Polyphenols from Parabarium huaitingii and their positive inotropic and anti-myocardial infarction effects in rats.
Eight phenolic compounds, including (-)-epicatechin (1) and seven proanthocyanidins (2-8), were obtained from the butanol extract of Parabarium huaitingii (PHB). Their chemical structures were identified based on analyses of mass spectra (MS), NMR, CD spectra, and partial acid catalyzed thiolytic degradation. The observation made by laser scanning confocal microscope found a significant increase of the concentration of intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.l]) in single myocytes when the PHB was added, while compounds 1 and 3 had the same physiological effect. Further investigations showed PHB had a dose-dependent positive inotropic effect on isolated right atria and papillary muscle of left ventricle of the rat, while having no significant influence on the spontaneous beating rate of the isolated right atria. The inotropic effect of PHB could be greatly abolished by pretreating the myocardium in [Ca.sup.2+]-free solution. These findings indicated that PHB could significantly increase [[[Ca.sup.2+]].sub.l] in myocytes, which was greatly dependent on the influx of extracellular [Ca.sup.2+]. Compounds 1 and 3 might be the effective ingredients of the inotropic effect of PHB. In addition, PHB could also significantly decrease the infarct size of the heart on acute myocardial infarction (AMI) model rats, which suggested its myocardial protective effect on ischemic myocardium. The positive inotropic effect of PHB, together with its myocardial protective effect on AMI, suggested that PHB had a promising potential for the prevention and treatment of heart failure, especially the one that was caused by AMI.
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Positive inotropic action
Parabarium huaitingii (Apocynaceae) is widely distributed in the southern and southwestern areas of China, which has been usually used as folk Medicine for treatment of hemiplegy and paralysis (Herbarium 2010). Up to now, studies on the bioactive constituents from the genus of Parabarium were scarce. During our investigations on bioactive constituents from herbal medicine, new phenylpropanoid-substituted epicatechin glycosides were obtained from Parabarium huaitingii (Tang et al. 2009).
In this paper, as our ongoing studies on the bioactive constituents from Parabarium huaitingii, eight phenolic constituents were obtained from the butanol extract of Parabarium huaitingii (PHB), including 7 proanthocyanidins. The positive inotropic effect and its relevant mechanism of PHB were evaluated in cellular and organ levels, and the myocardial protective effect of PHB was observed in vivo.
Materials and methods
The stems of Parabarium huaitingii were collected in 2006 from Guangxi Botanical Garden of Medicinal Plants. Guangxi province. P.R. China, and authenticated by Xiao-Hong Lu of Guangxi Botanical Garden of Medicinal Plants. A voucher specimen (no. 061220) has been deposited at the Institute of Traditional Chinese Medicine & Natural Products. Jinan University. Guangzhou. China.
All chemicals used in this study, such as EtOH, EtOAc. and n-BuOH were analytical grade and were purchased from Shandong Yuwang Industry Co.. Ltd. Methanol for analytical grade and chromatographic grade was purchased from Jiangsu Hanbon Science & Techonology Co.. Ltd. Methanol-[d.sub.4] for NMR measurement was purchased from ARMAR Chemicals (Switzerland).
Precoated silica gel TLC plates were products of Qingdao Marine Chemical Factory. Reverse-phase (Rp) [C.sub.18] columns (5 [micro]m. 4.6 mm x 250 mm; 10 mm x 250 mm; Purospher[R] Star) were purchased from phenomenex corporation. Diaion HP20 was purchased from Mitsubishi Chemical Corporation (Japan). Toyopear HW40C was product of Tosoh Corporation.
Optical rotations were obtained on a P-1020 digital polarime-ter (Jasco Corporation). Circular dichroism spectra were measured on a J-810 Circular dichroism spectrometer (Jasco Corporation). All NMR spectra were recorded on a Bruker AV 400 spectrometer. ESI-MS were recorded on a Finnigan LCQ Advantage Max ion trap mass spectrometer (Thermo Electron Corp.). HPLC for analysis was performed on a Dionex HPLC system equipped with a Dionex P-680 quaternary pump, a Dionex PDA-100 diode-array detector (DAD), a Dionex TCC-100 oven and a Dionex ASP-100 autosampling system (Dionex). Preparative HPLC was carried out on a Varian instrument equipped with UV detector.
