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Assessment of the renal protection and hepatotoxicity of rhubarb extract in rats
Journal of Ethnopharmacology 124 (2009) 18–25
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Assessment of the renal protection and hepatotoxicity of rhubarb extract in rats Jiabo Wang, Yanling Zhao, Xiaohe Xiao ∗ , Huifang Li, Haiping Zhao, Ping Zhang, Cheng Jin China Military Institute of Chinese Materia Medica, 302 Military Hospital, Beijing 100039, PR China
a r t i c l e
i n f o
Article history: Received 5 November 2008 Received in revised form 7 April 2009 Accepted 8 April 2009 Available online 17 April 2009 Keywords: Chronic renal failure (CRF) Rhubarb Renal protection Toxicity Anthraquinones High performance liquid chromatography (HPLC)
a b s t r a c t Aim of the study: Rhubarb is well used to treat chronic renal failure (CRF) in China and Japan, but recent studies reported that the anthraquinone derivatives contained in rhubarb had nephrotoxicity. In this investigation an attempt was made to assess the value and toxic potential of rhubarb to treat CRF. Materials and methods: Histopathologic and biochemical tests combined with toxicokinetic analysis were performed to investigate the nephrotoxic potential and protective effect of rhubarb extract. Results: In normal rat groups, no death was observed and no renal lesion was found after repetitive administration of rhubarb for 3 weeks. The survival rate, pathologic conditions and biochemical indexes of CRF rats treated with rhubarb at two dosages were all improved and significant amelioration was found in the low dosage group compared to the untreated CRF group. Rhein was the mainly absorbable anthraquinone derivative into systemic circulation after oral administration and the area under curve of rhein in CRF groups was lower than that in normal groups at same dosage. Conclusions: After 3 weeks of administration of rhubarb extract, there was evidence of protective effect to CRF rats, while incidences of hepatotoxicity with minimal to mild hyaline droplets were also observed in normal rats. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Anthraquinones (AQs) are the active components present in the root and rhizoma of rhubarb, which have wide pharmacological effects and also some toxic concerns on genetic toxicity (Mueller et al., 1998a,b) and nephrotoxicity (Yan et al., 2006). Despite the positive in vitro results of genetic toxicity, results from in vivo studies in rodents showed lack of mutagenic activity (Mengs et al., 1997; National Toxicology Program, 2001). However, the nephrotoxicity of AQs has been revealed through both in vitro and in vivo studies. Two in vitro studies reported that emodin, one of the anthraquinones contained in rhubarb, could induce lesion on human proximal tubular epithelial cell line HK-2 cells (Wang et al., 2007a) and repress the proliferation of rat kidney glomerular mesangial cells (MC) through inhibition on c-myc mRNA expression (Liu et al., 1996). In 2001, the National Toxicology Program reported that exposure of rats to emodin resulted in an increase in incidence of renal tubule hyaline droplets and severities of renal tubule pigmentation in both male and female animals. On the other hand, rhubarb has been well used in China and Japan to treat chronic renal failure (CRF) and reveals desirable prospects in clinic (Mitsuma et al., 1984, 1987; Huang, 1993; Li and Wang, 2005). And there were few clinical reports in China about the increase of renal risk of rhubarb in treatment of
∗ Corresponding author at: 100#, the 4th Ring Road, 302 Military Hospital, Beijing 100039, PR China. Tel.: +86 10 66933322; fax: +86 10 63879915. E-mail addresses: [email protected], pharm [email protected] (X. Xiao). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.04.018
