, and fungicidal activities [5 – 8], these compounds are of considerable pharmaceutical interests. In continuation of our studies on antioxidant agents from traditional Chinese medicines, a bioassay-guided fractionation technique was used to separate components from 90% EtOH extract of P. macrophyllus which displayed significant antioxidant
properties in both 1,1-diphenyl-2-picrylhydrazyl (DPPH z) and 2,20 -azinobis (3-ethylbenzthiazoline-6-sulfonic acid (ABTSzþ) radical scavenging assays and yielded a new flavonoid podocarflavone A (1) and 15 known flavonoids (Figure 1). Herein, the isolation, structural elucidation, and evaluation of antioxidant and cytoprotective activities of these flavonoids were reported. 2.
Results and discussion
The EtOAc-soluble portion of 90% EtOH extract from the aerial parts of P. macrophyllus was fractionated by chromatography over silica gel and Sephadex LH-20 and preparative reversed-phase HPLC to afford compounds 1 – 16. Podocarflavone A (1) was obtained as a yellowish amorphous solid. The
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
Journal of Asian Natural Products Research O
OH
HO
OH
O
4'' 5''
3''
6''
O
3'
2''
4'
2'
7
8a
1'
O
6
5
6'
O
OH O 2 R1=H R2=H 4 R1=CH3 R2=CH3
OH O
OH RO
O
O
OH
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O
OH
O
O 9 R=H 10 R=X
OCH3
OCH3
OH OCH3
OH
OH
7 R=H 8 R=X
O
3
OH HO
5 R=H 6 R=X
HO
O
OH
OR OH
O
HO
4
1
RO
O
H3CO
OH
HO
O OH
RO OH
OH
O
O
O
O
OH
O
H3CO HO
OY
14
OH
OH
OH
OH
12 R=H 13 R=CH3
11
HO
O
HO
O
O
HO
3
4a
OH
OR2
R1O
5'
2
OH
OH
OH
1''
HO
223
O 16
HO HO
O OH
OX Y=
OH
O 15
HO X=
O HO HO
OH
Figure 1. Structures of compounds 1 –16.
molecular formula was established as C21H14O6 by HR-ESI-MS showing a pseudo-molecular ion [M – H] – peak at m/z 361.0709. The UV spectrum showed characteristic absorptions of flavonoids, with maxima at 268 and 349 nm [9]. The IR spectrum showed characteristic absorption bands of hydroxyl (3333 cm21), conjugated carbonyl (1612 cm21), and aromatic ring (1507 and 1466 cm21). The 1 H NMR (600 MHz, DMSO-d6) spectrum (Table 1) exhibited typical signals of OH 4'' 3''
5''
2''
6''
HO
8a
7
O
2'
3'
OH
1' 6' 5'
4a
OH
O
Figure 2. Key HMBC (H ! C) and 1H – 1H COSY ( ) correlations of 1.
apigenin skeleton with two aromatic protons at d 6.73 (1H, s) and 6.26 (1H, s) which were assigned as H-3 and H-6 of rings C and A, and two pairs of orthocoupled aromatic protons at d 7.63 (2H, d, J ¼ 8.0 Hz) and 6.78 (2H, d, J ¼ 8.0 Hz) which were assigned as H-20 ,60 and H-30 ,50 of ring B. In addition, signals at d 7.27 (2H, d, J ¼ 8.0 Hz) and dH 6.88 (2H, d, J ¼ 8.0 Hz) were due to a 400 -hydroxyphenyl group [10]. The 13C NMR spectrum (Table 1) displayed signals for 21 carbons, which included the signals of a carbonyl carbon (dC 182.3) and 20 sp2 carbons. The above NMR data of 1 were very similar to those of apigenin (5),[11] except for the presence of the signals for one 4-hydroxyphenyl group. The 4-hydroxyphenyl moiety was attached to C-8 in 1, which was supported by the HMBC correlations from H-200 (dH 7.27) to C-8, C-400 , C-600 (Figure 2). Thus, the structure of 1 was determined to be 8-(400 -hydroxyphenyl)5,7,40 -trihydroxyflavone.
