Bioavailability of andrographolide and protection against carbon tetrachloride-induced oxidative damage in rats
Haw-Wen Chena, Chin-Shiu Huangb, Chien-Chun Lic,d, Ai-Hsuan Lina, Yu-Ju Huanga, Tsu-Shing Wange, Hsien-Tsung Yaoa,*, Chong-Kuei Liia,b,**
aDepartment of Nutrition, China Medical University, Taichung, Taiwan
bDepartment of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan cSchool of Nutrition, Chung Shan Medical University, Taichung, Taiwan
dDepartment of Nutrition, Chung Shan Medical University Hospital, Taichung, Taiwan eDepartment of Biomedical Science, Chung Shan Medical University, Taichung, Taiwan
* Corresponding author.
** Correspondence to: C.-K. Lii, Department of Nutrition, China Medical University, 91, Hsueh-Shih Rd., Taichung 404, Taiwan. Fax: +886 422062891.
E-mail addresses: [email protected] (H.-T. Yao), [email protected] (C.-K. Lii).
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Abbreviations
ARE, antioxidant response element; EMSA, electrophoretic mobility shift assay; GCLC, glutamate cysteine ligase catalytic subunit; GCLM, glutamate cysteine ligase modifier subunit; GSH, glutathione; GSSG, glutathione disulfide; GST, glutathione S-transferase; HO, heme oxygenase; Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid-reactive substances.
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Abstract
Andrographolide, a bioactive diterpenoid, is identified in Andrographis paniculata. In this study, we investigated the pharmacokinetics and bioavailability of andrographolide in rats and studied whether andrographolide enhances antioxidant defense in a variety of tissues and protects against carbon tetrachloride-induced oxidative damage. After a single 50-mg/kg administration, the maximum plasma concentration of andrographolide was 1 µM which peaked at 30 min. The bioavailability of andrographolide was 1.19%. In a hepatoprotection study, rats were intragastrically dosed with 30 or 50 mg/kg andrographolide for 5
consecutive days. The results showed that andrographolide up-regulated glutamate cysteine ligase (GCL) catalytic and modifier subunits, superoxide dismutase (SOD)-1, heme
oxygenase (HO)-1, and glutathione (GSH) S-transferase (GST) Ya/Yb protein and mRNA expression in the liver, heart, and kidneys. The activity of SOD, GST, and GSH reductase was also increased in rats dosed with andrographolide (p<0.05). Immunoblot analysis and EMSA revealed that andrographolide increased nuclear Nrf2 contents and Nrf2 binding to DNA, respectively. After the 5-day andrographolide treatment, one group of animals was intraperitoneally injected with carbon tetrachloride (CCl4) at day 6. Andrographolide pretreatment suppressed CCl4-induced plasma aminotransferase activity and hepatic lipid peroxidation (p<0.05). These results suggest that andrographolide is quickly absorbed in the intestinal tract in rats with a bioavailability of 1.19%. Andrographolide protects against chemical-induced oxidative damage by up-regulating the gene transcription and activity of antioxidant enzymes in various tissues.
Keywords Andrographolide; Antioxidant enzymes; Bioavailability; Carbon tetrachloride; Hepatoprotection; Nrf2 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Introduction
Oxidative stress is generated when the balance between oxidation and antioxidation is disrupted. Under this condition, reactive oxygen species (ROS) are overproduced, which leads to oxidation of cellular macromolecules and damage to cellular functions. Oxidative stress is known to be associated with the development of chronic human diseases including cardiovascular disease, cancer, cataracts, and neurodegenerative diseases (Cooke et al., 2003). This explains why antioxidant phytocompounds such as flavonoids, organosulfur compounds, terpenoids, and carotenoids in fruits and vegetables display chemoprevention against ROS-related diseases (Hollman and Katan, 1997; Rahman, 2001; Boeing et al., 2012; Tsai et al., 2012). Large cohort studies have demonstrated an inverse correlation between total fruit and vegetable intake and risk of CVD (Hung et al., 2004) and gastric and esophageal cancer (Jeurnink et al., 2012). Similar biological activities of many herbs have also been attributed to their rich contents of flavonoids, terpenoids, and carotenoids (Moon et al., 2006).