nephritic diseases. The possibility of an association between concurrent renal protection and lesion with the use of rhubarb to treat CRF needs to be assessed. Former studies have focused on the safety of the components contained in rhubarb, such as emodin, but a single component could not represent the whole of the rhubarb and the decision could not be applied to such phytomedicines processed with crude materials. Furthermore, such evaluations were only performed on normal animals and the differences between normal and morbid animals were not considered. The harms of a drug to renal failure animals might be obviously distinct to normal ones. It is, therefore, necessary to assess the value and toxic potential of rhubarb to treat CRF based on comparative investigation on both normal and CRF animals by histopathologic and biochemical tests. Besides, toxicokinetic studies were performed because the changes of a drug’s side effects from normal animals to morbid ones might be correlated with the alteration of its metabolism. Some experimental animal models of chronic renal failure, such as 5/6 nephrectomy/bipolar resection and adenine-induced renal failure models, were well documented (Yokozawa et al., 1986; Terzi et al., 1992). In this study, adenine-induced CRF model was selected because of the convenience of operation and high achievement ratio compared to nephrectomy and other chemical inducement (Lacour et al., 2005), and adenine was administered intragastricly at 250 mg kg−1 of body weight per day for 2 weeks instead of using an adenine content in diet of 0.75%. Rhubarb is the peeled and dried root of Rheum palmatum L., Rheum tanguticum Maxim. ex Balf. or Rheum officinale
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Baill. (family: Polygonaceae) stipulated in Chinese Pharmacopoeia (PPRC, 2005). Rhubarb is also officially listed in European and Japanese Pharmacopoeia (European Pharmacopoeia, 2001; Japanese Pharmacopoeia, 2006). Rheum palmatum was researched in this paper for it is stipulated in all of the three pharmacopoeias mentioned above and is more commonly used than other species. There are various kinds of constituents contained in rhubarb, which can be classified as anthraquinones, dianthrones, stilbens, anthocyanins, flavonoids, polyphenols, organic acids, and chromones (Kashiwada et al., 1989). Among them, anthraquinone derivatives, including emodin, chrysophanol, rhein, aloe-emodin, physcion, and their glucosides, are accepted as important active components with various pharmacological effects such as purgation, antibacterial, anti-inflammation, antitumor activity and nephric protection (Yokozawa et al., 1985; Dictionary of Traditional Chinese Medicine, 1997). The blood concentrations of these five AQs were determined to describe the pharmacokinetic course of rhubarb. 2. Materials and methods 2.1. Plant material and chemicals The dried root and rhizoma of Rheum palmatum were collected in Lixian county of Gansu province of China in November of 2006 and were identified by Professor Xiaohe Xiao, a taxonomist at PLA Institute of Traditional Chinese Material Medica. A voucher specimen (Rh200611W) was deposited in the institute. Rhubarb was grinded and decocted twice with water for 30 min each. The resulting extracts were decanted, filtered and evaporated to dryness under reduced pressure. This dried extract was re-dissolved and dispersed in warm water and then cooled for intragastric administration to rats. Aloe-emodin, rhein, emodin, chrysophanol and physcion were also supplied by National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) and the chemical structure of these anthraquinone derivatives are shown in Fig. 1. Methanol (HPLC grade, Fisher USA), aether, hydrochloric acid and phosphoric acid (AR, Beijing Chemical Regents Inc., PR China) were used for the mobile phase of chromatographic analysis and blood sample preparation. Adenine (>98%, Bioszume Inc., Beijing) was used to copy the chronic renal failure on rats. 2.2. Animals and study design Male and female Sprague–Dawley (S.D.) rats (License No.: SCXK2000-0010) of approximately 140 g were obtained from NICPBP. Just prior to the experiment, 120 Sprague–Dawley (S.D.)
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Fig. 1. Structures of rhein, emodin, aloe-emodin, chrysophanol and physcion.