224 Table 1.
Y. Qiao et al. 1
H and 13C NMR spectral data (d, 600 and 150 MHz) for compound 1 in DMSO-d6. 1
Position
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2 3 4 4a 5 6 7 8 8a
dH (J in Hz) 6.73, s
6.26, s
1
dC 163.8 102.6 182.3 103.4 161.6 99.6 160.2 108.7 156.6
Although biflavonoids incorporating a C –C connection to C-8 of a flavone are well known, the number of known compounds involving C-8 connection to a simple unfused benzene ring is relatively small [12]. This 8-aryl flavone is first found in P. macrophyllus. In addition, 15 known compounds were isolated and identified as amentoflavone (2) [13], hinokiflavone (3) [14], isoginkgetin (4) [15], apigenin (5), apigenin-7-O-(-D glucopy-ranoside (6), quercetin (7) [11], quercetin-3-O-(-D -glucopyranoside (8) [16], luteolin (9) [17], luteolin-7-O-(-D glucopyranoside (10) [18], tricin (11) [11], salvigenin (12) [19], ladanein (13) [20], norartocarpesin (14) [21], avicularin (15) [22], and pedalitin-30 -O-glucoside (16) [23] by spectroscopic analyzes and by comparison of data with those reported. Many studies have shown that the major molecular mechanism involved in H2O2induced cardiotoxicity is the generation of reactive oxygen species (ROS). Therefore, removing excess ROS or suppressing their generation using antioxidants may be effective in preventing H2O2 cardiotoxicity [23]. In this article, compounds were subjected to in vitro antioxidative test using the DPPHz, ferrous ions, and ABTSzþ radical scavenging assay [23]. As shown in Table 2, all the compounds exhibited a moderate to good antioxidant activity. The results indicated that 2, 3, 7, and 8 had significant antioxidant activity. Although 2
Position
dH (J in Hz)
0
1 20 ,60 30 ,50 40 100 200 ,600 300 ,500 400 5-OH
7.63, d (8.0) 6.78, d (8.0) 7.27, d (8.0) 6.88, d (8.0)
dC 121.7 128.7 116.3 161.6 123.1 132.6 115.1 156.6
13.13, s
was previously described as a poor radical scavenger and a moderate antioxidant, due probably to its metal-chelating potential [24], it appeared e contrario as a good antioxidant in our own evaluation (Table 2). This shows the lack of a standardized evaluation of antioxidant potentials and, therefore, the difficulty to compare results based on different assays [12]. In addition, those compounds were subsequently evaluated for their cytoprotective effects against H2O2-induced myocardial toxicity in H9c2 cells using the modified MTT colorimetric method. The cytoprotective effect of 16 flavonoids against H2O2-induced myocardial toxicity is summarized in Table 2. It should be noted that compounds 2 and 7 –10 showed strong cytoprotective activity. The effects of decreasing the creatine kinase (CK) and lactate dehydrogenase (LDH) levels of compounds on cell damage induced by H2O2 were further investigated. The results (Table 3) showed that all of these compounds at a concentration of 0.05 mM displayed decreasing effects of the CK and LDH levels compared with the H2O2 group. Compound 7 possessed a free hydroxyl group at C-3 and was more potent than compound 2, while 2 and 7 were both active. This suggested that the presence of a free hydroxyl group at C-3 played an important role in terms of cardioprotective activities. One potential mechanism for H2O2induced cell damage is the secondary
Journal of Asian Natural Products Research
225
Table 2. DPPHz, ferrous ions, and ABTSzþ radical scavenging activities of some compounds.
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Compound 1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a 12a 13a 14a 15a 16a a-Tocopherola
DPPHz EC50b (mM)
Fe2þ EC50c (mM)
ABTSzþ EC50d (mM)
Cytoprotection EC50 (mM)
9.80 0.92 1.85 20.37 30.55 21.34 0.83 0.75 3.94 6.12 19.97 79.89 73.21 28.89 7.89 51.17 0.3
21.8 20.1 19.3 111.0 115.6 117.2 3.2 3.8 12.0 1.1 54.90 115.68 114.51 55.60 23.5 27.6 2.4
41.5 30.8 39.4 430.0 19.88 13.09 17.5 14.6 105.3 56.46 76.34 95.09 104.58 34.56 23.46 86.12 32.1
13.35 8.26 10.53 None 22.34 24.30 4.57 7.60 5.53 5.04 20.45 None None None 9.30 None None
a
Each experiment was carried out at least three times independently. Effective concentration needed to scavenge (DPPHz) free radical to 50%. Effective concentration needed to chelate ferrous ions to 50%. d Effective concentration needed to scavenge ABTSzþ cation radical to 50%. b c
generation of ROS. The anti-ROS activity of 2 and 7 was analyzed. As shown in Table 4, H2O2 significantly increased the intracellular level of ROS. Pretreatment with 2 or 7 (5, 10, and 20 mM) significantly inhibited the elevated intracellular concentration of ROS by H2O2. Table 3. Effects of compounds on the release of CK and LDH levels on cell damage induced by H2O2. Compound (0.05 mM)
CK (U/ml)
LDH (U/ml)
1 2 3 5 6 7 8 9 10 11 15 H2O2 Control
1250 ^ 120 950 ^ 83 1000 ^ 880 1610 ^ 142 1650 ^ 150 900 ^ 62 1050 ^ 85 1100 ^ 105 1200 ^ 108 1600 ^ 130 1300 ^ 108 1700 ^ 155 300 ^ 35
560 ^ 50 450 ^ 8 520 ^ 45 730 ^ 58 725 ^ 60 430 ^ 30 485 ^ 35 450 ^ 40 459 ^ 41 740 ^ 68 530 ^ 45 750 ^ 65 400 ^ 40
This study concludes that flavonoids with OH groups at 30 ,40 -positions in the Bring and a double bond between C-2 and C-3 displayed important roles for their protective effects against H2O2-induced cardiotoxicity. In addition, compounds 2 and 7 showed cardioprotective effect by inhibiting the H2O2-induced intracellular level of ROS and may act as a promising therapeutic agent for preventing the cardiotoxicity.