To protect against ROS insult, an effective defense mechanism is critical. The inherent antioxidant defense system is composed of antioxidants including vitamin E, glutathione (GSH), and vitamin C and antioxidant enzymes including glutamate cysteine ligase (GCL), GSH peroxidase, GSH reductase, catalase, superoxide dismutase (SOD), heme oxygenase (HO), and GSH S-transferase (GST). GSH, a tripeptide, assists in the clearance of ROS and maintains the redox homeostasis (Lu, 2009). GCL catalyzes the rate-limiting step in GSH synthesis. It is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed by distinct genes (Franklin et al., 2009). SOD, both Cu/Zn- and Mn-SOD, quenches the superoxide anion and generates H2O2, and the H2O2 is then
decomposed to H2O by catalase and GSH peroxidase. HO is responsible for degrading free heme into Fe2+, carbon monoxide, and biliverdin, the latter being subsequently catabolized into bilirubin by biliverdin reductase (Ryter et al., 2006). GST catalyzes the conjugation of 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
GSH with a variety of electrophilic xenobiotics and also displays selenium-independent GSH peroxidase activity (Reddy et al., 1981). In fact, both HO and GST are recognized as not only antioxidant enzymes but also phase II drug metabolizing enzymes. Higher
antioxidant enzyme activity promises better protection of animals against oxidative injury. Most antioxidant enzymes are inducible, and the nuclear factor erythroid 2-related 2 (Nrf2) plays a key role in up-regulating their transcription (Baird and Dinkova-Kostova, 2011). The transcription factor Nrf2 positively regulates the basal and inducible expression of a large battery of genes including not only the familiar antioxidant and phase II
detoxification enzymes, but also the genes that control seemingly disparate processes such as immune and inflammatory responses, tissue remodeling and fibrosis, carcinogenesis and metastasis, and even cognitive dysfunction and addictive behavior (Baird and Dinkova-Kostova, 2011; Hybertson et al., 2011). Under unstressed conditions, Nrf2 is retained in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), which is constantly
ubiquitinated and rapidly degraded through the proteasome pathway (Katoh et al., 2005). In response to oxidative and electrophilic stress, Nrf2 is released from Keap1 and quickly translocates into the nucleus, where the free Nrf2 binds to the antioxidant response element (ARE). The ARE is found in many antioxidant enzyme genes including GCLC, GCLM, SOD, cytosolic GSH peroxidase, gastrointestinal GSH peroxidase, GSH reductase, GST, and HO-1 (Taguchi et al., 2011). An increase of GSH content and antioxidant enzyme expression and activity ameliorate oxidative insults and prevent the incidence of oxidative-related diseases (Dai et al., 2007; Kumar et al., 2012; Venkateshappa et al., 2012).
Andrographolide, a diterpene lactone, is the most active and abundant terpenoid of
Andrographis paniculata (Burm. f) (Pholphana et al., 2004). A. paniculata, a popular
medicinal herb in Asia, is used to treat infections, colds, fever, inflammation, and diarrhea.
In vivo and in vitro studies indicate that A. paniculata and andrographolide have diverse
physiological activities, including antioxidant, inflammatory, atherosclerosis, anti-75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
cancer, and hypoglycemic actions (Chao and Lin, 2010). The anticancer activity of andrographolide in a variety of cancer cells is attributed to its potency at inhibiting
proliferation, inducing apoptosis and cell-cycle arrest, and modulating the immune response against these cells (Varma et al., 2011). In streptozotocin-induced diabetic rats,
andrographolide and aqueous and ethanolic extracts of A. paniculata decrease the blood glucose level (Zhang and Tan, 2000; Husen et al., 2004) and induce glucose transporter 4 activity (Yu et al., 2003). Andrographolide suppresses intracellular adhesion molecule 1 expression in tumor necrosis factor -activated vascular endothelial cells and leads to an inhibition of monocyte adhesion to the endothelial cells (Chen et al., 2011). Recent works have also indicated that andrographolide pretreatment inhibits carbon tetrachloride (CCl4)- and cigarette smoke-induced mouse liver and lung injuries by suppressing inflammatory responses and increasing the GSH level and GSH peroxidase, GSH reductase, and HO-1 activity (Ye et al., 2011a; Guan et al., 2013). The modulatory effect of andrographolide on the antioxidation defense of tissues other than the liver and lung, however, is limited.
In this study, we firstly determined the pharmacokinetics and bioavailability of
andrographolide in rats. Thereafter, we examined the modulation by andrographolide of the antioxidant defense in red blood cells and tissues including liver, kidneys, and heart in rats. Finally, we investigated whether this modulation of antioxidant defense protects against CCl4-induced damage. 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
Materials and Methods
Chemicals and reagents
Andrographolide, NADPH, GSH, GSH disulfide (GSSG), Ellman’s reagent, 1-chloro-2,4-dinitrobenzene, pyrogallol, 2-thiobarbituric acid, 5,5’-dithiobis(2-nitrobenzoic acid), and methyl cellulose were obtained from Sigma (St. Louis, MO). TRIzol was purchased from Invitrogen (Carlsbad, CA). Carbon tetrachloride and acetonitrile were from Merck (Darmstadt, Germany). Fresh whole plants of A. paniculata were procured from Hualien, Taiwan. All other chemicals and reagents were of analytical grade and were obtained commercially.