Extraction and isolation
Slices of air-dried stems of Parabarium huaitingii (5.0 kg) was refluxed three times with 60% (v/v) aqueous EtOH (30 1) for 2h each time. The solvent was evaporated off under vacuum to yield 750 g crude extract (PH), which was suspended in [H.sub.2]O and partitioned with EtOAc and n-BuOH successively, to afford EtOAc (120 g), n-BuOH (PHB, 240g). and water-soluble (350g) fractions, respectively.
The n-BuOH extract (150 g) was directly chromatographed over an open Diaion HP20 column (7.0 cm x 90.0 cm), eluted with gradient MeOH-[H.sub.2]O (0%, 10%, 30%, 50%, 70%, and 100% of MeOH) to yield 10 fractions (A1-A10). The fraction A4 (13.6 g, 30%) was subjected to a Toyopear HW40C column [2.0 cm x 60.0 cm, eluted with MeOH-[H.sub.2]O (30%, 50%, 70%, and 100%)] to afford compounds 1 (80 mg) and 2 (25 mg). The fraction A7 (12.4 g, 50%) was applied to Toyopear HW40C column chromatography [2.0 cm x 60.0 cm, eluted with MeOH-[H.sub.2]O (30%, 50%, 70%, and 100%)) and preparative HPLC (25% MeOH-[H.sub.2]O) to yield compound 3 (15 mg). The fraction A5 (12.0 g, 50%) was applied to Rp-C18 open column chromatography (3.0 cm x 30.0 cm), eluted with gradient MeOH-[H.sub.2]O (10%, 20%, 30%, and 100% of MeOH) to afford subfraction A5-1 (6.0 g, 10%). A5-2 (0.5 g. 20%). and A5-3 (4.0 g. 30%), respectively. The subfraction A5-1 was subjected to Toyopear HW40C column chromatography [2.0 cm x 60.0 cm. eluted with MeOH-[H.sub.2]O (30%. 50%. and 70%)] and preparative HPLC to yield compound 4 (203 mg). 6 (28.0 mg). and 7 (60 mg). The subfraction A5-2 was subjected to Toyopear HW40C column chromatography [2.0 cm x 60.0 cm, eluted with MeOH-[H.sub.2]O (30%, 50%. and 70%)] and preparative HPLC to yield compounds 5 (40 mg) and 8 (49 mg).
[[[Ca.sup.2+]].sub.i] fluorescence measurement
All Wistar rats (weighting 250-300 g. male) used in the experiment were provided by the animal center of the Second Affiliated Hospital of Harbin Medical University. Single Wistar rat ventricular myocytes were isolated as previously described in detail (Yang et al. 2002). The effects of PH, PHB, and compounds 1-8 on the level of [[[Ca.sup.2+]].sub.i] in single myocytes were measured. KC1 was used as a positive control. Briefly, isolated ventricular myocytes were adhered to the cover slips of the chamber and incubated with a working solution containing Fluo-3/AM (20 mM) and Pluronic F-127 (0.03%) (Sigma) at 37 [degrees]C for 45min. After loading, extracellular Fluo-3/AM was removed by washing the myocytes once with standard Tyrode's solution. Fluorescent changes of the Fluo-3/AM-loaded cells were detected by the laser scanning confocal microscope (Fluoview-FV300, Olympus. Japan; excitation: 488 nm from an Ar ion laser; emission: 530 nm with 20x objective) were added between the 3rd and 4th image. The fluorescent intensities before ([F.sub.0]) and after ([F.sub.i]) the drug administration were recorded and the images were stored in disks. The change of [[[Ca.sup.2+]].sub.i] was represented by the ratio of fluorescence intensity of Fluo-3 over base ([F.sub.i]/[F.sub.0]).
