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Cite this: Food Funct., 2014, 5, 1369
Received 18th December 2013 Accepted 8th March 2014
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Protective effects of diphenylheptanes from Curcuma phaeocaulis Val. on H2O2 induced cell injury Yun-Fang Hao,†a Chuan-Li Lu,†ab Dong-Jun Li,c Liang Zhu,a Jian-Guo Jiang*a and Jin-Hua Piao*a
DOI: 10.1039/c3fo60687b www.rsc.org/foodfunction
Curcuma phaeocaulis Val. has been used as a health food in China for a long time. This research aimed to isolate and identify its active compounds with protective effects against hydrogen peroxideinduced PC12 cell death. 70% ethanol extracts of C. phaeocaulis were re-extracted and three fractions of water, petroleum ether and ethyl acetate were obtained. Three diphenylheptane compounds from the ethyl acetate fraction were identified for the first time from C. phaeocaulis, and compound III was considered to be a new structure. All of the three compounds displayed certain protective effects against toxicity in PC12 cells. For all concentrations, compound III displayed a more significant protective effect than ethanol extracts, the ethyl acetate fraction, and the other two compounds. At a concentration of 50 mg mL1, the survival rate of damaged PC-12 cells treated with compound III reached 84.7%. Diphenylheptanes were concluded to be the main compounds responsible for the health effects of C. phaeocaulis.
1. Introduction Curcuma phaeocaulis Val., belonging to the family Zingiberaceae, is mainly distributed in the southwest of China.1 Its dried rhizomes have been used for a long time as a health food due to their function of removing blood stasis and alleviating pain. In clinical practice in China, C. phaeocaulis, together with Curcuma kwangsiensis S.G. Lee et C.F. Liang and Curcuma wenyujin Y.H. Chen et C. Ling, is commonly prescribed for the treatment of tumors and cardiovascular diseases. It has been reported that methanol extracts of C. phaeocaulis could inhibit paw swelling, decrease serum haptoglobin a
College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640, China. E-mail: [email protected]; [email protected]; Fax: +86-20-87113843; Tel: +86-20-87113849
b
The Second Institute of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, China
c College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, 510642, China
† These authors contributed to the work equally and should be regarded as co-rst authors.
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concentration, and reduce cyclooxygenase-2 activity in mice with adjuvant arthritis.2 C. phaeocaulis was also reported to have signicant anti-inammatory activity, and furanodienone and curcumenol were considered to be the major active antiinammatory constituents.3–5 In addition, an ethanol extract of C. phaeocaulis could inhibit the proliferation of MCF-7 cells by inducing apoptosis, increasing ROS formation, regulating BcI-2 family protein expression, and activating caspases.6 C. phaeocaulis also showed memory improvement and anti-aging effects in mice.7 It also had signicant anti-platelet aggregation and anti-coagulant activity, which could be enhanced when it was pretreated with vinegar.8 Although C. phaeocaulis has been reported to have various bioactivities, most of the results were based on its crude extracts. Therefore, in the present research, we tried to extract and separate active constituents from C. phaeocaulis. Additionally, the protective effects of different fractions and compounds on hydrogen peroxide-induced PC12 cell injury were tested and compared in order to better understand the efficacy of C. phaeocaulis.
2.
Results and discussion
2.1. Structural identication of the isolated compounds Three compounds were obtained from the ethyl acetate fraction of C. phaeocaulis aer repeated isolation and purication (Fig. 1). The purities of the three compounds were more than 98% by HPLC analysis. Their chemical structures are shown in Fig. 2. These three compounds, all diphenylheptane derivatives, were identied from this plant for the rst time, and were characteristic constituents of curcuma plants. Compound I, a light yellow oil, displayed a purply red color when vanillin–sulfuric acid was used as the spray reagent. The molecular formula was inferred as C20H26O5 according to its EIMS, 1H and 13C NMR, and DEPT spectra. In the low eld of 1H NMR (400 MHz, DMSO-d6) (Table 1), it showed ve aromatic hydrogen signals at d 6.98 (2H, d, J ¼ 8.4 Hz), 6.81 (2H, d, J ¼ 8.4 Hz), 6.75 (1H, d, J ¼ 2.0 Hz), 6.72 (1H, d, J ¼ 8.4 Hz) and 6.61 Food Funct., 2014, 5, 1369–1373 | 1369
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Fig. 1
The extraction and separation procedure of C. phaeocaulis.
Chemical structures of the compounds isolated from C. phaeocaulis. I: 1-(4-hydroxy phenyl)-7-(4-hydroxy-3-methoxy phenyl)-3,5-dihydroxyheptane. II: 1,5-epoxy-3-hydroxy-1-(3,4-dihydroxy-5-methoxy phenyl)-7-(4-hydroxy-3-methoxy phenyl) heptane. III: 1-(4-hydroxy-3-methoxy phenyl)-7-(3,4-dihydroxy-5-methoxy phenyl)-3,5-dihydroxyheptane.