Animals and treatments
Seven-week-old Sprague-Dawley rats were purchased from the Bio LASCO
Experimental Animal Center (Taipei, Taiwan). The animals were fed a standard pelleted diet and were randomly assigned to the control, 30-mg/kg/day andrographolide, or 50-mg/kg/day andrographolide group (n=6 per group). Rats were housed in plastic cages in a room kept at 23 ± 1oC and 60 ± 5% relative humidity with a 12-hour light and dark cycle. Food and drinking water were available ad libitum. Andrographolide was suspended in 0.5% methyl cellulose and was intragastrically given (10 ml/kg) for 5 consecutive days. At the end of the experimental period, rats were fasted overnight and were then killed by exsanguination via the abdominal aorta while under carbon dioxide (CO2/O2, 70%/30%) anesthesia. Heparin was used as the anticoagulant.
Plasma and red blood cells were separated from the blood by centrifugation (1750 ×g) at 4oC for 20 min.Theliver, heart, and kidneys from each animal were excised, weighed, freeze-clamped in liquid nitrogen, and stored at -80oC. Animals in this study were treated on the basis of the animal ethics guidelines of the Institutional Animal Ethics Committee.
For CCl4 treatment, rats were intraperitoneally injected with 1 mL/kg CCl4 (50% in 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145
olive oil, v/v) after being intragastrically dosed with 0, 30, or 50 mg/kg/d andrographolide for 5 days (n=8 per group). Blood was drawn 24 and 48 h after CCl4 treatment with heparin as an anticoagulant and the plasma was prepared for transaminase activity assay. The rats were then sacrificed as described above and the liver was removed for lipid peroxide determination.
For the andrographolide pharmacokinetic study, 7-week-old male Sprague-Dawley rats cannulated in the jugular vein were purchased from the Bio LASCO Experimental Animal Center (Taipei, Taiwan). The animals were fed a standard rat diet and were randomly assigned to a group treated with the ethanolic extract of A. paniculata (APE-treated, n=4) and an andrographolide-treated group (n=3). APE was prepared as described previously (Chen et al., 2013). Food and drinking water were available ad libitum. A single dose of 50 mg/kg of andrographolide or 940 mg/kg APE (equivalent to 50 mg/kg andrographolide), which was suspended in 0.5% aqueous methyl cellulose, was orally administered (10 mL/kg) to each rat. Serial blood samples with EDTA as an anticoagulant were collected up to 12 h after dosing from each rat. To determine the bioavailability of andrographolide, a group of animal (n=3) were intravenously injected with andrographolide at a dose of 10 mg/kg.
Preparation of cellular subfractions
The frozen liver, heart, and kidneys were thawed and then
homogenized (1:4, w/v) in ice-cold 100 mM phosphate buffer (pH 7.4) containing 1.5% KCl and 1 mM phenylmethylsulfonyl fluoride (PMSF). The
homogenates were centrifuged at 10,000 xg for 30 min at 4oC. The supernatant was further ultracentrifuged at 105,000 xg for 1 h and the final cytosol and microsome fractions were used for enzyme activity and immunoblotting assays. The frozen red blood cells were thawed and then hemolyzed (1:40, v/v) with hypotonic 5 mM Tris-HCl buffer, pH 7.4. After 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
centrifugation at 10,000 xg for 10 min, the supernatant was used for enzyme activity determination. The protein content of the cytosolic and microsomal fractions was measured by using the Coomassie plus protein assay kit (Pierce, Rockford, IL).
Measurement of antioxidant enzyme activity
GSH peroxidase activity was determined with the coupled method by using hydrogen peroxide (H2O2) as a substrate (Lawrence and Burk, 1976). GSH reductase activity was measured as described by Bellomo et al. (1987). GST activity was expressed as the rate of GSH and 2,4-chloro-dinitrobenzene (CDNB) conjugate formation according to the method of Habig and Jakoby (1981). SOD activity was determined by measuring the degree of inhibition of pyrogallol oxidation according to the method of Marklund and Marklund (1974).One unit of SOD activity is defined as the amount of enzyme required to inhibit the rate of pyrogallol oxidation by 50%. Catalase activity was measured by using H2O2 as a substrate as described by Aebi et al. (1984).
SDS-PAGE and Western blotting
Equal amounts of cytosolic and microsomal proteins were electrophoresed in an SDS-PAGE, and proteins were then transferred to polyvinylidene fluoride membranes. After the nonspecific binding sites were blocked with 5% nonfat dry milk in 15 mM Tris/150 mM NaCl buffer (pH 7.4), the membranes were hybridized with anti-GCLC (Abcam), GCLM (Santa Cruz), HO-1 (Calbiochem), SOD1 (Gene Tex), GST Ya and Yb (Oxford), GAPDH (Millipore), and actin (Sigma) antibodies. The immunoreactive bands were detected by using an enhanced chemiluminescence plus Western blotting detection reagent (Amersham Biosciences, Boston, MA).