rats were randomized into six groups of 20 animals each. The groups shown in Table 1 were differentiated based on animal model and dosage. Three groups were treated with adenine (250 mg/kg of body weight per day) for 2 weeks to induce the chronic renal failure experimentally and the others received physiologic saline. The dosages of rhubarb were 3 and 20 g/kg of body weight per day od (counted on the quantity of crude material) and were equivalent to 6 and 40 times of the upper dose of human stipulated in Chinese Pharmacopoeia (0.5 g/kg), respectively. The extract of rhubarb was administered intragastricly. The dosage was set at high level to uncover any potential toxicity in order to investigate the renal risk of rhubarb rigorously. Feed and water were available ad libitum. The experiment lasted for 3 weeks. At the 7th, 14th and 21st experimental day, blood samples were collected after administration at eight time points (10, 20, 30, 50, 90, 150, 360 and 1440 min) for toxicokinetic analysis. Five AQs in specimens were determined by high performance liquid chromatography (HPLC). Necropsies were performed on all animals. Animals sacrificed midway were dissected in time for histologic examination. All experiments using rodents were performed in accordance with the applicable guidelines and regulations. All rats received humane care in compliance with the institutional animal care guidelines approved by the Ministry of Science and Technology of China. 2.3. Histopathologic examination and biochemical analysis Necropsies were performed on all study animals. Tissues for histopathologic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of approximately 5 m, and stained with hematoxylin and eosin. Biochemical analysis was performed on all studied animals. Biochemical parameters, serum urea nitrogen (SUN), creatinine (CREA), alanine aminotransferase (ALT), aspartate aminotrans-
Table 1 Survival and incidences of histopathologic lesions on kidney and liver in different groups. Animal model
Dosagea (g kg−1 )
Survivalb
N NL NH
Normal
0 3 20
20c 20 20
0d (0e ) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
CRF CRFL CRFH
CRF
0 3 20
6 16** 9
20 (3.3) 13 (2.2** ) 15 (2.5** )
20 (2.4) 14 (1.9* ) 14 (2.0)
Group
Renal tubule hyaline droplet
Renal tubule pigmentation
Glomeruli necrosis 0 (0) 0 (0) 0 (0) 20 (2) 10 (1.2** ) 14 (1.4* )
Hepatic hyaline droplets 0 (0) 8 (1.5** ) 15 (2** ) 15 (2.5) 15 (2.4) 14 (2.6)
N, normal rats group without treatment; NL, normal rats groups treated by rhubarb in low dosage (3 g kg−1 ); NH, normal rats groups treated by rhubarb in high dosage (20 g kg−1 ); CRF, CRF rats group without treatment; CRFL, CRF rats group treated by rhubarb in low dosage (3 g kg−1 ); CRFH, CRF rats group treated by rhubarb in high dosage (20 g kg−1 ). a Dosage was counted with the quantity of crude material. b Animals initially in the study of every group are 20. c Number of survived animals. d Number of animals with lesion. e Average severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. * p < 0.05, comparison between untreated and rhubarb-treated groups. ** p < 0.01, comparison between untreated and rhubarb-treated groups.
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ferase (AST), total protein (TP), albumin (ALB), calcium (CA), inorganic phosphate (P), total cholesterol (CHOL), alkaline phosphatase and total bile acids, were determined using the Hitachi clinical analyzer 7020 (Hitachi High-Technologies Corporation, Japan). 2.4. Specimen preparation for determination of anthraquinones For the determination of the aqueous extract of rhubarb, 0.15 g of the extract was dissolved in 10 ml of 2 M HCl and 20 ml of chloroform was added and the solution was kept on a water bath for 1 h. The hydrolyzed solution was extracted with 10 ml of chloroform four times and the combined extract was then evaporated to dryness. The residue was dissolved and transferred into a 50 ml volumetric flask by methanol. The solution was filtered through a 0.45 m filter before analysis. Blood specimens of about 0.3 ml were taken from the retroorbital sinus into heparinized polypropylene tubes. The blood was centrifuged (3000 × g, 10 min) and 100 l plasma was removed in a 1.5 ml tube. Then 10 l 3 M HCl water-solution and 10 l 120 g ml−1 danthron in methanol were added into the tube and stirred for 30 s. Aether of 1 ml was added and stirred for 2 min before centrifugation (10,000 × g, 10 min). The upper clear liquid of 800 l in the tube was transferred into a plain tube and was evaporated gently (60 ◦ C, stream of nitrogen) to dryness. The dry residue was reconstituted in 100 l of the mobile phase for the fluorescence analysis.