Fig. 2
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(1H, dd, J ¼ 8.4, 2.0 Hz) indicating that there were contraposition-disubstituted benzene and 1,2,4-trisubstituted benzene in the structure. Signals at d 3.80 (3H, s) indicated the existence of methoxyl. There were ve hydrogen signals at d 3.8–1.4. In the 13 C NMR (MeOH-d6, 100 MHz) (Table 1), there were 16 carbon signals. Combining this with information from DEPT and 1 H-NMR, the structure of the compound should include 20 carbon atoms. In the low eld, there were two benzene carbon signals [d 156.33, 148.85, 145.49, 135.23, 134.43, 130.27 (]CH– 2), 121.81, 116.12 (]CH– 3) and 113.27], while there were ve methylene signals (d 44.83, 40.85, 40.78, 32.31 and 31.83) in the high eld. From the above analysis, it can be concluded that compound I is a diphenylheptane. By comparing with previously reported spectral data,9 the structure of compound I was identied as 1-(4-hydroxy phenyl)-7-(4-hydroxy-3-methoxy phenyl)-3,5-dihydroxyheptane. Compound II, a light yellow oil, displayed a purply red color like compound I. Its molecular formula was inferred as C21H25O7 according to its EI-MS, 1H and 13C NMR, and DEPT spectra. In the low eld of 1H NMR (400 MHz, DMSO-d6) (Table 1), it showed four aromatic hydrogen signals at d 6.76 (1H, d, J ¼ 1.8 Hz), 6.69 (1H, d, J ¼ 8.0 Hz) and 6.62 (1H, dd, J ¼ 8.0, 1.8 Hz) indicated that there was 1,2,4-trisubstituted benzene in the structure. Signals at d 3.84 (3H, s) and 3.78 (3H, s) indicated the existence of methoxyl. In the 13C NMR (MeOH-d6, 100 MHz) (Table 1), there were 21 carbon signals. There were 2 benzene carbon signals at d 150–100: 149.7, 149.0, 146.6, 145.6, 135.6, 135.4, 134.6, 122.1, 116.3, 113.6, 108.2 and 103.0. The above data hinted that compound II was a diphenylheptane. By comparing with previously reported spectral data.10 the structure of compound II was identied as 1,5-epoxy-3-hydroxy-1-(3,4dihydroxy-5-methoxy phenyl)-7-(4-hydroxy-3-methoxy phenyl)heptane. Compound III, a light yellow powder, also showed a purply red color when vanillin-sulfuric acid was used as the spray reagent. Its molecular formula was inferred as C21H28O7 according to its EI-MS, 1H and 13C NMR, and DEPT spectra. In the low eld of 1H NMR (400 MHz, DMSO-d6) (Table 1), it showed ve aromatic hydrogen signals at d 6.77 (1H, d, J ¼ 2.0 Hz), 6.72 (1H, dd, J ¼ 8.0, 2.0 Hz) and 6.64 (1H, d, J ¼ 8.0 Hz) indicating that there was 1,2,4-trisubstituted benzene in the structure. Signals at d 3.83 (3H, s) and 3.81 (3H, s) indicated the existence of methoxyl. There were 21 carbon signals in the 13C NMR (MeOH-d6, 100 MHz) (Table 1), while there were two benzene carbon signals at d 150–100: 149.7, 149.0, 146.6, 145.6, 135.5, 134.7, 133.3, 122.1, 116.3, 113.4, 108.2 and 103.0. It showed nine carbon signals at d 80–30, which indicated compound III was also a diphenylheptane. Compound III and compound I showed the same saturated hydrogen and carbon signals. The difference is that the benzene ring in compound III showed 1,2,3,5-tetra-substituted benzene while compound I showed contraposition-disubstituted benzene. Compared with compound II, compound III had the same hydrogen and carbon signals in the aromatic region but different saturated hydrogen and carbon signals. Therefore it was determined to be 1-(4hydroxy-3-methoxy phenyl)-7-(3,4-dihydroxy-5-methoxy phenyl)3,5-dihydroxyheptane. No reports about compound III have
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1
H and
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13
C-NMR data for compounds I–III in CD3OD
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I
II
No
1
13
1 2 3 4 5 6 7 10 20 30 40 50 60 100 200 300 400 500 600 30 -OMe 300 -OMe
2.63–2.49 (4H, m) 1.74–1.54 (4H, m) 3.74 (2H, m) 1.58 (2H, m) 6.68 (2H, m) 1.74–1.54 (4H, m) 2.63–2.49 (4H, m)
32.3 40.8 71.0 44.8 71.0 40.9 31.8 135.2 113.3 148.9 145.5 116.1 121.8 134.4 130.3 116.1 156.3 116.1 139.3
H-NMR
6.75 (1H, d, J ¼ 8.4 Hz) 6.68 (1H, d, J ¼ 8.4 Hz) 6.61 (1H, dd, J ¼ 1.8, 8.4 Hz) 6.98 (2H, d, J ¼ 8.4 Hz) 6.67 (2H, d, J ¼ 8.4 Hz) 6.967 (2H, d, J ¼ 8.4 Hz) 6.98 (2H, d, J ¼ 8.4 Hz) 3.80 (3H, s)
C-NMR
56.4
1
III
H-NMR
1
75.4 41.4 65.8 39.5 72.7 39.5 32.4 134.6 108.2 146.6 135.4 149.7 103.0 135.5 113.6 149.0 145.6 116.3 122.1 56.9 56.6
2.68–2.48 (4H, m) 1.74–1.54 (4H, m) 3.80 (2H, m) 1.64 (2H, m) 6.35 (2H, m) 1.74–1.54 (4H, m) 2.68–2.48 (4H, m)
C-NMR
4.65 (1H, dd, J ¼ 1.8, 11.4 Hz) 1.87–1.75 (2H, m) 1.70–1.63(3H, m) 4.22 (1H, t, J ¼ 2.6 Hz) 1.70–1.63 (3H, m) 1.57–1.50 (2H, m) 3.88 (1H, m) 1.87–1.75 (2H, m) 1.70–1.63 (3H, m) 2.66 (2H, m) 6.54 (2H, brs)
6.54 (2H, brs) 6.76 (1H, d, J ¼ 1.8 Hz)
6.69 (1H, 6.62 (1H, 3.84 (3H, 6.68 (3H,
13
d, J ¼ 8.0 Hz) d, J ¼ 1.8, 8.0 Hz) s) s)
H-NMR
6.56 (2H, brs)
6.56 (2H, brs) 6.79 (1H, d, J ¼ 1.8 Hz)
6.65 6.63 3.85 6.80
(1H, d, J ¼ 8.0 Hz) (1H, d, J ¼ 1.8, 8.0 Hz) (3H, s) (3H, s)
13
C-NMR
32.4 39.8 71.2 45.0 71.2 39.8 32.8 134.7 108.2 146.6 135.4 149.7 103.0 135.4 113.4 149.0 145.6 116.3 122.0 56.6 56.8
been found in SciFinder scholar based on its structural formula, which means that compound III is a new compound.