Table 4. Effects of compounds 2 and 7 on the intracellular generation of ROS (mM). ROS positive rate (%)
Compounds 2 7 H2O2 (100 mM) Control
5 10 15 5 10 15
mM mM mM mM mM mM
45 25 20 40 22 18 49 15
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3. Experimental 3.1 General experimental procedures Melting points were measured with an X-6 micro-melting point apparatus (Analytical Instruments Co. Ltd, Shanghai, China) and are uncorrected. IR spectra were recorded on a Nicolet iN 10 Micro FT-IR spectrometer (Thermo Fisher Scientific, lnc., Waltham, MA, USA) by transmission mode. UV spectra were obtained on a Shimadzu UV2550 spectrophotometer (Shimadzu, Kyoto, Japan). NMR spectra were measured on a Bruker Avance DRX-600 spectrometer (Bruker, Zurich, Switzerland) operating at 600 MHz (1H) and 150 MHz (13C) with tetramethylsilane as the internal standard. HR-ESI-MS were carried out on a LTQOrbitrap XL (Thermo Fisher, Scientific, lnc.). All solvents used were of analytical grade (Laiyang Chemical Reagent Co. Ltd, Shandong, China). HPLC was performed on an Agilent 1100 G1310A isopump (Agilent, Palo Alto, CA, USA) equipped with an Agilent 1100 G1322A degasser, an Agilent 1100 G1314A VWD detector (210 nm) and a ZORBAX SB-C18 column (9.4 mm £ 250 mm, 5 mm). Silica gel (200– 300 mesh; Qingdao Haiyang Chemical Co. Ltd, Qingdao, China), C18 reversed-phase silica gel (YMC ODS-A gel, YMC Co. Ltd, Kyoto, Japan), MCI-gel (CHP20P, 75– 150 mm, Mitsubishi Chemical Industries Ltd, Tokyo, Japan), and Sephadex LH-20 (Pharmacia Biotek, Uppsala, Sweden) were used for column chromatography (CC). Thin layer chromatography (TLC) was carried out with high-performance TLC plates precoated with silica gel GF254 (Qingdao Haiyang Chemical Co. Ltd). Spots of TLC were visualized within iodine vapor or by spraying with H2SO4 –EtOH (1:9) followed by heating. 3.2
Plant material
The twigs and leaves of P. macrophyllus were collected from Xishuangbanna County, Yunnan Province, China, in July 2010. The plant material was identified by
Dr Tao Shen, Shandong University. A voucher specimen (PM01-2010-07) was deposited at the School of Medicine, Shandong University. 3.3 Extraction and isolation The air-dried and powdered plant material (6.0 kg) was extracted with 90% EtOH (4 £ 20 l, each for 5 days) at room temperature. The combined extracts were concentrated under reduced pressure to afford a dark gum (600 g), which was suspended in H2O, and partitioned successively with PE (petroleum ether, 5 £ 1 l), CH2Cl2 (5 £ 1 l), and n-BuOH (5 £ 1 l). The CH2Cl2 fraction extract (52.1 g) was divided by a silica gel column, eluted with a PE–EtOAc gradient (50:1 ! 1:1) to give eight fractions (Fr. 1– Fr. 8). Fr. 7 (10.1 g) was successively subjected to silica gel column eluted with a PE– acetone gradient (20:1 ! 1:1) to afford eight fractions (Fr. 7.1–Fr. 7.8). Fr. 7.3 (5.1 g) was further separated by Sephadex LH-20 CC (MeOH) to obtain five fractions (Fr. 7.3.1–Fr. 7.3.5). Fr. 7.3.1 (701.1 mg) was purified by preparative HPLC (MeOH– H2O, 75:25, 1.5 ml/min) to afford 1 (3.5 mg, tR 12.02 min), 5 (10.0 mg, tR 16.02 min), and 11 (8.5 mg, tR 18.33 min). Fr. 7.3.2 (520.2 mg) was purified by Sephadex LH-20 CC (EtOH) to obtain 12 (22.0 mg). Fr. 7.3.3 (454.0 mg) was purified by CC of reversed-phase C18 (MeOH – H2O, 30:70 ! 90:10) to afford 2 (22.0 mg), 3 (10.5 mg), and 4 (10.5 mg). Fr. 5 (4.0 g) was subjected to a silica gel column and eluted with a CH2Cl2 –MeOH to afford 9 (22.1 mg) and 14 (10.2 mg). Fr. 6 (3.3 g) was subjected to a Sephadex LH-20 column eluted with EtOH to afford 7 (20.5 mg) and 13 (13.2 mg). The n-BuOH extract (163.7 g) was divided by a silica gel column, eluted with a CH2Cl2 –MeOH gradient (100:1 ! 1:1) to give eight fractions (Fr. 9–Fr. 16). Fr. 11 (20.1 g) was successively subjected to silica gel column eluted with a CH2Cl2 –MeOH gradient (20:1 ! 4:1) and Sephadex LH-20 eluted with EtOH to afford
Journal of Asian Natural Products Research 6 (35.0 mg) and 10 (18.0 mg). Fr. 12 (12.1 g) was subjected to Sephadex LH-20 column eluted with EtOH to yield 8 (40.8 mg) and 15 (40.5 mg). Fr. 13 (9.1 g) was subjected to CC of reversed-phase C18 (MeOH – H2O, 30:70 ! 90:10) to afford 16 (20.3 mg).
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3.3.1
Podocarflavone A (1)
Yellowish amorphous solid (3.5 mg); m.p. 223 –2248C; UV (MeOH) lmax (log 1) nm: 208 (4.29), 268 (4.23), and 349 (3.96); IR nmax cm21: 3333, 1653, 1612, 1507, 1466, 1363, 1336, 1175, 1171, 1119, and 836; For 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS m/z 361.0709 [M – H] – (calcd for C21H13O6, 361.0712). 3.4 Antioxidant activity Some compounds were subjected to in vitro antioxidative testing using the DPPHz, ferrous ions, and ABTSzþ radical scavenging assay as previously reported. 3.4.1 DPPH free radical scavenging activity The free radical scavenging activity was measured by DPPHz. Briefly, 60 mM solution of DPPH in methanol was prepared and 100 ml of this solution was added to 100 ml of the test compound solution in methanol at different concentrations (0.125, 0.25, 0.5, 1.0, and 2.0 mM). After 30 min, the absorbance was measured at 517 nm. The capability to scavenge the DPPH radical was calculated using the following equation: DPPH scavenging effect ð%Þ ASample ¼ 12 £ 100; AControl where AControl is the absorbance of the control reaction and ASample is the absorbance in the presence of test compound. 3.4.2 Ferrous metal ions chelating activity The test compound in 100 ml was added to a solution of 1 mM FeCl2 (5 ml). The reaction
227
was initiated by the addition of 1 mM ferrozine (15 ml). Then, the mixture was shaken vigorously and left at room temperature for 10 min. The absorbance was measured at 570 nm. The inhibition percentage of ferrozine–Fe2þ complex formation was calculated by using the formula given below: Metal chelating effect ð%Þ ASample ¼ 12 £ 100; AControl where AControl is the absorbance of control and ASample is the absorbance in the presence of the test compound.