RT-PCR 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197
Frozen liver and kidneys were homogenized in TRIzol reagent (1:20, w/v) to extract the total RNA. RNA extracts were then suspended in nuclease-free water and were frozen at -20oC until the RT-PCR analysis was performed (Yang et al., 2011). An amount of 0.4 μg total RNA was reverse transcribed by use of Moloney murine leukemia virus reverse transcriptase (Promega) in the presence of 150 μM of each dNTP, 40 units RNase inhibitor, and 250 nmol oligo(dT) in a final volume of 20 μL. cDNA was amplified in a thermocycler in a reaction volume of 50 μL containing 20 μL of cDNA, BioTaq PCR buffer, 50 μmol of each dNTP, 1.25 mM MgCl2, and 1 unit of BioTaq DNA polymerase (BioLine). The sequences for the PCR primers were as follows: for GCLC (forward: 5’
-CCTTCTGGCACAGCACGTTG-3’; reverse: 5’-TAAGACGGC ATCTCGCTCCT-3’), GCLM (forward: 5’-CTGACATTGAAGCCCAGGAG-3’; reverse: 5’-ACATTGCCAAAC CACCACA-3’), HO-1 (forward: 5’-AGCATGTCCCAGGATTTGTC-3’; reverse: 5’-AAG GCGGTCTTAGCCTCTTC-3’), SOD1 (forward: 5’-GCAGGGCGTCATTCACTT-3’; reverse: TTCTCGTGGACCACCATA-3’), GST Ya (forward:
5’-CCATCACCATCTTCCAGGAG-3’; reverse: 5’-CCTGCTTCACCACCTTCTTG-3’), and GST Yb (forward: TGGCACTCACAGGGAGGACC-3’; reverse:
5’-TTAAAGATGAGACAGGCCTGGG-3’). The PCR amplicons were then electrophoresed in 1%-agarose gels containing 40 mM Tris/20 mM glacial acetic acid/2 mM EDTA buffer.
Nuclear extraction and electrophoretic mobilityshift assay (EMSA)
Nuclear proteins of liver were extracted as described by Tian et al. (2004) with some modifications. Briefly, frozen liver (50 mg) was homogenized (1:18, w/v) in ice-cold hypotonic buffer containing 10 mM HEPES, 10 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 4 μg/mL leupeptin, and 20 μg/mL aprotinin, pH 7.9. The homogenates were placed in an ice bath for 15 min. After centrifugation of the samples at 600 xg for 10 min, the supernatant was mixed with 100 μL 10% Nonidet P-40 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223
and was allowed to sit in an ice bath for an additional 10 min. Crude nuclei were prepared by centrifugation at 5000 xg for 5 min and were resuspended in 100 μL of hypertonic buffer containing 10 mM HEPES, 400 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 4 μg/mL leupeptin, 20 μg/mL aprotinin, and 25% glycerol at 4oC for 45 min. Nuclear proteins were then collected by centrifugation at 12,000 xg for 15 min.
The DNA binding activity of extracted nuclear proteins was measured by EMSA (Yang et al., 2011). The LightShift Chemiluminescent EMSA Kit (Pierce Chemical Company, Rockford, IL) and biotin-labeled double-stranded rHO-1 ARE consensus oligonucleotides (5′-AACCATGACACAGCATAAAA-3′) were used to measure Nrf2 binding to DNA. Unlabeled double-strandedARE oligonucleotide and a mutant double-stranded oligonucleotide were used to confirm the protein-binding specificity.
Determination of GSH content and lipid peroxidation
GSH content in the liver and red blood cells was determined by high-performance liquid chromatography-mass spectrometry (HPLC/MS, Hewlett Packard) as described by Guan et al. (2003). Briefly, 100 μL of liver cytosolic fraction and red blood cell
hemolysate were reacted with 200 μL of 10 mM Ellman’s reagent by gentle mixing, followed by the addition of 60 μL of 20% 5-sulfosalicylic acid. After centrifugation at 10,000 xg for 10 min at 4oC, the supernatant was used to analyze GSH content. For heart and kidneys, frozen tissues were thawed and then homogenized (1:4, w/v) in ice-cold 100 mM phosphate buffer (pH 7.4) containing 1.5% KCl and 1 mM PMSF. The homogenates were mixed with equal volumes of 5% trichloroacetic acid solution to precipitate the protein. After centrifugation at 5,000 xg for 5 min, 100 μL of the supernatant was mixed with 200 μL of 20 mM Tris/20 mM EDTA and then with 20 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
μL of 10 mM 5,5’-dithiobis(2-nitrobenzoic acid) solution. GSH content was determined by reading the absorbance at 412 nm.
The lipid peroxidation in red blood cells and tissues was determined by measuring the thiobarbituric acid-reactive substances (TBARS) in a fluorescence spectrophotometer (Hitachi F4500) as described by Fraga et al. (1988).
Biochemical assays
Plasma aspartate aminotransferase and alanine aminotransferase were measured by use of commercial kits (DiaSYS, Holzheim, Germany).