73.2%) at 60 mg mL1 and little cytotoxicity (cell survival > 92%) at 10 to 40 mg mL1. The water fraction exhibited no cytotoxicity (cell survival > 95%) at all of the test concentrations.
2.2
2.3. Protective effects
Cytotoxicity assay
As shown in Fig. 3, the ethanol extract of C. phaeocaulis showed no obvious cytotoxicity (cell survival > 95%) at 10 to 50 mg mL1, while the cell survival decreased sharply to 86.1% at 60 mg mL1. The ethyl acetate fraction also did not exhibit signicant cytotoxicity (cell survival > 95%) at 10 to 40 mg mL1, while the cell survival was 85.7 to 72.3% at 50 to 60 mg mL1. The petroleum ether fraction exhibited moderate cytotoxicity (cell survival was
Cytotoxic effects of the ethanol extracts and their polar fractions of C. phaeocaulis on PC-12 cells. Ethyl acetate fraction (EZ-EA), petroleum ether fraction (EZ-PE), and water fraction (EZ-W) from 70% ethanol extract (EZ-Z). Fig. 3
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Yousef et al.11 reported that Curcumin could successfully restore liver function and inhibit oxidative injury induced by paracetamol. The results of this research show that samples prepared from C. phaeocaulis also exhibited certain protective effects against H2O2 induced cell injury in a concentration-
Fig. 4 Protective effects of the ethanol extracts, their polar fractions and three compounds isolated from C. phaeocaulis against hydrogen peroxide-induced cell death in PC-12 cells. Ethyl acetate fraction (EZEA), petroleum ether fraction (EZ-PE), and water fraction (EZ-W) from 70% ethanol (EZ-Z). Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with control.
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dependent manner (Fig. 4). The protective effect of the three compounds and four extracts from C. phaeocaulis showed no obvious difference at low concentrations (#20 mg mL1). Both EZ-Z and EZ-EA exhibited signicant protective effects. EZ-Z and EZ-EA could raise the PC12 cell survival rate from 49.2 to 74.6% and 51.5 to 71.7% at concentrations ranging from 10 to 50 mg mL1, respectively. EZ-PE did not exhibit a signicant protective effect at low concentrations (#30 mg mL1), but could slightly raise the cell survival rate (56.6%) at a concentration of 50 mg mL1. EZ-W almost had no protective effect at all concentrations. Fig 4 shows that compound III had the highest protective activity at all concentrations. At a concentration of 20 mg mL1 of compound III, PC-12 cells survival rate was 54.7%, and at 50 mg mL1 it reached 83.3%. Compound I and compound II also exhibited obvious protective effects, especially at higher concentrations. At 50 mg mL1, the survival rate of cells treated by compound I was 78.6%, second only to compound III. Compound II also showed a high activity at 50 mg mL1, where the cell survival rate was 70.6%. These results suggest that diphenylheptanes may be the active compounds responsible for the protective effect of C. phaeocaulis against hydrogen peroxide-induced PC12 cell injury. In conclusion, the ethanol extract of C. phaeocaulis and its ethyl acetate fraction showed signicant chemical protective effects. Three diphenylheptane compounds, 1-(4-hydroxy phenyl)-7-(4-hydroxy-3-methoxy phenyl)-3,5-dihydroxyheptane (I), 1,5-epoxy-3-hydroxy-1-(3,4-dihydroxy-5-methoxy phenyl)-7(4-hydroxy-3-methoxy phenyl)heptane (II), and 1-(4-hydroxy-3methoxy phenyl)-7-(3,4-dihydroxy-5-methoxy phenyl)-3,5-dihydroxyheptane (III), were isolated from the ethyl acetate fraction, which were identied for the rst time from C. phaeocaulis, and compound III was a new compound. The three compounds displayed a greater protective effect against H2O2 induced cell injury than the ethanol extracts at a concentration of 50 mg mL1, and compound III showed the highest activity at almost all concentrations. The results obtained in this work might contribute to a better understanding of the biological activity of C. phaeocaulis.
3.
Materials and methods
3.1. Plant material The dried rhizome of C. phaeocaulis, derived from Sichuan province, was purchased from the Qingping market for Chinese medicinal material in Guangzhou, China. A voucher specimen was deposited in the department of Natural Products Studies, School of Light Chemistry and Food Science, South China University of Technology. 3.2. Reagents Methanol, chloroform, petroleum ether, concentrated sulfuric acid, glacial acetic acid, vanillin, and ethyl acetate were analytical grade and absolute ethanol was food grade. All chemicals were purchased from East Giant experiment instrument company (Guangzhou, China).