Measurement of the spontaneous beating rate and the contractile force of the isolated heart muscle
The Wistar rat was sacrificed by a blow on the head and the heart was rapidly removed (Nasa et al. 1992). Right atria and papillary muscle of the left ventricle were quickly dissected and placed in the 5 ml organ bath containing the K-H solution (NaCl 118 mM, KC1 4.7 mM, [MgSO.sub.4] 1.1 mM. [KH.sub.2] [PO.sub.4] 1.2 mM. [CaCl.sub.2] 1.5 mM, [NaHCO.sub.3] 25 mM, and glucose 10 mM, pH 7.35-7.45). The [Ca.sup.2+]-free solution was prepared by omitting [CaCl.sub.2] in the normal K-H solution. The solution was gassed with 95% [O.sub.2] + 5% [CO.sub.2] and maintained at 37 [degrees]C. One end of the tissue was secured to the tissue holder, and the other end was connected to a force transducer by a silk thread. An electronic stimulation at a frequency of 2 Hz by rectangular pulses (5 ms duration, 20% above threshold) was given to the left ventricle muscle, while the right atria had a spontaneous beating in this condition. The resting tension of the tissue was set at 0.4 g. The tissue was equilibrated for 30 min, and the solution in the organ bath was changed every 5 min. PHB (2, 4, 8, 16, 32, 64 x [10.sup.-1] mg/ml) was cumulatively added into the organ bath with 3 min interval in PHB groups while distilled water was added in the same way in control groups. Contractile force of the right atria and papillary muscle, and the spontaneous beating rate of the right atria of the whole process were recorded by the force transducer (MedLab BL-420E + recording system, Chengdu, taimeng).
Measurement of PHB's effect on myocardial infarction model rats
Wistar rats were randomly divided into 2 groups, the control group and the PHB group. For the PHB group, 1.5 ml of PHB was given to the rat through the tail vein 5 min before the ligation was conducted. All rats were performed left anterior descending coronary artery ligation (Pfeffer et al. 1979). In detail, the rat was anesthetized by i.p. sodium pentobarbital (40 mg/kg), and the temperature was kept at 37 [degrees]C by putting the animal on a heating pad. The rat was ventilated by a small animal ventilator at the frequency of 54/min and a tidal volume of 3 ml, and the standard limb lead ECG was continuously recorded on a recorder (MedLab BL-420E + recording system, Chengdu, taimeng). The chest was surgically opened via the fourth rib intercostal space. The heart was exposed and the left anterior descending (LAD) coronary artery was encircled by a segment of saline soaked 5-0 sutures near its origin from the left coronary artery. Myocardial ischemia was confirmed by regional cyanosis of the myocardial surface and the elevation of the ST on ECG. After the surgeon, the chest was closed quickly. After 6 h of infarction, the heart of the rat was removed, washed out all the blood, and the ventricle was dissected. The ventricles were sliced into 2 mm thick sections, and then incubated in 1% triphenyltetrazolium chloride at 37 [degrees]C in 0.2 M Tris buffer (pH 7.4) for 15 min. While normal myocardium was stained brick red. the infarcted areas remained unstained. The size of the infarcted area was estimated by the weight as a percentage of the left ventricle (Stamm et al. 2001). During the surgeon, the incidence of ventricular premature beats (VE), ventricular tachycardia (VT), ventricular fibrillation (VF) and the first VE starting time of the electrocardiogram (ECG) were recorded as the parameters to evaluate the effect of lycopene on the ECG. The incidence and severity of the arrhythmia of the myocardial infarction model rats were evaluated by the standard arrhythmia score (Curtis and Walker 1988).
Data were presented as mean [+ or -] SD. Statistical comparison was performed by the student's t-test and an analysis of variance (ANOVA) followed by post hoc LSD test analysis for statistical comparison between every two groups. A value of p < 0.05 was considered significant.
Structural elucidation of phenolic compounds (1-8)
The chemical structures of compounds 1-8 were displayed in Fig. 1.
[FIGURE 1 OMITTED]
Compound 1 was obtained as a white amorphous powder. [[alpha]].sup.16] -40.4[degrees] (MeOH. c = 0.5). ESI MS gave an ion [[M+Na].sup.+] at m/z 313 and [[M-H].sup.-] at m/z 289. Based on [.sup.1]H. [.sup.13]CNMR data and optical value, 1 was identified as (-)-epicatechin (Wang et al. 2007).