3.4.3 ABTS radical cation decolorization assay The ABTSzþ cation radical was produced by the reaction between 7 mM ABTS in H2O and 140 mM potassium persulfate, stored in dark at room temperature for 12 h. Before usage, the ABTSzþ solution was diluted to get an absorbance of 0.700 ^ 0.025 at 734 nm. Then, 100 ml of ABTSzþ solution was added to 100 ml of the test compounds solution in methanol at different concentrations. After 30 min, the percentage inhibition at 734 nm was calculated for each concentration relative to a blank absorbance (methanol). The scavenging capability of ABTSzþ radical was calculated using the following equation: ABTS·þ scavenging effect ð%Þ ASample ¼ 12 £ 100; AControl where AControl is the initial concentration of the ABTSzþ and ASample is the absorbance of the remaining concentration of ABTSzþ in the presence of the test compounds.
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3.5 Myocardial protective bioassay 3.5.1 Cell viability measurements Rat cardiac H9c2 cells (ATCC, Rockville, MD, USA) were cultured in high Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum, 1% penicillin, and 5% CO2 at 378C. The cells were fed every 2– 3 days and subcultured once they reached 70 –80% confluence. Cells were plated at an appropriate density according to each experimental design. The tested compounds were dissolved in dimethylsulfoxide (0.1% DMSO) and serially diluted in phosphate-buffered saline (PBS) immediately prior to the experiment, and were added into the cells with different concentrations for 12 h followed by the addition of doxorubicin (1024 M) for another 3 h to make the cell injury model. After this incubation, cells were measured by a modified MTT assay. The H9c2 cells were treated with MTT solution (final concentration, 0.5 mg/ml) for 4 h at 378C in 96-well plates. The supernatants were removed carefully, followed by the addition of 100 ml DMSO to each well to dissolve the precipitate. Then, the absorbance was measured at 490 nm in a Model 680 microplate reader (BIO-RAD, California, USA). 3.5.2 Measurement of the levels of LDH and CK Cellular injury was determined by measuring the levels of LDH released into the cell culture medium. According to the manufacturer’s instruction (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), LDH was assayed by measuring the increase in the absorbance of nicotinamide adenine dinucleotide at 450 nm at 258C, using a Model 680 microplate reader (BIO-RAD). Cellular damage was evaluated by measuring the levels of CK release in the culture medium. After treatments, where indicated, a spectrophotometric CK enzyme assay was performed with the test
kits (Jiancheng Bioengineering Institute) and UV-2550 mode spectrophotometer (Shimadzu) at 660 nm. All data were expressed as mean ^ SD, and a value of p , 0.05 was considered statistically significant.
3.5.3 Determination of ROS generation Changes in intracellular ROS levels were determined by measuring the oxidative conversion of cell-permeable 20 ,70 -dichlorfluorescein-diacetate (DCFHDA) to fluorescent dichlorofluorescein (DCF) in a FACS-SCAN apparatus (FACSCalibur; BD Biosciences, California, USA). In the presence of ROS, DCFH reacts with ROS to form the fluorescent product DCF, which is trapped inside the cells. To obtain the dissociated microglia for the ROS assay, the culture medium was first removed and the cells were washed three times with PBS. The cells were incubated with DCFH-DA at 378C for 20 min. The cellular fluorescence was measured by flow cytometry analysis with a FACS-SCAN apparatus. The decrease in value compared with that of the control was viewed as the decrease of intracellular ROS.
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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Flavonoids from Podocarpus macrophyllus and their cardioprotective activities a
b
c
Yun Qiao , Wei-Wei Sun , Jian-Feng Wang & Ji-Dong Zhang
a
a
Department of Traditional Chinese Medicine, Qilu Hospital, Shandong University, Jinan, 250012, China b
Basic Medical College of Shandong University of Traditional Chinese Medicine, Changqing University Science & Technology Park, Jinan, 250355, China c
Department of Pain Managent, Qilu Hospital, Shandong University, Jinan, 250012, China Published online: 09 Dec 2013.