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3.3. Ethanol extracts from the rhizome of C. phaeocaulis A pre-prepared powder (3.0 kg) of C. phaeocaulis was extracted with 70% ethanol under reux (3 7 L, each 2.5 h). The leach liquor was combined and concentrated under reduced pressure at 45 C to give a residue (EZ-Z, 77.8 g), which was suspended in water (1 L) and then partitioned with petroleum ether (3 1 L) and ethyl acetate (3 1 L) successively to give a petroleum ether fraction (EZ-PE, 36.5 g), an ethyl acetate fraction (EZ-EA, 28.7 g) and a water remnant (EZ-W, 9.4 g), respectively. 3.4. Isolation and identication of chemical components The ethyl acetate fraction showed the best protective effect among the three extracts in the cell protective effect screening tests (Fig. 4). Therefore, the ethyl acetate fraction was chosen for the following isolation. The pre-prepared ethyl acetate fraction was subjected to silica gel column chromatography (Ø 6.5 1200 cm) with a gradient of chloroform–methanol12 to yield 12 sub-fractions (EE-1 to EE-12). EE-7 (eluted by chloroform– methanol 85 : 15) was further subjected to ODS-A chromatography and eluted with a gradient of methanol–water to give four sub-fractions (EE-7a to EE-7d). EE-7b (40% methanol eluted) was used for HPLC with methanol–water as an eluent to obtain compounds I–III. The extraction and separation procedures are shown in Fig. 1. The three compounds were detected by electrospray ionization ion trap multiple mass spectrometry (EI-MS) and nuclear magnetic resonance spectroscopy (NMR) in order to identify their structures. EI-MS was measured on a Bruker Esquire HCTplus LC/MS system using both the positive- and the negative-ion modes with a scan range of 200–2000. 1H and 13C NMR, and DEPT spectra were recorded on a Bruker Daltonics DRX-400 spectrometer. 3.5. Protective effects on H2O2 induced cell injury The PC12 cell line was obtained from Sun Yat-Sen University, Guangzhou, China, and maintained at 37 C in a humidied atmosphere containing 5% CO2 in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U mL1) and streptomycin (100 mg mL1).13 To produce oxidative stress, H2O2 was freshly prepared from 30% stock solution prior to each experiment. Exponential growth phase PC-12 cells, at a density of 105 cells per mL, were seeded in 96-well culture plates (100 mL per well). Aer incubation for 16 h, the supernatant media were discarded and replaced with equal volumes of fresh media containing different concentrations of samples. The cells were then incubated for 0.5 h. Then the H2O2 solution was added into the media with a nal concentration of 600 mM and incubated for 24 h at 37 C according to our previous work.14 Cell survival was evaluated by a 3-(4,5-dimethylthiazol-z-yl)2,5-diphenyl tetrazolium bromide (MTT) test15 on the basis that the succinate dehydrogenase in the mitochondria of living cells can cleave the tetrazolium ring of MTT to produce formazan.16–18 Briey, aer 4 h of incubation with MTT (0.5 mg mL1) at 37 C, cells were lysed in dimethylsulfoxide (DMSO)
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and the amount of MTT formazan was quantied by determining the absorbance at 492 nm.
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3.6. Statistic analysis Data were expressed as mean SD (n ¼ 5), and statistical comparisons were made by means of a one-way ANOVA test followed by Dunett's t-test. P < 0.05 was considered statistically signicant, and P < 0.01 indicated the presence of a highly signicant difference.
References 1 B. Sasikumar, Genetic resources of curcuma: diversity, characterization and utilization, Plant Genet. Resour., 2005, 3, 230–251. 2 Y. Sasaki, H. Goto, C. Tohda, F. Hatanaka, N. Shibahara, Y. Shimada, K. Terasawa and K. Komatsu, Effects of curcuma drugs on vasomotion in isolated rat aorta, Biol. Pharm. Bull., 2003, 26, 1135–1143. 3 C. Tohda, N. Nakayama, F. Hatanaka and K. Komatsu, Comparison of anti-inammatory activities of six curcuma rhizomes: a possible curcuminoid-independent pathway mediated by Curcuma phaeocaulis extract, J. Evidence-Based Complementary Altern. Med., 2006, 3, 255–260. 4 S. R. Naik, V. N. Thakare and S. R. Path, Protective effect of curcumin on experimentally induced inammation, hepatotoxicity and cardiotoxicity in rats: Evidence of its antioxidant property, Exp. Toxicol. Pathol., 2011, 63, 419–431. 5 G. Angel-Morales, G. Noratto and S. U. Mertens-Talcott, Standardized curcuminoid extract (Curcuma longa L.) decreases gene expression related to inammation and interacts with associated microRNAs in human umbilical vein endothelial cells (HUVEC), Food Funct., 2012, 3, 1286– 1293. 6 K. Tanaka, Y. Kuba, A. Ina, H. Watanabe and K. Komatsu, Prediction of cyclooxygenase inhibitory activity of curcuma rhizome from chromatograms by multivariate analysis, Chem. Pharm. Bull., 2008, 56, 936–940. 7 X. P. Chen, L. X. Pei, Z. F. Zhong, J. J Guo, Q. W. Zhang and Y. T. Wang, Anti-tumor potential of ethanol extract of Curcuma phaeocaulis Val. against breast cancer cells, Phytomedicine, 2011, 18, 1238–1243.