Compound 2 was obtained as a pale amorphous powder. [[alpha]].sup.16] + 29.4[degrees] (MeOH, c = 0.5). ESI MS gave an ion [[M+Na].sup.+] at m/z 601 and [[M-H].sup.-] at m/z 577, which suggested that it was a dimer of (epi) catechin. Based on [.sup.1]H and [.sup.13]C NMR data (Zhang et al. 2003), 2 was identified as procyanidin B2. Thiolytic degradation of 2 with benzyl mercaptan yielded epicatechin 4-benzylthioether (2a) and epicatechin (1), which further confirmed the deduction mentioned above.
Compound 3 was obtained as a pale amorphous powder. [[alpha]].sup.27] + 22.8[degrees] (MeOH, c = 2.0). ESI MS of 3 provided an ion [[M+Na].sup.+] at m/z 599 and [[M-H].sup.-] at m/z 575, two mass units lower than that of 2, which indicated that 3 was a doubly linked procyanidin of the A-type. Based on [.sup.1]H and [.sup.13]C NMR data (Kamiya et al. 2001), 3 was identified as proanthocyanidin A2.
Compound 4 was obtained as a yellow amorphous powder. [[alpha]].sup.16] + 65.4[degrees] (MeOH, c = 0.5). ESI MS gave an ion [[M+Na].sup.+] at m/z 887 and [[M-H].sup.-] at m/z 863, which suggested that it was a trimeric proanthocyanidin possessing one doubly linked structure. Thiolytic degradation of 4 with benzyl mercaptan yielded proanthocyanidin A2 4-benzylthioether (4a) and epicatechin (1). Consequently, 4 was identified as cinnamtannin B-1 based on its [.sup.1]H, [.sup.13]NMR, and CD spectral data (Jayaprakasha et al. 2006; Botha et al. 1981).
Compound 5 was obtained as a yellow amorphous powder. [[alpha]].sup.16] + 67.8[degrees] (MeOH, c = 0.5). ESI MS gave the same molecular formula as that of 4 (m/z 887 for [[M+Nal.sup.+] and 863 for [[M-H].sup.-]), which also suggested that 5 was a trimeric proanthocyanidin possessing one doubly linked structure. The [.sup.1]H and [.sup.13]C NMR spectra of 5 showed a close similarity to those of 4 except for a set of heterocyclic proton and carbon signals. Thiolytic degradation of 5 with benzyl mercaptan yielded epicatechin-(2[beta] [right arrow] O [right arrow] 7, 4[beta] [right arrow] 8)-ent-catechin 4-benzylthioether (5a) and epicatechin (1) (Fig. 2). Consequently, 5 was identified as aesculitannin B based on its [.sup.1]H, [.sup.13]NMR, and CD spectral data (Kamiya et al. 2001).
[FIGURE 2 OMITTED]
Compound 6 was obtained as a yellow amorphous powder. [[alpha]].sup.16] -43.0[degrees] (MeOH, c = 0.5). Analysis of the high-resolution ESI-TOF mass spectrum indicated that 6 has the molecular formula of [C.sub.60][H.sub.48][O.sub.24] (m/z 1153.2607 [[M+H].sup.+], calcd 1153.2614), which suggested that it was a tetrameric proanthocyanidin possessing one doubly linked structure. Thiolytic degradation of 6 with benzyl mercaptan yielded 4, proanthocyanidin A2 4-benzylthioether (4a), epicatechin 4-benzylthioether (2a), and epicatechin (1) (Fig. 3). Thus, 6 was identified as parameritannin A-1 (Kamiya et al. 2001), which was further confirmed by 2D NMR and CD spectral data.
[FIGURE 3 OMITTED]
Compound 7 was obtained as a yellow amorphous powder. [[alpha]].sup.16] + 131.4[degrees] (MeOH, c = 0.5). ESI MS gave the same molecular mass as that of 6 (m/z at 1175 for [[M+Na].sup.+] and 1151 for [[M-H].sup.-]), which suggested that 7 was also a tetrameric proanthocyanidin possessing one doubly linked structure. The [.sup.1]H and [.sup.13]C NMR spectra of 7 showed a close similarity to those of 6. Thiolytic degradation of 7 with benzyl mercaptan yielded identical products with those of 6, which indicated that the difference between 6 and 7 lay in the interflavonoid linked position between the upper and 2nd units. Thus, 7 was identified as epicatechin-(4[beta] [right arrow] 8)-epicatechin-(2[beta] [right arrow] 0 [right arrow] 7, [beta] [right arrow] 8)-epicatechin-(4[beta] [right arrow] 8)-epicatechin based on its [.sup.1]H, [.sup.13]C NMR, and CD spectral data (Kamiya et at 2003).