Plasma andrographolide determination
After administration, blood samples (200 L) were collected from the cannulated jugular vein of each rat at 0, 5, 10, 15, 30, 60, 120, 240, 360, 480, and 720 min after dosing. Plasma was separated from blood by centrifugation at 4000 xg for 10 min. An aliquot of 100 L plasma was mixed with 200 L acetonitrile and was then centrifuged at 10,000 xg for 15 min at 4oC. The supernatant was used to determine the concentration of andrographolide by LC/MS with some modifications as described by Gu et al. (2007). The HPLC system consisted of an Agilent 1100 series LC System (Palo Alto, CA) and an Agilent ZORBAX Extend-C18 (5 μm, 3.0×250 nm) interfaced to an Agilent MSD mass spectrometer equipped with an electrospray ionization source. The initial mobile phase composition was acetonitrile (solvent A) and ddH2O (solvent B). The following gradient system was used: 20% A (0-1 min), 20% A to 80% A (1–7 min), 80% A to 98% A (7–8 min), 98% A (8–11 min), 98% A to 20% A (11–12 min), and 20% A (12–18 min). The flow rate was 0.6 mL/min. The MS data acquisition was via selected ion monitoring. The negative ion of the testing compound was selected and the peak size was measured. Calibration standards of andrographolide were 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273
prepared by serial dilution of an andrographolide stock solution with blank plasma, yielding final concentrations of tolbutamide ranging from 0.24 to 500 g/mL of plasma.
Statistical analysis
Data are expressed as means ± SD. Statistical analysis was performed with SAS statistical software (Cary, NC). The significance of the differences among group means was determined by one-way ANOVA followed by Duncan’s test and the difference between mean values was determined by Student’s t-test. P values < 0.05 were taken to be statistically significant. 274 275 276 277 278 279 280 281 282
Results
Pharmacokinetics and bioavailability of andrographolide
Changes in plasma andrographolide concentrations in rats versus time after oral
administration are shown in Fig. 1. The concentration of andrographolide in the blood rapidly increased after dosing with 50 mg/kg of andrographolide and the maximum concentration of 0.35 µg/mL was reached at 30 min. The pharmacokinetic parameters of andrographolide calculated from these data are shown in Table 1. The area under the curve (AUC0-12 h), mean retention time, and half-life (t1/2) of andrographolide were 0.50 µg/mL・h, 1.24 h, and 2.49 h, respectively. The oral bioavailability of andrographolide was 1.19%. Compared with
administration of 50 mg/kg andrographolide, administration of the ethanolic extract of
Andrographis paniculata at 940 mg/kg (equivalent to 50 mg/kg andrographolide) resulted in a
greater AUC0-12 h, mean retention time, and t1/2 of andrographolide (p < 0.05). Also, the bioavailability of andrographolide in the extract was 4-fold higher, i.e., 5.15% (Table 1).
Effects of andrographolide on rat growth characteristics
After treatment for 5 days with 30 or 50 mg/kg andrographolide, the body weight; liver, heart, and kidney weights; and relative percentage of tissue weight were not significantly different from those in the control rats (Table 2). Thus, treatment with andrographolide did not appear to have an adverse effect on rat growth.
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Andrographolide increases antioxidant enzyme activities
Activities of catalase, SOD, GSH peroxidase, GSH reductase, and GSH S-transferase in the red blood cells, liver, heart, and kidneys were determined (Table 3). Compared with the values in the control rats, the SOD activity in the liver, kidneys, heart, and red blood cells was dose-dependently higher in rats treated with andrographolide (p < 0.05). The activity of catalase was dose-dependently increased by andrographolide in the heart (p < 0.05). An increase in GSH peroxidase activity with andrographolide treatment was noted only in the kidneys (p < 0.05). With the exception of the liver, andrographolide increased GSH reductase activity in the kidneys, heart, and red blood cells (p < 0.05). Regarding GSH S-transferase, enzyme activity was 21% and 79% higher in the liver of rats treated with 30 and 50 mg/kg/day andrographolide,
respectively (p < 0.05), than in the control rats. A 65% higher cardiac GST activity was also noted in rats treated with 30 mg/kg/day andrographolide (p < 0.05).
Andrographolide effects on GSH contents and lipid peroxidation
Next, changes in TBARS production and GSH levels were measured. As shown in Table 4, lipid peroxidation in the liver, kidneys, and red blood cells was dose-dependently decreased by andrographolide (p < 0.05). Andrographolide, however, did not significantly change TBARS production in heart tissue. An increase in GSH contents by andrographolide was noted only in rat heart (p < 0.05). After treatment for 5 days, a 37% and 42% increase in cardiac GSH content was 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322
noted in rats dosed with 30 and 50 mg/kg andrographolide, respectively.
Andrographolide induces antioxidant enzyme expression
Protein and mRNA levels of antioxidant enzymes in rat liver, kidneys, and heart were determined by immunoblotting and RT-PCR. Consistent with the changes shown in SOD and GST activities, immunoblots revealed that protein levels of SOD1 and two GST isozymes, i.e., GST Ya and GST Yb, in rat liver (Fig. 2A), kidneys (Fig. 3A), and heart (Fig. 4A) were up-regulated by treatment for 5 days with 30 and 50 mg andrographolide compared with that in the control rats. The protein expression of HO-1, GCLC, and GCLM was also higher in these tissues in andrographolide-treated rats than in the control rats. Consistent with the changes in protein levels, mRNA levels of GCLC, GCLM, GST Ya and Yb, SOD1, and HO-1 in the liver (Fig. 2B) and kidneys (Fig. 3B) were induced by andrographolide.
