Article pubs.acs.org/JAFC
Esterification Enhanced Intestinal Absorption of Ginsenoside Rh2 in Caco‑2 Cells without Impacts on Its Protective Effects against H2O2‑Induced Cell Injury in Human Umbilical Vein Endothelial Cells (HUVECs) Bing Zhang,†,§ Hui Ye,† Xue-Mei Zhu,† Jiang-Ning Hu,*,† Hong-Yan Li,† Rong Tsao,§ Ze-Yuan Deng,*,† Yi-Nan Zheng,# and Wei Li# †
State Key Laboratory of Food Science and Technology, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330047, China § Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario N1G 5C9, Canada # College of Chinese Material Medicine, Jilin Agricultural University, Changchun, Jilin 130118, China S Supporting Information *
ABSTRACT: Ginsenoside Rh2 and its octyl ester derivative (Rh2-O) were investigated for their transcellular transport in the Caco-2 cell system and their protective effect against oxidative stress in human umbilical vein endothelial cells (HUVECs). Results showed that the transport rates for apical-to-basolateral (AP-BL) flux of Rh2 (0.21 × 10−6 cm/s) was enhanced by the synthesis of its esterified derivative Rh2-O (1.93 × 10−6 cm/s) over the concentrations of 10−50 μM. In addition, both Rh2 and its esterified derivative Rh2-O exhibited similar protective effects against oxidative damage induced by H2O2. Pretreatment of Rh2 and Rh2-O significantly decreased the activation of caspase-3 known to play a key role in H2O2-induced cell apoptosis. These results were consistent with that of a flow cytometry assay analyzing HUVECs apoptosis. The present study demonstrated that the absorption of ginsenoside Rh2 in vitro can be significantly enhanced by synthesis of its ester derivative. Meanwhile, no significant discrepancy between Rh2 and Rh2-O on their bioactivities against the oxidative damage induced by H2O2 was observed, which means that esterification of Rh2 might have a higher bioavailability than Rh2 in vitro without impacts on pharmaceutical actions. KEYWORDS: ginsenosides, Rh2, octyl ester derivative, Caco-2 cells, absorption, P-glycoprotein, caspase-3, oxidative damage
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INTRODUCTION Panax ginseng C.A. Meyer is a functional food and a nutritional supplement or health tonic frequently utilized in oriental countries.1 Ginsenosides, a member of the triterpene saponins family, have been the focus of extensive studies for their antiaging, anti-inflammation, and antioxidation activities in the central nervous system, cardiovascular system, and immune system.2−4 Approximately 35 ginsenosides have been isolated and categorized on the basis of ginseng species and growth locations.5 Although many health-promoting benefits of ginsenosides have been proven, increasing pharmaceutical studies revealed that the functions of ginsenosides have been curtailed by its low oral bioavailability.6−8 Ginsenoside Rh2, first isolated from red ginseng, is a protopanaxadiol type of steroidal saponin and has an aglycone of dammarane skeleton.9 Gu et al. reported that the bioavailability of Rh2 was about 5% in rats and 16% in dogs after oral administration, which was attributed to its extremely low membrane permeability.10,11 It is therefore important to improve the oral bioavailability of ginsenoside. The oral absorption of poorly absorbed drugs could be enhanced through improving overall membrane permeability by designing lipophilic ester derivatives, such as diglyceride acylated norfloxacin, bupronolol, and phenytoin.12−14 Several acylated triterpenoid saponins isolated from the roots of Solidago © 2014 American Chemical Society