To cite this article: Yun Qiao, Wei-Wei Sun, Jian-Feng Wang & Ji-Dong Zhang (2014) Flavonoids from Podocarpus macrophyllus and their cardioprotective activities, Journal of Asian Natural Products Research, 16:2, 222-229, DOI: 10.1080/10286020.2013.861821 To link to this article: http://dx.doi.org/10.1080/10286020.2013.861821
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 2, 222–229, http://dx.doi.org/10.1080/10286020.2013.861821
Flavonoids from Podocarpus macrophyllus and their cardioprotective activities Yun Qiaoa, Wei-Wei Sunb*, Jian-Feng Wangc and Ji-Dong Zhanga* a
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Department of Traditional Chinese Medicine, Qilu Hospital, Shandong University, Jinan 250012, China; bBasic Medical College of Shandong University of Traditional Chinese Medicine, Changqing University Science & Technology Park, Jinan 250355, China; cDepartment of Pain Managent, Qilu Hospital, Shandong University, Jinan 250012, China (Received 18 July 2013; final version received 30 October 2013) One new 8-aryl flavone, podocarflavone A (1), together with 15 previously reported flavonoids were isolated from the twigs and leaves of Podocarpus macrophyllus. Their structures were established on the basis of extensive spectroscopic analysis and by the comparison with spectroscopic data reported in the literature. Antioxidant capacities of the isolated substances were determined using the 1,1-diphenyl-2-picrylhydrazyl, ferrous ions, and 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical in vitro assays, and their cytoprotective activities were also tested on H2O2-induced apoptosis in H9c2 cardiomyocytes. The results showed that those flavonoids exhibited significant cardioprotective effects by decreasing the H2O2-induced death of H9c2 cell, and the levels of lactate dehydrogenase and creatine kinase, and by inhibiting the elevated intracellular concentration of reactive oxygen species. Keywords: Podocarpus macrophyllus; flavonoids; cardioprotective effect; H2O2induced apoptosis
1. Introduction Podocarpus macrophyllus var. maki is a small- to medium-size evergreen tree of the family Podocarpaceae distributed throughout Australia to the tropical and subtropical areas of eastern Asia [1]. A number of flavonoids, phenolics, sesquiterpenoids, norditerpene dilactones, triterpenoids, and steroids have been isolated from this plant [2 – 4]. Because of their antioxidant, antitumor, insecticidal, antifeedant, allelopathic, and fungicidal activities [5 – 8], these compounds are of considerable pharmaceutical interests. In continuation of our studies on antioxidant agents from traditional Chinese medicines, a bioassay-guided fractionation technique was used to separate components from 90% EtOH extract of P. macrophyllus which displayed significant antioxidant
properties in both 1,1-diphenyl-2-picrylhydrazyl (DPPH z) and 2,20 -azinobis (3-ethylbenzthiazoline-6-sulfonic acid (ABTSzþ) radical scavenging assays and yielded a new flavonoid podocarflavone A (1) and 15 known flavonoids (Figure 1). Herein, the isolation, structural elucidation, and evaluation of antioxidant and cytoprotective activities of these flavonoids were reported. 2.
Results and discussion
The EtOAc-soluble portion of 90% EtOH extract from the aerial parts of P. macrophyllus was fractionated by chromatography over silica gel and Sephadex LH-20 and preparative reversed-phase HPLC to afford compounds 1 – 16. Podocarflavone A (1) was obtained as a yellowish amorphous solid. The
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
Journal of Asian Natural Products Research O
OH
HO
OH
O
4'' 5''
3''
6''
O
3'
2''
4'
2'
7
8a
1'
O
6
5
6'
O
OH O 2 R1=H R2=H 4 R1=CH3 R2=CH3
OH O
OH RO
O
O
OH
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O
OH
O
O 9 R=H 10 R=X
OCH3
OCH3
OH OCH3
OH
OH
7 R=H 8 R=X
O
3
OH HO
5 R=H 6 R=X
HO
O
OH
OR OH
O
HO
4
1
RO
O
H3CO
OH
HO
O OH
RO OH
OH
O
O
O
O
OH
O
H3CO HO
OY
14
OH
OH
OH
OH
12 R=H 13 R=CH3
11
HO
O
HO
O
O
HO
3
4a
OH
OR2
R1O
5'
2
OH
OH
OH
1''
HO
223
O 16
HO HO
O OH
OX Y=
OH
O 15
HO X=
O HO HO
OH
Figure 1. Structures of compounds 1 –16.
molecular formula was established as C21H14O6 by HR-ESI-MS showing a pseudo-molecular ion [M – H] – peak at m/z 361.0709. The UV spectrum showed characteristic absorptions of flavonoids, with maxima at 268 and 349 nm [9]. The IR spectrum showed characteristic absorption bands of hydroxyl (3333 cm21), conjugated carbonyl (1612 cm21), and aromatic ring (1507 and 1466 cm21). The 1 H NMR (600 MHz, DMSO-d6) spectrum (Table 1) exhibited typical signals of OH 4'' 3''
5''
2''
6''
HO
8a
7
O
2'
3'
OH
1' 6' 5'
4a
OH
O
Figure 2. Key HMBC (H ! C) and 1H – 1H COSY ( ) correlations of 1.
apigenin skeleton with two aromatic protons at d 6.73 (1H, s) and 6.26 (1H, s) which were assigned as H-3 and H-6 of rings C and A, and two pairs of orthocoupled aromatic protons at d 7.63 (2H, d, J ¼ 8.0 Hz) and 6.78 (2H, d, J ¼ 8.0 Hz) which were assigned as H-20 ,60 and H-30 ,50 of ring B. In addition, signals at d 7.27 (2H, d, J ¼ 8.0 Hz) and dH 6.88 (2H, d, J ¼ 8.0 Hz) were due to a 400 -hydroxyphenyl group [10]. The 13C NMR spectrum (Table 1) displayed signals for 21 carbons, which included the signals of a carbonyl carbon (dC 182.3) and 20 sp2 carbons. The above NMR data of 1 were very similar to those of apigenin (5),[11] except for the presence of the signals for one 4-hydroxyphenyl group. The 4-hydroxyphenyl moiety was attached to C-8 in 1, which was supported by the HMBC correlations from H-200 (dH 7.27) to C-8, C-400 , C-600 (Figure 2). Thus, the structure of 1 was determined to be 8-(400 -hydroxyphenyl)5,7,40 -trihydroxyflavone.