Nrf2 is an important transcription factor that modulates the transcription of many antioxidant enzyme genes. To confirm the activation of this key factor in rat liver by
andrographolide, changes in nuclear Nrf2 contents and Nrf2 binding activity to oligonucleotides harboring the ARE were examined. Compared with the control rats, the rats treated with both 30 and 50 mg/kg andrographolide showed increased nuclear translocation of Nrf2 (Fig. 5A).
Moreover, EMSA revealed that andrographolide enhanced the binding activity of nuclear Nrf2 protein to the ARE consensus sequences (Fig. 5B).
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Andrographolide alleviates CCl4-induced hepatotoxicity
We used CCl4 to induce oxidative damage and to investigate the protective effects of andrographolide. In the control rats, CCl4 administration led to increases in plasma alanine aminotransferase and aspartate aminotransferase (p < 0.05; Table 5). The maximum increase in these aminotransferases in plasma was reached at 24 h, after which concentrations slightly decreased at 48 h. Compared with that in the control rats, the increase in plasma
aminotransferase activities by CCl4 was significantly suppressed by pretreatment with both 30 and 50 mg/kg andrographolide (p < 0.05). Furthermore, hepatic TBARs production was dose-dependently suppressed by andrographolide pretreatment (p < 0.05), and the GSH/GSSG ratio was dose-dependently increased (p < 0.05).
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Discussion
A large body of evidence indicates that the inhibition of abnormal ROS production is a key factor contributing to the health benefit of various phytochemicals present in fruits, vegetables, and herbs (Surh et al., 2008). In the present study, we examined protection by andrographolide against chemical-induced oxidative damage. In addition, we determined the pharmacokinetic parameters and bioavailability of andrographolide in rats. The results indicated that
andrographolide protects against CCl4-induced hepatotoxicity by up-regulating antioxidant enzyme expression via Nrf2 activation. Similar changes in antioxidant defense were found in heart and kidneys and also red blood cells. After an orally given dose of 50 mg/kg of
andrographolide, the maximum plasma andrographolide concentration was 1 µM and the bioavailability was 1.19%.
Oxidative damage is involved in the pathogenesis of many chronic diseases, including metabolic syndrome, cancer, neurodegenerative diseases, and cardiovascular diseases. This oxidative damage is therefore an attractive therapeutic target for preventing oxidative-stress-related diseases (Halliwell, 2012). HO-1 is an inducible enzyme that catalyzes the degradation of free heme into ferrous iron, carbon monoxide, and biliverdin. HO-1-null mice exhibit a
significant increase in plasma lipid hydroperoxides and have severe aortitis and coronary arteritis with mononuclear cellular infiltration and fatty streak formation (Ishikawa et al., 2012). In contrast, induction of HO-1 expression attenuates the formation of foam cells induced by treatment with oxidized low-density lipoprotein (Tsai et al., 2010). Increasing cellular 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
antioxidant enzyme activity and GSH content is, therefore, a promising strategy for
counteracting the oxidative damage caused by a variety of chemicals, including cigarette smoke, acetaminophen, ethanol, and pesticides (Wu et al., 2010; Wu et al., 2012a; Wu et al., 2012b; Micale et al., 2013). For example, one study showed that butein and phloretin increase cellular GSH content and HO-1 expression and ameliorate tert-butyl hydroperoxide-induced damage in rat primary hepatocytes and CCl4-induced hepatotoxicity in rats (Yang et al., 2011). In another study, sulforaphane was shown to up-regulate endothelial HO-1 and GCLC and GCLM
expression and decrease oxidized low-density lipoprotein-induced oxidative stress. These effects of sulforaphane led to the inhibition of NFκB activation, inhibition of NFκB-driven intracellular adhesion molecule 1 and vascular adhesion molecule 1 expression, and a decrease in monocyte adhesion to the vascular endothelium (Huang et al., 2013). In a study in mice, the decrease in acetaminophen-induced liver damage by acanthoic acid, a diterpenoid found in Acanthopanax
koreanum, was partly attributed to an increase of SOD and GSH peroxidase activity (Wu et al.,
2010). Also in mice, a polyphenol-rich extract from apple was shown to have effective free radical–scavenging activity, to increase hepatic SOD activity and GSH content, and to
ameliorate CCl4-induced acute hepatoxicity (Yang et al., 2010). In the present study, 5 days of andrographolide treatment had potent effects on up-regulating GCLC, GCLM, SOD-1, HO-1, and GST isozyme expression and SOD, GST, and GSH reductase activity in the liver, heart, and kidneys. In addition, a similar increase in SOD and GSH reductase activity by andrographolide was found in the red blood cells.