virgaurea subsp. virgaurea in low concentration activated the metabolism of endothelial cells, which enhanced the permeability of the blood vessel walls for better absorption of the saponin into tissues.15 We speculate that the novel ester derivative of Rh2, the octyl ester of Rh2 (Rh2-O), may have excellent oral bioavailability. However, the underlying mechanism of oral absorption for Rh2 or Rh2-O remains unclear. Endothelial cells (EC) play a pivotal role in the regulation of cardiovascular health and the elasticity of vessels in human. The inflammatory responses triggered by EC oxidative injury are considered as the first stage of atherosclerosis (AS) and could ultimately cause endothelial cell death through apoptosis.16 Oxidative stress, mainly caused by the excessive accumulation of reactive oxygen species (ROS), is a critical pathogenic factor in EC injury.17 Plants-sourced antioxidants are so far increasingly studied to counteract the injury caused by oxidative stress.18 Zhu et al. reported that ginsenoside Rg1 protected rat cardiomyocyte from oxidative injury via its antioxidant ability.19 A ginsenoside compound K, the isomer of Rh2, reportedly regulated inflammatory signaling through Received: Revised: Accepted: Published: 2096
October 21, 2013 January 25, 2014 February 13, 2014 February 13, 2014 dx.doi.org/10.1021/jf404738s | J. Agric. Food Chem. 2014, 62, 2096−2103
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inhibition of ROS.20 In addition, Wang et al. found that Rh2 also acted against the doxorubicin-induced cardiotoxicity.21 Unfortunately, such good health benefits of ginsenosides including cardiovascular protection shown in vitro attenuated, to some extent, in vivo due to their poor bioavailabilities. To our best knowledge, there is no information on whether the bioactivities of ginsenosides are enhanced or decreased through synthesis of their esterified derivatives. In this study, first, to exam whether Rh2 ester has better bioavailability than Rh2, the transepithelial transport and absorption mechanisms of Rh2 and its derivative were studied in the Caco-2 system. Caco-2 cell monolayers have been generally accepted as an in vitro model for prediction of drug absorption across the human intestine and for mechanistic studies of intestinal drug transport due to their morphologic and functional similarities to human small intestinal epithelial cells.22−24 Subsequently, an oxidative injury model was established using human umbilical vein endothelial cells (HUVECs) exogenously exposed to hydrogen peroxide (H2O2). The protective effects of Rh2 and its derivative on oxidative injury were evaluated to exam whether Rh2 ester with an enhanced absorption rate had similar or even better pharmaceutical function than Rh2. The present study will provide useful information on the pharmaceutical and preclinical research of ginsenosides and their structure modification.
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well and incubated for 4 h in the dark. The medium was then removed, and the MTT−formazan crystals were solubilized by incubating with 0.15 mL of DMSO with gentle shaking for 10 min; the absorbance was determined at 490 nm in a microplate reader. Cells incubated without the test compounds were used as controls. In each MTT assay, every sample was tested in five replicates. Transepithelial Transport Experiments across Caco-2 Monolayer. For transport experiments, Caco-2 cells were seeded onto the 6-well transwell inserts coated with type-I collagen at a density of 4 × 105 cells/well to generate Caco-2 monolayers. Medium was replaced every 2−3 days, and the monolayers used for the experiments were cultured to differentiate for 21−27 days after postseeding. The integrity of the cell layer and the full development of the tight junctions were monitored before every experiment by measurement of transepithelial electrical resistance (TEER) of filter-grown cell monolayers with millicell-ERS equipment. Only a monolayer with a TEER value of >300 Ω·cm2 was used for the transepithelial transport experiments. The transport of Rh2 and Rh2-O across Caco-2 monolayers was investigated using the methods described previously with minor modifications.27 Briefly, the cell monolayers were gently rinsed twice with warm HBSS (pH 7.4, 37 °C) before the experiments. Cell monolayers were then incubated at 37 °C for 30 min in the transport buffer. To measure the apical-to-basolated (AP-BL) transport, 1.5 mL