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8 C. Q. Mao, H. Xie and T. L. Lu, Studies on antiplatelet aggregation and analgesic action of Curcuma phaeocaulis, J. Chin. Med. Mater., 2000, 23, 212–213. 9 A. Yokosuka, Y. Mimaki, H. Sakagami and Y. Sashlda, New diarylheptanoids and diarylheptanoid glucosides from the rhizomes of Tacca chantrieri and their cytotoxic activity, J. Nat. Prod., 2002, 65, 283–289. 10 H. Kikuzaki and N. Nakatani, Cyclic diarylheptanoids from rhizomes of Zingiber officinale, Phytochemistry, 1996, 43, 273–277. 11 M. L. Yousef, S. A. M. Omar, M. I. El-Guendia and L. A. Abdelmegid, Potential protective effects of quercetin and curcumin on paracetamol-induced histological changes, oxidative stress, impaired liver and kidney functions and haematotoxicity in rat, Food Chem. Toxicol., 2010, 48, 3246–3261. 12 Q. M. Liu, H. Y. Zhao, X. K. Zhong and J. G. Jiang, Eclipta prostrata L. phytochemicals: isolation, structure elucidation, and their antitumor activity, Food Chem. Toxicol., 2012, 50, 4016–4022. 13 S. L. Hqang, J. A. Lin, P. H. Shih, C. T. Yeh and G. C. Yen, Procellular survival and neuroprotection of citrus avonoid: the actions of hesperetin in PC12 cells, Food Funct., 2012, 3, 1082–1090. 14 H. Y. Zhao, L. Yang, J. Wei, M. Huang and J. G. Jiang, Bioactivity evaluations of ingredients extracted from the owers of Citrusaurantium L. var. amara Engl, Food Chem., 2012, 135, 1725–2181. 15 K. N. C. Murthy, G. K. Jayaprakasha and B. S. Patil, Citrus limonoids and curcumin additively inhibit human colon cancer cells, Food Funct., 2013, 5, 803–810. 16 V. D. La, J. Labrecque and D. Grenier, Cytoprotective effect of proanthocyanidin-rich cranberry fraction against bacterial cell wall-mediated toxicity in macrophages and epithelial cells, Phytother. Res., 2009, 23, 1449–1452. 17 J. Tavakkol-Afshari, A. Brook and S. H. Mousavi, Study of cytotoxic and apoptogenic properties of saffron extract in human cancer cell lines, Food Chem. Toxicol., 2008, 46, 3443–3447. 18 C. L. Lu, Y. M. Li, G. Q. Fu, L. Yang, J. G. Jiang, L. Zhu, F. L. Lin, J. Chen and X. S. Lin, Extraction optimization of daphnoretin from root bark of Wikstroemia indica (L.) C.A. and its anti-tumor activity test, Food Chem., 2011, 124, 1500–1506.
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Protective effects of diphenylheptanes from Curcuma phaeocaulis Val. on H2O2 induced cell injury Yun-Fang Hao,†a Chuan-Li Lu,†ab Dong-Jun Li,c Liang Zhu,a Jian-Guo Jiang*a and Jin-Hua Piao*a
DOI: 10.1039/c3fo60687b www.rsc.org/foodfunction
Curcuma phaeocaulis Val. has been used as a health food in China for a long time. This research aimed to isolate and identify its active compounds with protective effects against hydrogen peroxideinduced PC12 cell death. 70% ethanol extracts of C. phaeocaulis were re-extracted and three fractions of water, petroleum ether and ethyl acetate were obtained. Three diphenylheptane compounds from the ethyl acetate fraction were identified for the first time from C. phaeocaulis, and compound III was considered to be a new structure. All of the three compounds displayed certain protective effects against toxicity in PC12 cells. For all concentrations, compound III displayed a more significant protective effect than ethanol extracts, the ethyl acetate fraction, and the other two compounds. At a concentration of 50 mg mL1, the survival rate of damaged PC-12 cells treated with compound III reached 84.7%. Diphenylheptanes were concluded to be the main compounds responsible for the health effects of C. phaeocaulis.
1. Introduction Curcuma phaeocaulis Val., belonging to the family Zingiberaceae, is mainly distributed in the southwest of China.1 Its dried rhizomes have been used for a long time as a health food due to their function of removing blood stasis and alleviating pain. In clinical practice in China, C. phaeocaulis, together with Curcuma kwangsiensis S.G. Lee et C.F. Liang and Curcuma wenyujin Y.H. Chen et C. Ling, is commonly prescribed for the treatment of tumors and cardiovascular diseases. It has been reported that methanol extracts of C. phaeocaulis could inhibit paw swelling, decrease serum haptoglobin a
College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640, China. E-mail: [email protected]; [email protected]; Fax: +86-20-87113843; Tel: +86-20-87113849
b
The Second Institute of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, China
c College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, 510642, China
† These authors contributed to the work equally and should be regarded as co-rst authors.
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concentration, and reduce cyclooxygenase-2 activity in mice with adjuvant arthritis.2 C. phaeocaulis was also reported to have signicant anti-inammatory activity, and furanodienone and curcumenol were considered to be the major active antiinammatory constituents.3–5 In addition, an ethanol extract of C. phaeocaulis could inhibit the proliferation of MCF-7 cells by inducing apoptosis, increasing ROS formation, regulating BcI-2 family protein expression, and activating caspases.6 C. phaeocaulis also showed memory improvement and anti-aging effects in mice.7 It also had signicant anti-platelet aggregation and anti-coagulant activity, which could be enhanced when it was pretreated with vinegar.8 Although C. phaeocaulis has been reported to have various bioactivities, most of the results were based on its crude extracts. Therefore, in the present research, we tried to extract and separate active constituents from C. phaeocaulis. Additionally, the protective effects of different fractions and compounds on hydrogen peroxide-induced PC12 cell injury were tested and compared in order to better understand the efficacy of C. phaeocaulis.
2.