224 Table 1.
Y. Qiao et al. 1
H and 13C NMR spectral data (d, 600 and 150 MHz) for compound 1 in DMSO-d6. 1
Position
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2 3 4 4a 5 6 7 8 8a
dH (J in Hz) 6.73, s
6.26, s
1
dC 163.8 102.6 182.3 103.4 161.6 99.6 160.2 108.7 156.6
Although biflavonoids incorporating a C –C connection to C-8 of a flavone are well known, the number of known compounds involving C-8 connection to a simple unfused benzene ring is relatively small [12]. This 8-aryl flavone is first found in P. macrophyllus. In addition, 15 known compounds were isolated and identified as amentoflavone (2) [13], hinokiflavone (3) [14], isoginkgetin (4) [15], apigenin (5), apigenin-7-O-(-D glucopy-ranoside (6), quercetin (7) [11], quercetin-3-O-(-D -glucopyranoside (8) [16], luteolin (9) [17], luteolin-7-O-(-D glucopyranoside (10) [18], tricin (11) [11], salvigenin (12) [19], ladanein (13) [20], norartocarpesin (14) [21], avicularin (15) [22], and pedalitin-30 -O-glucoside (16) [23] by spectroscopic analyzes and by comparison of data with those reported. Many studies have shown that the major molecular mechanism involved in H2O2induced cardiotoxicity is the generation of reactive oxygen species (ROS). Therefore, removing excess ROS or suppressing their generation using antioxidants may be effective in preventing H2O2 cardiotoxicity [23]. In this article, compounds were subjected to in vitro antioxidative test using the DPPHz, ferrous ions, and ABTSzþ radical scavenging assay [23]. As shown in Table 2, all the compounds exhibited a moderate to good antioxidant activity. The results indicated that 2, 3, 7, and 8 had significant antioxidant activity. Although 2
Position
dH (J in Hz)
0
1 20 ,60 30 ,50 40 100 200 ,600 300 ,500 400 5-OH
7.63, d (8.0) 6.78, d (8.0) 7.27, d (8.0) 6.88, d (8.0)
dC 121.7 128.7 116.3 161.6 123.1 132.6 115.1 156.6
13.13, s
was previously described as a poor radical scavenger and a moderate antioxidant, due probably to its metal-chelating potential [24], it appeared e contrario as a good antioxidant in our own evaluation (Table 2). This shows the lack of a standardized evaluation of antioxidant potentials and, therefore, the difficulty to compare results based on different assays [12]. In addition, those compounds were subsequently evaluated for their cytoprotective effects against H2O2-induced myocardial toxicity in H9c2 cells using the modified MTT colorimetric method. The cytoprotective effect of 16 flavonoids against H2O2-induced myocardial toxicity is summarized in Table 2. It should be noted that compounds 2 and 7 –10 showed strong cytoprotective activity. The effects of decreasing the creatine kinase (CK) and lactate dehydrogenase (LDH) levels of compounds on cell damage induced by H2O2 were further investigated. The results (Table 3) showed that all of these compounds at a concentration of 0.05 mM displayed decreasing effects of the CK and LDH levels compared with the H2O2 group. Compound 7 possessed a free hydroxyl group at C-3 and was more potent than compound 2, while 2 and 7 were both active. This suggested that the presence of a free hydroxyl group at C-3 played an important role in terms of cardioprotective activities. One potential mechanism for H2O2induced cell damage is the secondary
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Table 2. DPPHz, ferrous ions, and ABTSzþ radical scavenging activities of some compounds.
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Compound 1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a 12a 13a 14a 15a 16a a-Tocopherola
DPPHz EC50b (mM)
Fe2þ EC50c (mM)
ABTSzþ EC50d (mM)
Cytoprotection EC50 (mM)
9.80 0.92 1.85 20.37 30.55 21.34 0.83 0.75 3.94 6.12 19.97 79.89 73.21 28.89 7.89 51.17 0.3
21.8 20.1 19.3 111.0 115.6 117.2 3.2 3.8 12.0 1.1 54.90 115.68 114.51 55.60 23.5 27.6 2.4
41.5 30.8 39.4 430.0 19.88 13.09 17.5 14.6 105.3 56.46 76.34 95.09 104.58 34.56 23.46 86.12 32.1
13.35 8.26 10.53 None 22.34 24.30 4.57 7.60 5.53 5.04 20.45 None None None 9.30 None None
a
Each experiment was carried out at least three times independently. Effective concentration needed to scavenge (DPPHz) free radical to 50%. Effective concentration needed to chelate ferrous ions to 50%. d Effective concentration needed to scavenge ABTSzþ cation radical to 50%. b c
generation of ROS. The anti-ROS activity of 2 and 7 was analyzed. As shown in Table 4, H2O2 significantly increased the intracellular level of ROS. Pretreatment with 2 or 7 (5, 10, and 20 mM) significantly inhibited the elevated intracellular concentration of ROS by H2O2. Table 3. Effects of compounds on the release of CK and LDH levels on cell damage induced by H2O2. Compound (0.05 mM)
CK (U/ml)
LDH (U/ml)
1 2 3 5 6 7 8 9 10 11 15 H2O2 Control
1250 ^ 120 950 ^ 83 1000 ^ 880 1610 ^ 142 1650 ^ 150 900 ^ 62 1050 ^ 85 1100 ^ 105 1200 ^ 108 1600 ^ 130 1300 ^ 108 1700 ^ 155 300 ^ 35
560 ^ 50 450 ^ 8 520 ^ 45 730 ^ 58 725 ^ 60 430 ^ 30 485 ^ 35 450 ^ 40 459 ^ 41 740 ^ 68 530 ^ 45 750 ^ 65 400 ^ 40
This study concludes that flavonoids with OH groups at 30 ,40 -positions in the Bring and a double bond between C-2 and C-3 displayed important roles for their protective effects against H2O2-induced cardiotoxicity. In addition, compounds 2 and 7 showed cardioprotective effect by inhibiting the H2O2-induced intracellular level of ROS and may act as a promising therapeutic agent for preventing the cardiotoxicity.