of mixture containing Rh2 or Rh2-O over 10−50 μM (final concentrations) in transport buffer was added to the AP side of the transwell insert and 2.6 mL of HBSS was added to the BL chamber. The plates were then put in an incubator at 37 °C. At a designated time point (30 min interval), 0.4 mL aliquots of solution were collected from the BL side and then replaced with an equal volume of transport buffer. In the direction of BL-AP, Rh2 or Rh2-O dissolved in 2.6 mL of transport buffer over 10−50 μM (final concentrations) was added to the BL side and 1.5 mL of the HBSS to the AP side, and 0.4 mL of the samples was collected from the AP side. All experiments were performed in triplicates. For ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA)-modulated transport experiments, the cell monolayers were pretreated with 2.5 mM EGTA in both AP and BL sides for 15 min at 37 °C after the initial 30 min of preincubation. Cell monolayers were then washed three times with transport buffer. AP to BL transport was initiated in the same way as described above. In the present study, the inhibitory effects of MK-517 and verapamil on Rh2 and Rh2-O flux by Caco-2 monolayers were examined by addition of 100 μM of each inhibitor to the AP side, regardless of where samples were loaded. Thereafter, the transport study was performed according to the method described above. Superoxide Radical Scavenging Capability of Rh2 and Rh2-O in a Cell-Free System. The superoxide radical scavenging capability of Rh2 and Rh2-O was assayed by a photochemical system (Analytik Jena, Berlin, Germany); the principle of this method was photochemiluminescence (PCL), which is based on the photoinduced autoxidation inhibition of luminol by antioxidants mediated from the radical anion superoxide. The PCL assay was based on the method previously described.28 Trolox is used as reference compound to evaluate the superoxide radical scavenging capability, and all of the samples were quantified as equivalent units of trolox. Oxidative Stress Model Induced by H2O2. The HUVECs were incubated under the conditions described above. Before H2O2 treatment, cells were seeded in 96-well microstate plates at a density of 5 × 103 cells/well and grown to approximately 80% confluence. The plates were incubated at 37 °C for 24 h before further treatments. Then H2O2 solutions at various concentrations were added into each well and incubated at 37 °C for 1 h. Cell survival was evaluated by the MTT assay as described above. In this study, the cell survival rate decreased significantly (p < 0.05, data not shown) when the H2O2 concentration was >100 μM. Therefore, HUVECs treated with freshly prepared 100 μM H2O2 for 1 h were used as the oxidative stress model. Cytoprotective Assay. The HUVECs were seeded in 6-well microstate plates at a density of 4 × 105 cells/well and incubated as
MATERIALS AND METHODS
Materials and Chemicals. The human colon adenocarcinoma cell line, Caco-2, was purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). HUVECs were obtained from the College of Medicine, Nanchang University. Dulbecco’s modified Eagle’s medium (DMEM), nonessential amino acids (NEAA), penicillin−streptomycin (10000 IU/mL), Hank’s balanced salt solution (HBSS), trypsin, and ethylenediaminetetraacetic acid (EDTA) (0.25%/0.02%) in phosphate buffer saline (PBS) were all from Invitrogen Corp. (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Gibco BRL (N.V. Life Technologies, Paisley, UK). 