anthraquinone:danthron). The correlation coefficient (r) was also expressed. Accuracy was calculated as the percentage found on the standard curve, and the precision of the method, expressed as the relative standard deviation (R.S.D. = 100 S.D./mean), was also evaluated. 2.6. Statistics Statistical analysis was performed using an unpaired t-test. When variances were not homogeneous a Welch’s correction was employed. p-Values lower than 0.05 were considered significant. Values are expressed as means ± standard error (S.E.). 3. Results 3.1. Common changes All rhubarb-treated rats had a dosage-related body color change pattern from yellow to red, which appeared earlier in the study as the exposure concentration increased. Red to brown urine and reddish-brown staining of the anal area were noted for all the groups treated with rhubarb. Marked and durative diarrhea accompanied with emaciation and inactivity was observed in all CRF and normal rats administered 20 g/kg of rhubarb. The average urine pH value of CRF rats was about 8.5, while the value of normal ones was in the range of 7.0–7.5.
2.5. Chromatography and calibration
3.2. Survival and histopathologic analysis
Chromatographic analyses were performed using an Agilent 1100 chromatograph. The chromatographic system consisted of a solvent degasser, a quaternary gradient pump before column, fluorescence detector (FLD), diode array detector (DAD) and a data station with analytical software Chemstation 8.03 (Agilent, Inc., USA). The method to analyze the anthraquinones in the aqueous extract of rhubarb was well established (PPRC, 2005) and it was used in this study. The mobile phase was a mixture of methanol–0.1% phosphoric acid in water (85:15, v:v). The flow rate was 1 ml min−1 . The injection volume was 10 l while the diode array detector (ultraviolet detector) was set at 254 nm. The chromatographic separation was performed on a Kromasil® C18 analytical column (250 mm × 4.6 mm i.d., 5 m) and maintained at 25 ◦ C. The standard solution of five AQs (16 g/ml of each) was prepared in methanol. Since the plasma concentrations of anthraquinones absorbed into the body were very low, a sensitive and rapid method with fluorescence analysis was developed and calibrated in this study. The chromatographic separation was performed on a Phenix® C18 analytical column (150 mm × 4.6 mm i.d., 5 m) and maintained at 40 ◦ C. Mobile phase consisting of a mixture of methanol–0.1% phosphoric acid in water (80:20, v:v) with the flow rate of 2 ml/min was employed. Fluorescence detection was performed with excitation wavelength at 256 nm and emission wavelength at 520 nm. For the calibration of fluorescence analysis, a mixed standard stock solution (1 mg ml−1 of five AQs each in methanol) was prepared. A calibration series of mixed standard solution were diluted as 10, 30, 50, 100, 200, and 300 g ml−1 (six samples for each concentration), all containing 10 g ml−1 danthron as internal standard. Transfer 10 l above mixed standard solutions into drug-free rat plasma (100 l) and following the sample preparation procedure was performed as described above. Six-level calibration series with six analyses at each concentration level were measured. The linearity of the calibration curve was tested and evaluated (y = kx + b, where x is the concentration ratio of an anthraquinone to danthron (internal standard) and y is the corresponding peak-area ratio
A summary of survival and histopathologic results for different animal groups is shown in Table 1. Typical histopathologic section photos are shown in Fig. 2. In normal groups, no death was observed and no renal lesion was found after repetitive administration of rhubarb for 3 weeks. However, minimal to mild hepatic lesion was observed in rhubarb-treated normal rats and it was dose dependent. In CRF groups, evident renal lesions were observed after modeling in histopathologic examination, principally involving the proximal tubules, some distal tubules and glomeruli, characterized by moderate to crucial necrosis of tubules, slight necrosis of glomeruli and lots of light brown uroliths (possibly 2,8dihydroxyadenine). Rats without treatment died successively and only 6/20 animals survived at the end of 3 weeks. The survival of two rhubarb-treated CRF groups both increased and a significant enhancement was found in the low dosage group compared to the untreated CRF group. The symptoms of renal and hepatic lesions found in rhubarb-treated CRF groups were similar to those in untreated group, as well as the extent of some renal lesions was significantly lessened. Based on those results, it could be concluded that there was no explicit evidence of renal toxicity in normal rats and there was evidence of a treatment-related renal protection of rhubarb to CRF rats. The phenomenon that the curative effect of rhubarb to chronic renal failure was not improved with dosage increase from 3 to 20 g/kg might be related to the marked and durative diarrhea, which led to electrolyte imbalance and consequently worsened renal disorder. Another phenomenon of mild erosion at gastric mucosa that was only observed in CRF rats might relate to the occurrence of the pharmacokinetic change of rhubarb and it would be discussed below. The phenomenon that a definite dosage-related increase of hepatic lesion of rhubarb was found in normal groups while a marginal increase of hepatic lesion was observed with dosage enhancement in CRF groups over the background led by adenine, might relate to the alteration of pharmacokinetics and hepatic metabolism induced by adenine and need be investigated further.