Results and discussion
2.1. Structural identication of the isolated compounds Three compounds were obtained from the ethyl acetate fraction of C. phaeocaulis aer repeated isolation and purication (Fig. 1). The purities of the three compounds were more than 98% by HPLC analysis. Their chemical structures are shown in Fig. 2. These three compounds, all diphenylheptane derivatives, were identied from this plant for the rst time, and were characteristic constituents of curcuma plants. Compound I, a light yellow oil, displayed a purply red color when vanillin–sulfuric acid was used as the spray reagent. The molecular formula was inferred as C20H26O5 according to its EIMS, 1H and 13C NMR, and DEPT spectra. In the low eld of 1H NMR (400 MHz, DMSO-d6) (Table 1), it showed ve aromatic hydrogen signals at d 6.98 (2H, d, J ¼ 8.4 Hz), 6.81 (2H, d, J ¼ 8.4 Hz), 6.75 (1H, d, J ¼ 2.0 Hz), 6.72 (1H, d, J ¼ 8.4 Hz) and 6.61 Food Funct., 2014, 5, 1369–1373 | 1369
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Fig. 1
The extraction and separation procedure of C. phaeocaulis.
Chemical structures of the compounds isolated from C. phaeocaulis. I: 1-(4-hydroxy phenyl)-7-(4-hydroxy-3-methoxy phenyl)-3,5-dihydroxyheptane. II: 1,5-epoxy-3-hydroxy-1-(3,4-dihydroxy-5-methoxy phenyl)-7-(4-hydroxy-3-methoxy phenyl) heptane. III: 1-(4-hydroxy-3-methoxy phenyl)-7-(3,4-dihydroxy-5-methoxy phenyl)-3,5-dihydroxyheptane.
Fig. 2
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(1H, dd, J ¼ 8.4, 2.0 Hz) indicating that there were contraposition-disubstituted benzene and 1,2,4-trisubstituted benzene in the structure. Signals at d 3.80 (3H, s) indicated the existence of methoxyl. There were ve hydrogen signals at d 3.8–1.4. In the 13 C NMR (MeOH-d6, 100 MHz) (Table 1), there were 16 carbon signals. Combining this with information from DEPT and 1 H-NMR, the structure of the compound should include 20 carbon atoms. In the low eld, there were two benzene carbon signals [d 156.33, 148.85, 145.49, 135.23, 134.43, 130.27 (]CH– 2), 121.81, 116.12 (]CH– 3) and 113.27], while there were ve methylene signals (d 44.83, 40.85, 40.78, 32.31 and 31.83) in the high eld. From the above analysis, it can be concluded that compound I is a diphenylheptane. By comparing with previously reported spectral data,9 the structure of compound I was identied as 1-(4-hydroxy phenyl)-7-(4-hydroxy-3-methoxy phenyl)-3,5-dihydroxyheptane. Compound II, a light yellow oil, displayed a purply red color like compound I. Its molecular formula was inferred as C21H25O7 according to its EI-MS, 1H and 13C NMR, and DEPT spectra. In the low eld of 1H NMR (400 MHz, DMSO-d6) (Table 1), it showed four aromatic hydrogen signals at d 6.76 (1H, d, J ¼ 1.8 Hz), 6.69 (1H, d, J ¼ 8.0 Hz) and 6.62 (1H, dd, J ¼ 8.0, 1.8 Hz) indicated that there was 1,2,4-trisubstituted benzene in the structure. Signals at d 3.84 (3H, s) and 3.78 (3H, s) indicated the existence of methoxyl. In the 13C NMR (MeOH-d6, 100 MHz) (Table 1), there were 21 carbon signals. There were 2 benzene carbon signals at d 150–100: 149.7, 149.0, 146.6, 145.6, 135.6, 135.4, 134.6, 122.1, 116.3, 113.6, 108.2 and 103.0. The above data hinted that compound II was a diphenylheptane. By comparing with previously reported spectral data.10 the structure of compound II was identied as 1,5-epoxy-3-hydroxy-1-(3,4dihydroxy-5-methoxy phenyl)-7-(4-hydroxy-3-methoxy phenyl)heptane. Compound III, a light yellow powder, also showed a purply red color when vanillin-sulfuric acid was used as the spray reagent. Its molecular formula was inferred as C21H28O7 according to its EI-MS, 1H and 13C NMR, and DEPT spectra. In the low eld of 1H NMR (400 MHz, DMSO-d6) (Table 1), it showed ve aromatic hydrogen signals at d 6.77 (1H, d, J ¼ 2.0 Hz), 6.72 (1H, dd, J ¼ 8.0, 2.0 Hz) and 6.64 (1H, d, J ¼ 8.0 Hz) indicating that there was 1,2,4-trisubstituted benzene in the structure. Signals at d 3.83 (3H, s) and 3.81 (3H, s) indicated the existence of methoxyl. There were 21 carbon signals in the 13C NMR (MeOH-d6, 100 MHz) (Table 1), while there were two benzene carbon signals at d 