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), mannitol, verapamil, and MK-571 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Rh2 and its ester derivative Rh2-O were kindly provided by Dr. Yinan Zheng, Jilin Agricultural University China. Transwell cell culture chambers (pore size = 0.4 μM, diameter = 24 mm) and the Millicell-ERS volt-ohmmeter with Ag/AgCl electrodes were purchased from Costar Corp (Cambridge, MA, USA) and from Nihon Millipore (Tokyo, Japan), respectively. The microplate reader was purchased from Thermo Fisher (Thermo Scientific Multiskan MK3, USA). Culture of Caco-2 Cells and HUVECs. Caco-2 cells were cultured in DMEM supplemented with FBS (10%, v/v), NEAA (1%, v/v), penicillin (100 U/mL), streptomycin (0.1 mg/mL), and glutamine (0.29 g/L). The cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C. The medium was replaced every 2 days, and the cells were passaged at about 90% confluence using a trypsin/EDTA solution (0.25%/0.02%) at a split ratio of 1:4. Caco-2 cells used in this study were between passage 40 and 60. HUVECs were grown in DMEM containing FBS (10%, v/v), penicillin (100 U/mL), streptomycin (0.1 mg/mL), and glutamine (0.29 g/L). Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. Cytotoxicity Assay. The cytotoxicity of Rh2 and Rh2-O to Caco-2 cells and HUVECs was quantified using MTT assays.25,26 The Rh2 and Rh2-O were first dissolved in DMSO and then diluted to desire concentration using PBS (ensuring the concentration of DMSO was 50 μM. Figure 1B shows that Rh2 and Rh2-O were nontoxic toward HUVECs at concentrations below 100 μM. Therefore, to ensure cell viability, we chose nontoxic concentration of 2 was considered as an active efflux. Therefore, the mechanism of permeation for Rh2-O in the translocation across Caco-2 cell monolayers may be passive diffusion with no active efflux involved. Effects of Various Compounds on Rh2 and Rh2-O Transport. In the present study, EGTA, verapamil, and MK-571 were investigated for their effects on the transport flux of Rh2 and Rh2-O across the Caco-2 cells. The drug molecule can use either a transcellular (passive or active) or paracellular (passive) route to cross intestinal epithelium into systemic circulation after oral administration.33 Generally, lipophilic molecules might have a significant transport by passive diffusion through the transcellular route, whereas hydrophilic molecules with low molecular weight cross the intestinal epithelium predominantly by paracellular passive diffusion process.34 Because of much lower surface area available to the molecules entering the intercellular space and tight junctions in the intercellular space, the efficiency of absorption through the paracellular route is lower than that of the transcellular route. Therefore, one of the first transport mechanisms to be determined for studying the processes involved in the absorption of compounds across monolayers was the contribution of the paracellular route. Manipulating tight junction integrity of Caco-2 monolayers, which was performed by a depletion of calcium concentration in the transport medium with a calcium chelator, can be used as an approach to define absorption routes.35 EGTA, a selective calcium chelator, is known to open the 2100
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reported that AS is characterized by the excessive apoptosis of vascular endothelial cells.39 Oxidative stress in the vascular wall is closely related to the excessive apoptosis of HUVECs.40 Therefore, inhibition of the excessive apoptosis of HUVECs may be considered as an efficient route in the prevention and control of AS. In the present study, HUVEC apoptosis was stimulated by H2O2, a commonly used oxidant. HUVECs control and pretreated cells were stained with Annexin V−FITC/PI and gated into four quadrants. The cells in the lower right (LR) and upper right (UR) were considered early apoptotic (Annexin+/PI−) and late apoptotic (Annexin+/PI+), respectively, whereas the cells in the lower left and upper left quadrants were considered live and necrotic, respectively. Extent of apoptosis was expressed as the total percentages in LR and UR quadrants. As shown in Figure 3, the apoptosis rate in untreated controls was 2.6 ± 1.1%, whereas the apoptosis rate elevated to 84.9 ± 4.3% after pretreatment with 100 μM H2O2-treated cells. In contrast, the apoptosis rates of HUVECs pretreated with 20 μM Rh2 markedly decreased to 63.5 ± 2.2%, and the apoptosis rates of HUVECs pretreated with Rh2-O (69.7 ± 3.9%) have no significant difference from that of pretreated