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Fig. 2. Typical histopathologic section photos of rats in different groups. There was no evidence of lesion on both kidney (a) and liver (e) of normal rats without treatment. No histopathologic change was observed on kidney (b), but a minimal to mild lesion on hepatic tissue (f) was found in normal rats after administering rhubarb. In CRF groups, evident renal lesions were observed in both untreated (c) and treated (d) rats. The light brown uroliths (pointed by arrows) were possibly generated by adenine. Hepatic lesions were also observed in both untreated (g) and treated (h) CRF rats. Mild erosion at gastric mucosa was observed in CRF groups (i), but was not found in normal groups.
Fig. 3. Comparison on the biochemical parameters of survived rats within different groups at the end of experiment. Significance of differences from the value of normal rats without administration of rhubarb (*p < 0.05, **p < 0.01). Significance of differences from the value of CRF rats without administration of rhubarb ( p < 0.05, p < 0.01).
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3.3. Biochemical analysis Those biochemical parameters changed significantly in the study groups are shown in Fig. 3. In normal animal groups, SUN and CREA values held constant in low dosage group while these two indexes increased 29% and decreased 12%, respectively in high dosage group at the end of 3 weeks administration of rhubarb, compared to untreated group. ALT and AST values increased significantly in both rhubarb-treated normal groups. TP, ALB and serum calcium values also increased significantly in low dosage group. In CRF animal groups, significant changes of many biochemical parameters mostly listed in Fig. 3 were observed after modeling with adenine for 2 weeks. For example, SUN and CREA values increased 7 and 3 times, respectively compared to normal. In the subsequent 3 weeks of treatment, the mortality of rhubarb-treated CRF groups decreased and the survivors’ SUN and CREA values were also significantly decreased comparing to those CRF ones’ without treatment, which demonstrated the curative effect of rhubarb on CRF. Other biochemical parameters changed in untreated CRF rats showed reconverting trends in rhubarb-treated groups, however, some parameters did not recover to normal levels: SUN, CREA, ALT and AST values were significantly higher; TP and ALB values were significantly lower than normal. 3.4. Chromatography Typical HPLC analyses are shown in Fig. 4. Five AQs and internal standard could be separated from endogenous substances in rat plasma within 8 min under this chromatographic condition and the analytical procedure is accurate and precise. The calibration curves were found to be linear between 4.680 and 187.2; 4.680 and 187.2; 4.680 and 187.2; 4.680 and 187.2; 4.680 and 187.2 for rhein, aloe-emodin, emodin, chrysophanol and physcion, respectively. The precision in 1 day and 1 week of AQs determination in rat plasma samples were found to be less than 5.28% and 8.73%, respectively. The mean recovery of five AQs (based on the determination of six spiked plasma samples) were verified from 91.47 to 94.26 (max SD = 6.43%, n = 6). 