150–100: 149.7, 149.0, 146.6, 145.6, 135.5, 134.7, 133.3, 122.1, 116.3, 113.4, 108.2 and 103.0. It showed nine carbon signals at d 80–30, which indicated compound III was also a diphenylheptane. Compound III and compound I showed the same saturated hydrogen and carbon signals. The difference is that the benzene ring in compound III showed 1,2,3,5-tetra-substituted benzene while compound I showed contraposition-disubstituted benzene. Compared with compound II, compound III had the same hydrogen and carbon signals in the aromatic region but different saturated hydrogen and carbon signals. Therefore it was determined to be 1-(4hydroxy-3-methoxy phenyl)-7-(3,4-dihydroxy-5-methoxy phenyl)3,5-dihydroxyheptane. No reports about compound III have
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1
H and
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13
C-NMR data for compounds I–III in CD3OD
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I
II
No
1
13
1 2 3 4 5 6 7 10 20 30 40 50 60 100 200 300 400 500 600 30 -OMe 300 -OMe
2.63–2.49 (4H, m) 1.74–1.54 (4H, m) 3.74 (2H, m) 1.58 (2H, m) 6.68 (2H, m) 1.74–1.54 (4H, m) 2.63–2.49 (4H, m)
32.3 40.8 71.0 44.8 71.0 40.9 31.8 135.2 113.3 148.9 145.5 116.1 121.8 134.4 130.3 116.1 156.3 116.1 139.3
H-NMR
6.75 (1H, d, J ¼ 8.4 Hz) 6.68 (1H, d, J ¼ 8.4 Hz) 6.61 (1H, dd, J ¼ 1.8, 8.4 Hz) 6.98 (2H, d, J ¼ 8.4 Hz) 6.67 (2H, d, J ¼ 8.4 Hz) 6.967 (2H, d, J ¼ 8.4 Hz) 6.98 (2H, d, J ¼ 8.4 Hz) 3.80 (3H, s)
C-NMR
56.4
1
III
H-NMR
1
75.4 41.4 65.8 39.5 72.7 39.5 32.4 134.6 108.2 146.6 135.4 149.7 103.0 135.5 113.6 149.0 145.6 116.3 122.1 56.9 56.6
2.68–2.48 (4H, m) 1.74–1.54 (4H, m) 3.80 (2H, m) 1.64 (2H, m) 6.35 (2H, m) 1.74–1.54 (4H, m) 2.68–2.48 (4H, m)
C-NMR
4.65 (1H, dd, J ¼ 1.8, 11.4 Hz) 1.87–1.75 (2H, m) 1.70–1.63(3H, m) 4.22 (1H, t, J ¼ 2.6 Hz) 1.70–1.63 (3H, m) 1.57–1.50 (2H, m) 3.88 (1H, m) 1.87–1.75 (2H, m) 1.70–1.63 (3H, m) 2.66 (2H, m) 6.54 (2H, brs)
6.54 (2H, brs) 6.76 (1H, d, J ¼ 1.8 Hz)
6.69 (1H, 6.62 (1H, 3.84 (3H, 6.68 (3H,
13
d, J ¼ 8.0 Hz) d, J ¼ 1.8, 8.0 Hz) s) s)
H-NMR
6.56 (2H, brs)
6.56 (2H, brs) 6.79 (1H, d, J ¼ 1.8 Hz)
6.65 6.63 3.85 6.80
(1H, d, J ¼ 8.0 Hz) (1H, d, J ¼ 1.8, 8.0 Hz) (3H, s) (3H, s)
13
C-NMR
32.4 39.8 71.2 45.0 71.2 39.8 32.8 134.7 108.2 146.6 135.4 149.7 103.0 135.4 113.4 149.0 145.6 116.3 122.0 56.6 56.8
been found in SciFinder scholar based on its structural formula, which means that compound III is a new compound.
73.2%) at 60 mg mL1 and little cytotoxicity (cell survival > 92%) at 10 to 40 mg mL1. The water fraction exhibited no cytotoxicity (cell survival > 95%) at all of the test concentrations.
2.2
2.3. Protective effects
Cytotoxicity assay
As shown in Fig. 3, the ethanol extract of C. phaeocaulis showed no obvious cytotoxicity (cell survival > 95%) at 10 to 50 mg mL1, while the cell survival decreased sharply to 86.1% at 60 mg mL1. The ethyl acetate fraction also did not exhibit signicant cytotoxicity (cell survival > 95%) at 10 to 40 mg mL1, while the cell survival was 85.7 to 72.3% at 50 to 60 mg mL1. The petroleum ether fraction exhibited moderate cytotoxicity (cell survival was
Cytotoxic effects of the ethanol extracts and their polar fractions of C. phaeocaulis on PC-12 cells. Ethyl acetate fraction (EZ-EA), petroleum ether fraction (EZ-PE), and water fraction (EZ-W) from 70% ethanol extract (EZ-Z). Fig. 3
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Yousef et al.11 reported that Curcumin could successfully restore liver function and inhibit oxidative injury induced by paracetamol. The results of this research show that samples prepared from C. phaeocaulis also exhibited certain protective effects against H2O2 induced cell injury in a concentration-
Fig. 4 Protective effects of the ethanol extracts, their polar fractions and three compounds isolated from C. phaeocaulis against hydrogen peroxide-induced cell death in PC-12 cells. Ethyl acetate fraction (EZEA), petroleum ether fraction (EZ-PE), and water fraction (EZ-W) from 70% ethanol (EZ-Z). Results are mean SD. *P < 0.05, **P < 0.01, statistically significant in comparison with control.