with Rh2. These results demonstrated that both Rh2 and Rh2-O possess similar protective effects against oxidative stress-related cellular apoptosis. Effects of Rh2 and Rh2-O on the NO, LDH, MDA, GSH-Px, and CAT of HUVECs. The results of effect of Rh2 and Rh2-O on the NO, LDH, MDA, GSH-Px and CAT levels of oxidative injury HUVECs are shown in Table 2. NO has gained recognition as a crucial modulator in vascular disease. It is derived from the action of eNOS and is usually regarded as an endothelial cell survival factor inhibiting cell apoptosis.41 As shown in Table 2, compared to control group, NO levels in the medium were markedly decreased by exposure to H2O2, whereas pretreatment of HUVECs cells with Rh2 or Rh2-O significantly inhibited H2O2-induced NO level decrease. LDH is an oxidoreductase enzyme that catalyzes the interconversion of pyruvate and lactate and is widely used as an indicator for cell damage. As shown in Table 2, LDH release in HUVECs was minimal in the control group (41.39 ± 3.97 U/L), and addition of 100 μM H 2O 2 caused a significant increase (95.25 ± 8.13 U/L). However, pretreatment of Rh2 and Rh2-O significantly attenuated the increase in LDH release induced by H2O2. Lipid peroxidation is the degradation of lipids that occurs as a result of oxidative damage and is a useful marker for oxidative stress. Generally, polyunsaturated lipids are susceptible to attack by ROS, resulting in the formation of MDA. Therefore, MDA can be employed as an indicator of lipid peroxidation or oxidative damage in cells.42 The MDA concentration was 4.58 ± 0.61 nmol/mg protein in the control group (Table 2) and markedly increased to 10.32 ± 1.13 nmol/mg protein after 1 h of exposure to 100 μM H2O2. In contrast, the MDA concentration significantly decreased to 7.92 ± 0.69 and 6.11 ± 0.74 nmol/mg protein, respectively, after pretreatment with 20 μM Rh2 and Rh2-O, respectively. To protect cells from the oxidative damage caused by ROS, organisms have developed several defense mechanisms to remove ROS including antioxidant enzymes GSH-PX and CAT. Both GSH-PX and CAT represent the most important H2O2 detoxification system in endothelial cells. GSH-Px enzymes reduce peroxides to alcohols using glutathione, thus preventing the formation of free radical damaging cells, whereas CAT directly catalyzes the decomposition of hydrogen peroxide (H2O2) to water and oxygen. As shown in Table 2, the activities
Figure 3. Analysis of HUVEC apoptosis by flow cytometry using Annexin V−FITC and PI. Quadrant analysis of fluorescence intensity of gated cells in Annexin V−FITC and PI channels was from 20000 events. Different letters above the bars present significant difference (P < 0.05). (A) Control; (B) H2O2 group (100 μM); (C) Rh2 group (20 μM); (D) Rh2-O group (20 μM).
of GSH-Px and CAT significantly decreased in the H2O2 group compared to the control, whereas pretreating with Rh2 and Rh2-O significantly increased the GSH-PX and CAT activities in the HUVECs. These results jointly indicated that enhancement of endogenous antioxidant preservation and attenuation of lipid peroxidation may represent major mechanisms of cellular protection against oxidative damage by ginsenoside Rh2 and its derivative Rh2-O. In addition, as depicted in Table 2, no significant difference was observed in the NO, LDH, MDA, GSH-Px, and CAT levels between the Rh2 and Rh2-O groups. These results further indicated that the synthesis of esterified derivative of Rh2 caused little impact on its bioactive protection against HUVEC oxidative damage. Effects of Rh2 and Rh2-O on Caspase-3 Activation of HUVECs. Cell apoptosis is induced mainly via two pathways, the mitochondrial apoptotic pathway and the death receptor pathway. Both pathways converge at caspase-3 activation. Caspase-3 is one of the downstream effectors of the caspase family and regarded as a major executor caspase that leads to 2101
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Table 2. Effect of Rh2 and Rh2-O on the Nitrite, LDH, MDA, GSH-Px, and CAT Activities in HUVECsa group control DMSO H2O2 Rh2 + H2O2 Rh2-O + H2O2