3.5. Toxicokinetic analysis After intragastric administration of water extract of Rheum palmatum L., the plasma concentrations of AQs were followed through time by HPLC. Rhein could be detected in all animal groups in plasma. Aloe-emodin could be detected in high dosage group and other AQs, emodin, chrysophanol and physicion, could be detected at few sampling points. This is consistent with the literature (Lee et al., 2003). Hence, rhein was intensively investigated to evaluate the toxicokinetics of rhubarb in this study. Average concentration of rhein versus time profiles are shown in Fig. 5 and pharmacokinetic parameters are shown in Fig. 6. At same dosage, the values of AUC (area under curve), Cpeak (peak concentration), t1/2 Ka (half time of absorption) and t1/2  (half time of elimination) of CRF groups were generally lower than those of normal groups. While the dosage increased, the AUC and Cpeak values did not increase proportionally in both CRF and normal groups. This might be on account of non-linear pharmacokinetic course, or of the indigestion led by severe diarrhea induced by rhubarb itself at high dosage. Comparing to normal groups, the decline of AUC or Cpeak in CRF groups might be on account of the acceleration of elimination, showed by the shortening of t1/2  . A possible reason behind this is the urine alkalization of CRF rats, which resulted in accelerated elimination of acidic anthraquinones. And of note, urinary volume was markedly increased in CRF rat groups compared with normal rat groups, which could also accelerate the elimination
Fig. 4. Typical chromatograms from the aqueous extract of Rheum palmatum L. and the plasma samples. (a) A chromatogram of mixed standard solution of five AQs. (b) A representative chromatogram from the extract of rhubarb. (c) A chromatogram of plasma sample contained standards of five AQs and danthron as internal standard. (d) A representative chromatogram of a rat plasma after administrated the extract of rhubarb which demonstrated that rhein was the most massively and frequently absorbed anthraquinone. (e) A chromatogram of blank plasma sample with internal standard. (1) Aloe-emodin; (2) rhein; (3) danthron; (4) emodin; (5) chrysophanol; (6) physcion.
of anthraquinones. According to the results in histologic test, the shortening of the half time of absorption (t1/2 Ka ) in CRF groups might be on account of the mild erosion at gastric mucosa found in CRF rats, which led to acceleration of absorption.
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Fig. 5. Average plasma concentration of rhein versus time profiles after oral administration of Rheum palmatum extract. Observed data of CRF and normal rats are differentiated with filled and opened symbols, respectively. High and low dosage groups are differentiated with square and circle, respectively.
4. Discussion A recent in vitro study showed that emodin, one of the anthraquinones contained in rhubarb, could induce lesion on
human proximal tubular epithelial cell line HK-2 cells (Wang et al., 2007a). In a comprehensive feeding study (National Toxicology Program, 2001), increased incidences of hyaline droplets of renal tubule epithelial cells were observed in F344/N rats receiving over
Fig. 6. Comparison on pharmacokinetic parameters of survived rats within different groups at the different time points of experiment. Significance of differences from the value of normal rats administered low dosage of rhubarb (**p < 0.01). Significance of differences from the value of normal rats administered high dosage of rhubarb ( p < 0.01).