Table 4. Effects of compounds 2 and 7 on the intracellular generation of ROS (mM). ROS positive rate (%)
Compounds 2 7 H2O2 (100 mM) Control
5 10 15 5 10 15
mM mM mM mM mM mM
45 25 20 40 22 18 49 15
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3. Experimental 3.1 General experimental procedures Melting points were measured with an X-6 micro-melting point apparatus (Analytical Instruments Co. Ltd, Shanghai, China) and are uncorrected. IR spectra were recorded on a Nicolet iN 10 Micro FT-IR spectrometer (Thermo Fisher Scientific, lnc., Waltham, MA, USA) by transmission mode. UV spectra were obtained on a Shimadzu UV2550 spectrophotometer (Shimadzu, Kyoto, Japan). NMR spectra were measured on a Bruker Avance DRX-600 spectrometer (Bruker, Zurich, Switzerland) operating at 600 MHz (1H) and 150 MHz (13C) with tetramethylsilane as the internal standard. HR-ESI-MS were carried out on a LTQOrbitrap XL (Thermo Fisher, Scientific, lnc.). All solvents used were of analytical grade (Laiyang Chemical Reagent Co. Ltd, Shandong, China). HPLC was performed on an Agilent 1100 G1310A isopump (Agilent, Palo Alto, CA, USA) equipped with an Agilent 1100 G1322A degasser, an Agilent 1100 G1314A VWD detector (210 nm) and a ZORBAX SB-C18 column (9.4 mm £ 250 mm, 5 mm). Silica gel (200– 300 mesh; Qingdao Haiyang Chemical Co. Ltd, Qingdao, China), C18 reversed-phase silica gel (YMC ODS-A gel, YMC Co. Ltd, Kyoto, Japan), MCI-gel (CHP20P, 75– 150 mm, Mitsubishi Chemical Industries Ltd, Tokyo, Japan), and Sephadex LH-20 (Pharmacia Biotek, Uppsala, Sweden) were used for column chromatography (CC). Thin layer chromatography (TLC) was carried out with high-performance TLC plates precoated with silica gel GF254 (Qingdao Haiyang Chemical Co. Ltd). Spots of TLC were visualized within iodine vapor or by spraying with H2SO4 –EtOH (1:9) followed by heating. 3.2
Plant material
The twigs and leaves of P. macrophyllus were collected from Xishuangbanna County, Yunnan Province, China, in July 2010. The plant material was identified by
Dr Tao Shen, Shandong University. A voucher specimen (PM01-2010-07) was deposited at the School of Medicine, Shandong University. 3.3 Extraction and isolation The air-dried and powdered plant material (6.0 kg) was extracted with 90% EtOH (4 £ 20 l, each for 5 days) at room temperature. The combined extracts were concentrated under reduced pressure to afford a dark gum (600 g), which was suspended in H2O, and partitioned successively with PE (petroleum ether, 5 £ 1 l), CH2Cl2 (5 £ 1 l), and n-BuOH (5 £ 1 l). The CH2Cl2 fraction extract (52.1 g) was divided by a silica gel column, eluted with a PE–EtOAc gradient (50:1 ! 1:1) to give eight fractions (Fr. 1– Fr. 8). Fr. 7 (10.1 g) was successively subjected to silica gel column eluted with a PE– acetone gradient (20:1 ! 1:1) to afford eight fractions (Fr. 7.1–Fr. 7.8). Fr. 7.3 (5.1 g) was further separated by Sephadex LH-20 CC (MeOH) to obtain five fractions (Fr. 7.3.1–Fr. 7.3.5). Fr. 7.3.1 (701.1 mg) was purified by preparative HPLC (MeOH– H2O, 75:25, 1.5 ml/min) to afford 1 (3.5 mg, tR 12.02 min), 5 (10.0 mg, tR 16.02 min), and 11 (8.5 mg, tR 18.33 min). Fr. 7.3.2 (520.2 mg) was purified by Sephadex LH-20 CC (EtOH) to obtain 12 (22.0 mg). Fr. 7.3.3 (454.0 mg) was purified by CC of reversed-phase C18 (MeOH – H2O, 30:70 ! 90:10) to afford 2 (22.0 mg), 3 (10.5 mg), and 4 (10.5 mg). Fr. 5 (4.0 g) was subjected to a silica gel column and eluted with a CH2Cl2 –MeOH to afford 9 (22.1 mg) and 14 (10.2 mg). Fr. 6 (3.3 g) was subjected to a Sephadex LH-20 column eluted with EtOH to afford 7 (20.5 mg) and 13 (13.2 mg). The n-BuOH extract (163.7 g) was divided by a silica gel column, eluted with a CH2Cl2 –MeOH gradient (100:1 ! 1:1) to give eight fractions (Fr. 9–Fr. 16). Fr. 11 (20.1 g) was successively subjected to silica gel column eluted with a CH2Cl2 –MeOH gradient (20:1 ! 4:1) and Sephadex LH-20 eluted with EtOH to afford
Journal of Asian Natural Products Research 6 (35.0 mg) and 10 (18.0 mg). Fr. 12 (12.1 g) was subjected to Sephadex LH-20 column eluted with EtOH to yield 8 (40.8 mg) and 15 (40.5 mg). Fr. 13 (9.1 g) was subjected to CC of reversed-phase C18 (MeOH – H2O, 30:70 ! 90:10) to afford 16 (20.3 mg).