NO (μmol/L) 32.58 30.36 12.83 23.24 20.69
± ± ± ± ±
3.15 3.21 1.54* 2.12# 2.36#
LDH (U/L) 41.39 42.86 95.25 70.84 75.28
± ± ± ± ±
MDA (nmol/mg protein)
3.97 3.52 8.13* 5.99# 5.45#
4.58 4.03 10.32 7.92 6.11
± ± ± ± ±
GSH-PX (U/mg protein)
0.61 0.55 1.13* 0.69# 0.74#
167.92 171.35 108.63 253.75 246.28
± ± ± ± ±
18.25 20.12 9.65* 21.84 18.33#
CAT (U/mg protein) 40.38 46.25 15.97 57.28 50.19
± ± ± ± ±
2.68 3.14 1.06* 1.98# 3.83#
Values are the mean ± SD, n = 3. *, statistically significant differences between the untreated and the H2O2 group (p < 0.05); #, statistically significant differences between the H2O2- and the drug-treated group (p < 0.05). a
apoptosis.16 Caspase-3 activity was expressed as the fold of control group in this study; a lower value means better inhibitory activity. As depicted in Figure S1 (Supporting Information), an increase in caspase-3 activity was obvious in cells incubated with 100 μM H2O2 for 1 h. However, when the cells were pretreated with 20 μM Rh2 or Rh2-O for 2 h prior to the addition of 100 μM H2O2, the caspase-3 activation was significantly inhibited, which was consistent with the above results that Rh2 and Rh2-O protected HUVECs from oxidative damage and markedly decreased the HUVEC apoptosis. Furthermore, the inhibitory effects on caspase-3 activation had no significant difference between Rh2 and its esterified derivative as well. These studies demonstrated that Rh2 or Rh2-O can effectively restrain cell apoptosis by preventing caspase-3 activation. In conclusion, the transport of the ginsenoside Rh2 across the intestinal epithelium was evidenced as very poor, whereas its esterified derivative Rh2-O was verified to possess a better absorption in vitro. Because verapamil had a significant effect on the Papp values of Rh2 and a negligible effect on that of Rh2-O, the transport mechanisms for both Rh2 and Rh2-O across Caco-2 monolayers were speculated to be transcellular passive diffusion, with an active efflux pump (P-gp) involved for Rh2 and without an active efflux involved for Rh2-O. In addition, both Rh2 and Rh2-O exhibited protective effects against HUVEC oxidative damage stimulated by H2O2 and effectively suppressed the activation of caspase-3 and HUVEC apoptosis. Meanwhile, we found that the synthesis of esterified derivative of Rh2 caused an ignorable impact on its bioactivities against HUVEC oxidative injury. Therefore, it is noteworthy that the findings of the present study may provide useful strategy for molecular structure modification of ginsenosides to improve oral absorption of compounds in vitro without affecting their bioactivities against the oxidative injury of HUVECs. Our further work will examine whether the intestinal absorption of Rh2 and its oral bioavailabilities are enhanced in vitro through esterification of Rh2, as well as the effects of structure modification on its bioactivities in vivo.
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Funding
This work was supported by the National Natural Science Foundation of China (Grant 31360370), the Youth Foundation of Jiangxi Educational Committee (Grant GJJ13023), the Open Project Program of State Key Laboratory of Food Science and Technology, Nanchang University (Grants SKLF-KF-201211, SKLF-QN-201109); and the Young Teachers Development Programs of Jiangxi Province and Graduate Innovation Special Fund of Jiangxi Province (Grant YC2012-B007). Notes
The authors declare no competing financial interest.
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ASSOCIATED CONTENT
S Supporting Information *
Effect of Rh2 and Rh2-O on H2O2-induced caspase-3 activation in HUVECs. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*(J.-N.H.) Phone: +86 791 88304449-8230. E-mail:
[email protected]. *(Z.-Y.D.) Phone/fax: +86 791 88304402. E-mail:
[email protected]. 2102
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dx.doi.org/10.1021/jf404738s | J. Agric. Food Chem. 2014, 62, 2096−2103