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CCl4 is primarily metabolized by the cytochrome p450 system, especially the cytochrome
p450 2E1, which generates the reactive metabolites trichloromethyl free radical (·CCl
3) and
proxy trichloromethyl radical (·OOCCl
3). The production of CCl4-derived free radicals triggers
the chain oxidation of membrane lipids and proteins and ultimately disrupts membrane integrity (Diaz Gomez and Castro, 1980). Owing to the rich content of cytochrome p450 enzymes in the
hepatocytes, CCl4 is commonly used as a hepatotoxicant. Increases in lipid peroxidation and
enzyme leakage and a decrease in antioxidant defense are the common consequences of exposure to CCl4. In addition, leukocyte infiltration, collagen accumulation, and fibrosis are induced after long-term exposure to CCl4. Recently, andrographolide was reported to protect against CCl4-induced liver toxicity. Studies have shown that andrographolide helps to return the CCl4–CCl4-induced decrease in hepatic antioxidant enzyme activity and GSH content to control levels (Kapil et al., 1993; Maiti et al., 2010; Ye et al., 2011a).In the present study, the activity and expression of most of the antioxidant enzymes was comprehensively examined before CCl4 treatment. These results showed that andrographolide effectively up-regulates SOD1, GCLC, GCLM, GST Ya and GST Yb, and HO-1 mRNA and protein expression (Fig. 2) as well as SOD and GST activity in liver tissue (Table 3). The CCl4-induced increase in plasma aminotransferase activity and hepatic lipid peroxidation (Table 5) were attenuated by andrographolide pretreatment. Moreover, the decreases in GSH/GSSG ratio induced by CCl4 were reversed. These findings clearly indicate that andrographolide is a potent chemopreventive agent against oxidant insult.
We were also interested in knowing whether the modulation of antioxidant defense by 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412
andrographolide was tissue specific.As shown, changes in SOD1, GCLC, GCLM, GST Ya and Yb, and HO-1 expression and in SOD, GSH reductase, and GST activity were noted not only in the liver but also in the kidneys and heart. In addition, andrographolide increased SOD and GSH reductase activity in the red blood cells. Saranya and colleagues (2011) reported that
andrographolide improves the antioxidation capacity in the gastric mucosa layer and reduces ethanol-induced gastric ulcers. In cigarette smoke–induced lung injury and nicotine-induced brain damage models, animals that receive andrographolide have higher GSH content and GSH peroxidase, GSH reductase, and HO-1 activity than do control rats and have less tissue damage (Das et al., 2009; Guan et al., 2013). These findings support that andrographolide displays physiological activity in a systemic manner.
Expression of antioxidant enzyme genes is susceptible to the cellular redox status. A number of transcription factors including Nrf2, activating protein 1, nuclear factor kappa B, and forkhead box O have been demonstrated to play key roles in the transcription of numerous antioxidant genes (Baird and Dinkova-Kostova, 2011; Dansen, 2011). Among those, Nrf2 is the transcription factor most well known to be involved in antioxidation, anti-inflammation, and modulation of the phase II drug metabolism of phytochemicals (Surh et al., 2008). Under oxidative or electrophilic stress, Nrf2-Keap1 disassociation arises from Nrf2 phosphorylation or Keap1 thiol modification, leading to stabilization of Nrf2, an increase in Nrf2 nuclear
translocation, and activation of Nrf2-dependent genes including SOD-1, GSH peroxidase, GSH reductase, GCLC, GCLM, HO-1, and GST (Zhang and Hannink, 2003; Taguchi et al., 2011). 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432
The increase in nuclear Nrf2 content in the liver (Fig. 5A) and in Nrf2 binding activity to DNA (Fig. 5B) in the present study suggest that the modulation of antioxidant enzyme gene expression by andrographolide is at least in part through Nrf2 activation.