Compound 8 was obtained as a yellow amorphous powder. [[alpha]].sup.16] + 30.8[degrees] (MeOH, c = 0.5). ESI MS showed the same molecular mass as that of 6, which suggested that 8 was also a tetrameric proanthocyanidin possessing one doubly linked structure. The [.sup.1]H NMR spectrum of 8 showed to be exceedingly complex, presumably due to the effects of dynamic rotational isomerism at ambient temperature (Tarascou et al. 2007). Thiolytic degradation of 8 with benzyl mercaptan yielded identical products with those of 6 and 7, which indicated that differences between 6 and 8 lay in the interflavonoid linked position between the upper and 2nd units. Based on its [.sup.1]H, [.sup.13]C NMR, and CD spectral data, 8 was identified as epicatechin-(4[beta] [right arrow] 6)-epicatechin-(2[beta] [right arrow] O [right arrow] 7, [beta] [right arrow 8)-epicatechin-(4[beta] [right arrow] 8)-epicatechin (Kamiya et al. 2003).
The effects of PH, PHB, and compounds 1-8 on [[[Ca.sup.2+]].sub.i] of ventricular myocytes
Fig. 4 showed the pharmacological activities of PH (1 [gl.sup.-1]), PHB (1 [gl.sup.-1]), and compounds 1-8 (4 x [10.sup.-3] mol [l.sup.-1]) on [[[Ca.sup.2+]].sub.i] in rat cardiac myocytes evaluated by confocal laser scanning microscopy. It indicated that PHB, 1, and 3 exhibited significant increases of [[[Ca.sup.2+]].sub.i] on ventricle myocytes, which did not display significant difference (p > 0.05) compared with the positive control (KC1). PH. 2, and 4-8 also exhibited increasing of [[[Ca.sup.2+]].sub.i] in different levels. Fig. 5 showed the time course of the increase of [[[Ca.sup.2+]].sub.i] of ventricular myocytes after they were exposed to PHB and 1.
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
Effects of PHB on spontaneous beating rate and contractile force of the isolated heart muscle
Fig. 6 suggested that PHB could increase the contractile force of the right atria in a dose-dependent manner. The maximum contractile force of right atria caused by PHB was 0.7 g, which was 170% of the original level. This inotropic effect arrived to a plateau when the accumulating concentration of PHB was 16 x [10.sup.-2] mg/ml on right atria. The positive inotropic effect of PHB on right atria could be greatly abolished by pretreating the muscle in [Ca.sup.2+]-free solution. The contractile force did not change significantly in control groups.
[FIGURE 6 OMITTED]
Fig. 7 showed that the spontaneous beating rate of the right atria maintained in a level of 237 beat/min and did not change significantly in the whole process.
[FIGURE 7 OMITTED]
A positive inotropic effect of PHB on papillary muscle was also observed in the experiment; the detailed data is not shown.
Effects of PHB on myocardial infarction model rats
Fig. 8 showed that the infarct size of the PHB was significantly smaller than that of the control group on myocardial infarction model rats. The percent of infarct size of the PHB group and the control group were 31.70 [+ or -]6.79% and 25.48 [+ or -]5.84%, respectively. The arrhythmia core of PHB group in myocardial infarction model rats was 4.57 [+ or -] 1.62, while the control group was 4.17 [+ or -] 0.41, which did not show significant difference.
[FIGURE 8 OMITTED]
Proanthocyanidin, also called condensed tannin, is one of the major groups of plant polyphenols, which is widely distributed in nature, such as vegetables, fruits, and beverages. Epidemiological studies showed that consumption of diets rich in proantho-cyanidins have beneficial effects in human health. Increasing evidences demonstrate that proanthocyanidins exert extensive biological and pharmacological activities including antioxidative, antidiabetic, antitumor, anti-inflammatory, antivirus, and especially cardioprotective activities. The latest research proves that Crataegus oxycantha extract, which is aboundant in proanthocyanidins, reduce the oxidative stress and exert cardioprotective effect in the reperfused myocardium by inhibition of apoptotic pathways (Swaminathan et al. 2010). Additionally, it is also reported that (- )-epicatechin, compound 1 in this study, demonstrates the capacity to confer cardioprotective effects in myocardial ischemic injury. Moreover, protection is sustained over time and preserves left ventricle structure and function (Yamazaki et al. 2010).