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17000 ppm of emodin in diet for 16 days, over 321 ppm for 14 weeks and 280 ppm for 2 years. And it was reported that the total anthraquinones extracted from rhubarb could induce swell and denaturation of renal tubule epithelial cells in Sprague–Dawley (S.D.) rats with 13 weeks of administration at dosage of 4.5 g/kg of body weight per day counted with the quantity of the extract (Yan et al., 2006). Another 6-month study showed that the critical dosage of rhubarb extract inducing above renal lesion in rats was 10 g/kg of body weight per day counted with the quantity of crude material, and such lesion could be recovered after drug withdrawal (Wang et al., 2007b). In this study, however, no renal lesion was found after repetitive administration of rhubarb to normal rats for 3 weeks at the dosage of 3 and 20 g/kg of body weight per day (counted with the quantity of crude material), and no additional rhubarb-related renal lesion was found in CRF rats over which was induced by adenine. In addition to this, there were few clinical reports in China about the increase of renal risk of rhubarb in treatment of nephritic diseases unless it was misused (Xiao, 2002b). Therefore, data suggest that the possibility of renal lesion with the use of rhubarb to treat CRF would be little when the dosage was properly controlled. Beyond the effect on kidney, an incidence of minimal to mild hyaline droplets in hepatic tissues indicated by histopathologic examination and biochemical analysis was observed in normal rats after administration of rhubarb at dosage of 3 g/kg for 3 weeks and the lesion was consequently worsened while the dosage increased. Such hepatic injury was also reported in the above-mentioned literatures: the 6-month study on rhubarb extract and the 2-year study on emodin, though it could be recovered after drug withdrawal (Wang et al., 2007b). But to adenine-induced CRF rats, the enhancement of such hepatic lesion was marginal with the increase of dosage when the lesion background induced by adenine was subtracted. In addition, the present toxicokinetic results showed that the values of AUC (area under curve, of plasma rhein) of CRF groups were significantly lower than those of normal groups at same dosage. Hence, an intuitionistic relationship between hepatic lesion and rhein, or other anthraquinones, could be included and the plasma level of rhein might be valuable to indicate the toxicity of rhubarb. However, it was reported that rhubarb and emodin could protect animals against hepatic injury induced by tetrachloromathane (Zhou and Jiang, 1991; Zhan et al., 2001). The divergence of the effect on liver of rhubarb is rarely concerned and requires further investigation. Rhubarb has been used to treat renal diseases in China for several decades and reveals desirable prospects to treat CRF (Xiao et al., 2002a; Wang et al., 2006). In a systemic review of 18 randomized or quasi-randomized trials from 15 Chinese journals, rhubarb showed a positive effect on relieving uremic symptoms, lowering serum creatinine, improving hemoglobin levels, and adjusting disturbance of lipid metabolism in 1322 patients with chronic renal disease, but the effect on reducing the number of deaths and the progression to end-stage kidney failure was not confirmed because of the small sample size (Li et al., 2004). In a prospective open-label trial involving 151 patients, the beneficial effect of rhubarb on the prevention of progression in chronic renal disease is more marked than that of captopril and their therapeutic effects are additive (Zhang et al., 1993). The combined treatment with rhubarb and an angiotensin-convertion enzyme (ACE) inhibitor seems to be quite promising (Li, 1996), but the interaction between them remains in uncertainty. In the present study, we found that the curative effect of rhubarb on CRF induced by adenine was positive in reducing the death number of rats and protecting the renal tissue. However, this curative effect was weakened by severe diarrhea, and side effect of rhubarb itself occurring at very high dosage, which would lead to electrolyte imbalance and indigestion, and consequently worsen the kidney disorder of CRF bodies and cause the decline of bioavailability, respectively. In addition, the alteration of phar-