Determining the pharmacokinetic parameters of a phytochemical is required for
understanding its potent chemopreventive activity in vivo. In humans, most phytochemicals are poorly absorbed and quickly metabolized and excreted, which leads to poor bioavailability. In fact, plasma concentration of a phytochemical rarely exceeds 1 µM after the consumption of 10 to 100 mg (Scalbert and Williamson, 2000). Regarding andrographolide, a recent study reports that the maximum concentration and time to achieve the maximum concentration are 0.66 µM and 29.8 min, respectively, after 120 mg/kg of this diterpenoid is given orally (Ye et al., 2011b). In this study, after a single dose of 50 mg/kg, andrographolide was detected early in the plasma. The time to reach the maximum concentration and the maximum concentration were 30 min and 1 µM (0.35 µg/mL), respectively (Table 1). These findings suggest that andrographolide quickly passes through enterocytes and enters the blood circulation, where it is delivered to the liver and extrahepatic tissues. This explains the similar induction by andrographolide of antioxidant enzyme mRNA and protein expression and activity in the liver, heart, and kidneys. Moreover, the short mean retention time, i.e., 1.24 h, further indicates that the biotransformation and excretion of this diterpenoid is fast. It is interesting to note that the pharmacokinetic parameters of andrographolide differ when andrographolide is given as an ethanolic extract of A. paniculata. The higher AUC0-12 h, mean retention time, and plasma half-life of the ethanolic extract suggest 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452
that the metabolism or excretion of andrographolide are likely to be changed by other compounds present in A. paniculata (Table 1). This leads to better bioavailability when andrographolide is given in a mixture rather than in a purified form (1.19% vs. 5.15%). This result can be explained by a similar study (Cheng et al., 2014). It has been
demonstrated that the absorption, distribution, metabolism, and excretion of geniposide are affected by the other compounds present in the extract of Gardenia fruits. This characteristic accounts for the bioavailability of geniposide is 32.3% when the extract of Gardenia fruits is administrated compared with 4.23% when a pure compound is administrated.
It is interesting to answer whether the 1 µM concentration reached in plasma is sufficient to trigger diverse physiological responses such as those noted in vitro. Recently, a concentration of 1 µM andrographolide was reported to effectively induce HO-1 expression and inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and invasion in MCF-7 breast cancer cells (Chao et al., 2013). In vascular endothelial cells, andrographolide in the range of 2.5 to 7.5 µM suppresses tumor necrosis factor α–induced intercellular adhesion molecule 1 expression, which subsequently attenuates endothelial cell adhesion to monocytes (Yu et al., 2010). These findings clearly support that the effective concentration of andrographolide in inhibiting inflammation and tumorigenesis and enhancing antioxidant defense is approachable after oral administration.
In conclusion, pharmacokinetic study showed that andrographolide is quickly absorbed and 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472
metabolized in rats. A plasma andrographolide concentration of approximately 1 µM is reached at 30 min after administration and the bioavailability is 1.19%. After treatment for 5 days, andrographolide activates Nrf2 and the expression of numerous Nrf2-dpendent antioxidant genes and attenuates CCl4-induced liver damage. Similar changes in antioxidant defense are observed in the heart and kidneys. It is recognized that increased oxidative stress in accumulated fat is an early indicator of metabolic syndrome. Therefore, modulation of the redox state in adipose tissue is a potentially therapeutic target for obesity-associated metabolic syndrome. Based on the results of the present study, andrographolide has excellent antioxidant activity and it might be used in the management of oxidative stress-mediated diseases.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgments
This work was supported by grants NSC 101-2313-B-039-005-MY3 from the National Science Council and CMU101-ASIA-02 from the China Medical University, Taiwan.
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Legends
Figure 1. Changes in plasma andrographolide (AND) concentrations after oral administration. Rats were orally dosed with 50 mg/kg AND or 940 mg/kg of an ethanolic extract of
Andrographis paniculata (APE, equivalent to 50 mg/kg andrographolide). Serial blood samples
were collected up to 12 h and plasma andrographolide was determined. The values represent the means±SD, n = 3 to 4.
Figure 2. Andrographolide (AND) increases antioxidant enzyme expression in rat liver. Rats were intragastrically administered 30 or 50 mg/kg/day andrographolide for 5 days, after which the animals were sacrificed and the liver tissues removed. (A) Equal amounts of cytosolic and microsomal (for HO-1) proteins were electrophoresed by SDS-PAGE and protein levels were detected by Western blot. Three to four rats for each group are shown. (B) Pooled RNA samples of each group were used to determine antioxidant enzyme mRNA levels by RT-PCR.
Figure 3. Induction of GCLC, GCLM, GST Ya, GST Yb, SOD-1, and HO-1 protein (A) and mRNA (B) levels in rat kidney by andrographolide (AND). Animals were treated as stated in the legend for Fig. 2. Protein and mRNA expression were measured by Western blot and RT-PCR, respectively.
Figure 4. Cardiac GCLC, GCLM, GST Ya, GST Yb, SOD-1, and HO-1 protein expression after treatment with andrographolide (AND) for 5 days. Animals were treated as stated in the legend to Fig. 2. The heart was removed and tissue homogenate of the ventricles was prepared for immunoblotting assay.
Figure 5. Effect of andrographolide (AND) on Nrf2 activation in rat liver. After treatment of rats with 30 or 50 mg/kg andrographolide for 5 consecutive days, nuclear protein extracts were 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689
prepared from liver tissues as described in the Materials and Methods. Immunoblots of nuclear extracts were probed with Nrf2 antibody (A). For Nrf2 nuclear protein DNA-binding activity, aliquots of nuclear extracts (8 μg) were used for EMSA (B). To confirm the specificity of the nucleotide, 200-fold cold probe (biotin-unlabeled ARE binding site) and biotin-unlabeled double-stranded mutant ARE oligonucleotide (2 ng) were included in the EMSA. Three rats of each group are shown.
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