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dependent manner (Fig. 4). The protective effect of the three compounds and four extracts from C. phaeocaulis showed no obvious difference at low concentrations (#20 mg mL1). Both EZ-Z and EZ-EA exhibited signicant protective effects. EZ-Z and EZ-EA could raise the PC12 cell survival rate from 49.2 to 74.6% and 51.5 to 71.7% at concentrations ranging from 10 to 50 mg mL1, respectively. EZ-PE did not exhibit a signicant protective effect at low concentrations (#30 mg mL1), but could slightly raise the cell survival rate (56.6%) at a concentration of 50 mg mL1. EZ-W almost had no protective effect at all concentrations. Fig 4 shows that compound III had the highest protective activity at all concentrations. At a concentration of 20 mg mL1 of compound III, PC-12 cells survival rate was 54.7%, and at 50 mg mL1 it reached 83.3%. Compound I and compound II also exhibited obvious protective effects, especially at higher concentrations. At 50 mg mL1, the survival rate of cells treated by compound I was 78.6%, second only to compound III. Compound II also showed a high activity at 50 mg mL1, where the cell survival rate was 70.6%. These results suggest that diphenylheptanes may be the active compounds responsible for the protective effect of C. phaeocaulis against hydrogen peroxide-induced PC12 cell injury. In conclusion, the ethanol extract of C. phaeocaulis and its ethyl acetate fraction showed signicant chemical protective effects. Three diphenylheptane compounds, 1-(4-hydroxy phenyl)-7-(4-hydroxy-3-methoxy phenyl)-3,5-dihydroxyheptane (I), 1,5-epoxy-3-hydroxy-1-(3,4-dihydroxy-5-methoxy phenyl)-7(4-hydroxy-3-methoxy phenyl)heptane (II), and 1-(4-hydroxy-3methoxy phenyl)-7-(3,4-dihydroxy-5-methoxy phenyl)-3,5-dihydroxyheptane (III), were isolated from the ethyl acetate fraction, which were identied for the rst time from C. phaeocaulis, and compound III was a new compound. The three compounds displayed a greater protective effect against H2O2 induced cell injury than the ethanol extracts at a concentration of 50 mg mL1, and compound III showed the highest activity at almost all concentrations. The results obtained in this work might contribute to a better understanding of the biological activity of C. phaeocaulis.
3.
Materials and methods
3.1. Plant material The dried rhizome of C. phaeocaulis, derived from Sichuan province, was purchased from the Qingping market for Chinese medicinal material in Guangzhou, China. A voucher specimen was deposited in the department of Natural Products Studies, School of Light Chemistry and Food Science, South China University of Technology. 3.2. Reagents Methanol, chloroform, petroleum ether, concentrated sulfuric acid, glacial acetic acid, vanillin, and ethyl acetate were analytical grade and absolute ethanol was food grade. All chemicals were purchased from East Giant experiment instrument company (Guangzhou, China).
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3.3. Ethanol extracts from the rhizome of C. phaeocaulis A pre-prepared powder (3.0 kg) of C. phaeocaulis was extracted with 70% ethanol under reux (3 7 L, each 2.5 h). The leach liquor was combined and concentrated under reduced pressure at 45 C to give a residue (EZ-Z, 77.8 g), which was suspended in water (1 L) and then partitioned with petroleum ether (3 1 L) and ethyl acetate (3 1 L) successively to give a petroleum ether fraction (EZ-PE, 36.5 g), an ethyl acetate fraction (EZ-EA, 28.7 g) and a water remnant (EZ-W, 9.4 g), respectively. 3.4. Isolation and identication of chemical components The ethyl acetate fraction showed the best protective effect among the three extracts in the cell protective effect screening tests (Fig. 4). Therefore, the ethyl acetate fraction was chosen for the following isolation. The pre-prepared ethyl acetate fraction was subjected to silica gel column chromatography (Ø 6.5 1200 cm) with a gradient of chloroform–methanol12 to yield 12 sub-fractions (EE-1 to EE-12). EE-7 (eluted by chloroform– methanol 85 : 15) was further subjected to ODS-A chromatography and eluted with a gradient of methanol–water to give four sub-fractions (EE-7a to EE-7d). EE-7b (40% methanol eluted) was used for HPLC with methanol–water as an eluent to obtain compounds I–III. The extraction and separation procedures are shown in Fig. 1. The three compounds were detected by electrospray ionization ion trap multiple mass spectrometry (EI-MS) and nuclear magnetic resonance spectroscopy (NMR) in order to identify their structures. EI-MS was measured on a Bruker Esquire HCTplus LC/MS system using both the positive- and the negative-ion modes with a scan range of 200–2000. 1H and 13C NMR, and DEPT spectra were recorded on a Bruker Daltonics DRX-400 spectrometer. 3.5. Protective effects on H2O2 induced cell injury The PC12 cell line was obtained from Sun Yat-Sen University, Guangzhou, China, and maintained at 37 C in a humidied atmosphere containing 5% CO2 in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U mL1) and streptomycin (100 mg mL1).13 To produce oxidative stress, H2O2 was freshly prepared from 30% stock solution prior to each experiment. Exponential growth phase PC-12 cells, at a density of 105 cells per mL, were seeded in 96-well culture plates (100 mL per well). Aer incubation for 16 h, the supernatant media were discarded and replaced with equal volumes of fresh media containing different concentrations of samples. The cells were then incubated for 0.5 h. Then the H2O2 solution was added into the media with a nal concentration of 600 mM and incubated for 24 h at 37 C according to our previous work.14 Cell survival was evaluated by a 3-(4,5-dimethylthiazol-z-yl)2,5-diphenyl tetrazolium bromide (MTT) test15 on the basis that the succinate dehydrogenase in the mitochondria of living cells can cleave the tetrazolium ring of MTT to produce formazan.16–18 Briey, aer 4 h of incubation with MTT (0.5 mg mL1) at 37 C, cells were lysed in dimethylsulfoxide (DMSO)
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and the amount of MTT formazan was quantied by determining the absorbance at 492 nm.
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3.6. Statistic analysis Data were expressed as mean SD (n ¼ 5), and statistical comparisons were made by means of a one-way ANOVA test followed by Dunett's t-test. P < 0.05 was considered statistically signicant, and P < 0.01 indicated the presence of a highly signicant difference.
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