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3.3.1
Podocarflavone A (1)
Yellowish amorphous solid (3.5 mg); m.p. 223 –2248C; UV (MeOH) lmax (log 1) nm: 208 (4.29), 268 (4.23), and 349 (3.96); IR nmax cm21: 3333, 1653, 1612, 1507, 1466, 1363, 1336, 1175, 1171, 1119, and 836; For 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS m/z 361.0709 [M – H] – (calcd for C21H13O6, 361.0712). 3.4 Antioxidant activity Some compounds were subjected to in vitro antioxidative testing using the DPPHz, ferrous ions, and ABTSzþ radical scavenging assay as previously reported. 3.4.1 DPPH free radical scavenging activity The free radical scavenging activity was measured by DPPHz. Briefly, 60 mM solution of DPPH in methanol was prepared and 100 ml of this solution was added to 100 ml of the test compound solution in methanol at different concentrations (0.125, 0.25, 0.5, 1.0, and 2.0 mM). After 30 min, the absorbance was measured at 517 nm. The capability to scavenge the DPPH radical was calculated using the following equation: DPPH scavenging effect ð%Þ ASample ¼ 12 £ 100; AControl where AControl is the absorbance of the control reaction and ASample is the absorbance in the presence of test compound. 3.4.2 Ferrous metal ions chelating activity The test compound in 100 ml was added to a solution of 1 mM FeCl2 (5 ml). The reaction
227
was initiated by the addition of 1 mM ferrozine (15 ml). Then, the mixture was shaken vigorously and left at room temperature for 10 min. The absorbance was measured at 570 nm. The inhibition percentage of ferrozine–Fe2þ complex formation was calculated by using the formula given below: Metal chelating effect ð%Þ ASample ¼ 12 £ 100; AControl where AControl is the absorbance of control and ASample is the absorbance in the presence of the test compound.
3.4.3 ABTS radical cation decolorization assay The ABTSzþ cation radical was produced by the reaction between 7 mM ABTS in H2O and 140 mM potassium persulfate, stored in dark at room temperature for 12 h. Before usage, the ABTSzþ solution was diluted to get an absorbance of 0.700 ^ 0.025 at 734 nm. Then, 100 ml of ABTSzþ solution was added to 100 ml of the test compounds solution in methanol at different concentrations. After 30 min, the percentage inhibition at 734 nm was calculated for each concentration relative to a blank absorbance (methanol). The scavenging capability of ABTSzþ radical was calculated using the following equation: ABTS·þ scavenging effect ð%Þ ASample ¼ 12 £ 100; AControl where AControl is the initial concentration of the ABTSzþ and ASample is the absorbance of the remaining concentration of ABTSzþ in the presence of the test compounds.
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3.5 Myocardial protective bioassay 3.5.1 Cell viability measurements Rat cardiac H9c2 cells (ATCC, Rockville, MD, USA) were cultured in high Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum, 1% penicillin, and 5% CO2 at 378C. The cells were fed every 2– 3 days and subcultured once they reached 70 –80% confluence. Cells were plated at an appropriate density according to each experimental design. The tested compounds were dissolved in dimethylsulfoxide (0.1% DMSO) and serially diluted in phosphate-buffered saline (PBS) immediately prior to the experiment, and were added into the cells with different concentrations for 12 h followed by the addition of doxorubicin (1024 M) for another 3 h to make the cell injury model. After this incubation, cells were measured by a modified MTT assay. The H9c2 cells were treated with MTT solution (final concentration, 0.5 mg/ml) for 4 h at 378C in 96-well plates. The supernatants were removed carefully, followed by the addition of 100 ml DMSO to each well to dissolve the precipitate. Then, the absorbance was measured at 490 nm in a Model 680 microplate reader (BIO-RAD, California, USA). 3.5.2 Measurement of the levels of LDH and CK Cellular injury was determined by measuring the levels of LDH released into the cell culture medium. According to the manufacturer’s instruction (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), LDH was assayed by measuring the increase in the absorbance of nicotinamide adenine dinucleotide at 450 nm at 258C, using a Model 680 microplate reader (BIO-RAD). Cellular damage was evaluated by measuring the levels of CK release in the culture medium. After treatments, where indicated, a spectrophotometric CK enzyme assay was performed with the test
kits (Jiancheng Bioengineering Institute) and UV-2550 mode spectrophotometer (Shimadzu) at 660 nm. All data were expressed as mean ^ SD, and a value of p , 0.05 was considered statistically significant.
3.5.3 Determination of ROS generation Changes in intracellular ROS levels were determined by measuring the oxidative conversion of cell-permeable 20 ,70 -dichlorfluorescein-diacetate (DCFHDA) to fluorescent dichlorofluorescein (DCF) in a FACS-SCAN apparatus (FACSCalibur; BD Biosciences, California, USA). In the presence of ROS, DCFH reacts with ROS to form the fluorescent product DCF, which is trapped inside the cells. To obtain the dissociated microglia for the ROS assay, the culture medium was first removed and the cells were washed three times with PBS. The cells were incubated with DCFH-DA at 378C for 20 min. The cellular fluorescence was measured by flow cytometry analysis with a FACS-SCAN apparatus. The decrease in value compared with that of the control was viewed as the decrease of intracellular ROS.
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