The potential of proanthocyanidins against heart failure has received considerable attention recently, and the study on the mechanism of action is mainly focused on the anti-oxidation and cardio-protection. We investigated whether the underlying mechanism was involved. The observation made by laser scanning confocal microscope showed PHB could significantly increase the concentration of [[[Ca.sup.2+]].sub.i] in single myocytes. Compounds 1 and 3, which presented the same physiological effect on myocytes, might be the two bioactive ingredients. This phenomenon reminded us PHB might have a positive inotropic effect on myocardium since [Ca.sup.2+] plays a crucial role in the excitation-contraction coupling in cardiac myocytes. Further investigations in organ level convinced that when PHB was added in the organ bath accumulatively, the PHB had an inotropic effect on isolated right atria and papillary muscle of left ventricle muscle, without disturbing the spontaneous beating rate of the right atria. The inotropic effect of PHB was abolished by pretreating the myocardium in [Ca.sup.2+]-free solution, which suggested that the inotropic effect of PHB was greatly dependent on the influx of extracellular [Ca.sup.2+]. To the best of our knowledge, this is the first time that proanthocyanidins has demonstrated a significant positive inotropic effect on isolated myocardial tissue.
In addition, the myocardial protective effect of proanthocyanidins on myocardial infarction was also observed by our study. As was proved by research, the large infarcts left by AMI gradually should lead to the remodeling and dilatation of myocardium. Clinical researches also supported that AMI made patients more susceptible to heart failure due to left ventricular systolic dysfunction (Dargie 2005). Thus, the importance of medical intervention after AMI has been emphasized these years as an effective way to prevent the occurrence of heart failure (Dargie 2005). As PHB could significantly limit the infarct size of AMI, and (-)-epicatechin, 1 in this study, could preserve left ventricle structure and function. That means PHB might be more efficacious than other traditional positive inotropic drugs that were used to treat heart failure.
In summary, our findings revealed that PHB, which contained abundant proanthocyanidins, had positive inotropic effect on isolated atria and papillary muscle of left ventricle. The results suggested that PHB had a promising effect for the prevention and treatment of heart failure, especially the one that was caused by AMI. According to the results presented, further studies need to be undertaken to validate these results in animal model and to investigate the key underlying mechanisms of action of PHB, which are likely complex.
The authors are grateful to Miaomiaojiang, Yang Yu, and Weihua Jiao for NMR measurements. Thanks are also expressed to Huinan Zhao, Institute of Traditional Chinese Medicine & Natural Products, Jinan University (Guangzhou) for high-resolution ESI-TOF mass spectral measurements. This work is supported by Key Laboratory of Bio-pharmaceutical-engineering (Harbin Medical University), Ministry of Education.
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J.S. Tang (a), (d), (1), Y.L Hou (b), (1), H. Gao (a), X. Chen (b), S.C Sun (b), T.Z. Guo (b), H. Kobayashi (c), W.C. Ye (a), X.S. Yao (a), (d), *
(a) Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou 510632, China
(b) State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Department of Pharmacology, Harbin Medical University, Harbin 150086, China
(c) Institute of Molecular and Cellular Bioscience, The University of Tokyo, Bunkyo-ku 113-0032, Japan
(d) College of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, China
* Corresponding author at: Institute of Traditional Chinese Medicine and Natural Products. Jinan University, West 601, Huangpu Avenue, Guangzhou 510632, P.R. China. Tel.: +86 20 85225849; fax: +86 20 85225849. E-mail address: email@example.com (X.S. Yao).
(1) Both authors contribute equally to this work.
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|Author:||Tang, J.S.; Hou, Y.L.; Gao, H.; Chen, X.; Sun, S.C.; Guo, T.Z.; Kobayashi, H.; Ye, W.C.; Yao, X.S.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||May 15, 2011|
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