macokinetic course in CRF rats compared to normal ones, involving the decline of plasma concentration and acceleration of elimination, indicated the decrease of exposure and shortening of half life of rhubarb in plasma. The effect of histopathologic condition on rhubarb pharmacokinetics remains uncertain. These results indicate that a well-designed clinical trial of rhubarb with long-term follow-up to treat chronic renal failure is warranted. Acknowledgement This work was financially supported by the National Key Technologies R&D Program of China (No. 2004BA721A14). References Dictionary of Traditional Chinese Medicine, 1st ed., 1997. New Medical College of Jiangsu, People’s Published of Shanghai, p. 102. European Pharmacopoeia, 4th ed., 2001. European Directorate for the Quality of Medicines, Council of Europe, Strasbourg, p. 1858. Huang, K.C., 1993. The Pharmacology of Chinese Herbs. CRC Press, Boca Raton, FL, pp. 233–234. Japanese Pharmacopoeia, 15th ed., 2006. Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Tokyo, p. 1344. Kashiwada, Y., Nonaka, G.I., Nishioka, I., 1989. Studies on Rhubarb (Rhei Rhizoma). XV. Simultaneous determination of phenolic constituents by high-performance liquid chromatography. Chemical and Pharmaceutical Bulletin 37, 999–1004. Lacour, B., Lucas, A., Auchère, D., Ruellan, N., De Serre Patey, N.M., Drueke, T.B., 2005. Chronic renal failure is associated with increased tissue deposition of lanthanum after 28-day oral administration. Kidney International 67, 1062–1069. Lee, J.H., Kim, J.M., Kim, C., 2003. Pharmacokinetic analysis of rhein in Rheum undulatum L. Journal of Ethnopharmacology 84, 5–9. Li, L., 1996. End-stage renal disease in China. Kidney International 49, 287–301. Li, X., Wang, H., 2005. Chinese herbal medicine in the treatment of chronic kidney disease. Advances in Chronic Kidney Diseases 12, 276–281. Li, Z., Qing, P., Ji, L., Su, B.H., He, L., Fan, J.M., 2004. Systematic review of rhubarb for chronic renal failure. Chinese Journal of Evidence-Based Medicine 4, 468–473. Liu, Z.H., Li, L.S., Hu, W.X., Zhou, H., 1996. Effect of emodin on c-myc proto-oncogene expression in cultured rat mesangial cells. Acta Pharmacology Sinica 17, 61–63. Mengs, U., Krumbiegel, G., Völkner, W., 1997. Lack of emodin genotoxicity in the mouse micronucleus assay. Mutation Research 393, 289–293. Mitsuma, T., Terasawa, K., Yokozawa, T., Oura, H., 1984. Rhubarb therapy in patients with chronic renal failure (Part 1). Journal of Medical and Pharmaceutical Society for Wakan-Yaku 1, 266–278. Mitsuma, T., Yokozawa, T., Oura, H., Terasawa, K., 1987. Rhubarb therapy in patients with chronic renal failure (Part 2). Japanese Journal of Nephrology 29, 195–207. Mueller, S.O., Lutz, W.K., Stopper, H., 1998a. Factors affecting the genotoxic potency ranking of natural anthraquinones in mammalian cell culture systems. Mutation Research 414, 125–129. Mueller, S.O., Stopper, H., Dekant, W., 1998b. Biotransformation of the anthraquinones emodin and chrysophanol by cytochrome P450 enzymes: bioactivation to genotoxic metabolites. Drug Metabolism and Disposition 26, 540–546. National Toxicology Program, 2001. Technical report on the toxicology and carcinogenesis studies of emodin in F344/N rats and B6C3F1 mice. Series No. 493. NIH Publication No. 01-3952. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. Pharmacopoeia of the People’s Republic of China, First Div., 2005. China Chemical Industry Press, Beijing, pp. 17–18. Terzi, F., Beaufils, H., Laouari, D., Burtin, M., Kleinknecht, C., 1992. Renal effect of antihypertensive drugs depends on sodium diet in the excision remnant kidney model. Kidney International 42, 354–363. Wang, C.F., Wu, X.D., Chen, M., Duan, W.G., Sun, L.X., Yan, M., Zhang, L.Y., 2007a. Emodin induces apoptosis through caspase 3-dependent pathway in HK-2 cells. Toxicology 231, 120–128. Wang, Q.X., Wu, C.Q., Liao, M.Y., 2007b. Study on the toxicity of rhubarb and main components contained in it. Journal of Toxicology 21, 301–302. Wang, X.H., Wang, X.Y., Xiong, M.X., Zhang, F.F., Wang, N.N., Li, J., He, D.Y., Yang, J.W., 2006. Administration of rhubarb extract attenuates renal fibrosis in mice. Chinese Journal of Nephrology 22, 360–363. Xiao, W., Deng, H.Z., Ma, Y., 2002a. Summarization of the clinical and laboratory study on the rhubarb in treating chronic renal failure. Chinese Journal of Chinese Materia Medica 27, 4–7, 25. Xiao, X.R., 2002b. The correct application of rhubarb on chronic renal failure. Liaoning Journal of Traditional Chinese Medicine 29, 311–312. Yan, M., Zhang, L.Y., Sun, L.X., Jiang, Z.Z., Xiao, X.H., 2006. Nephrotoxicity study of total rhubarb anthraquinones on Sprague Dawley rats using DNA microarrays. Journal of Ethnopharmacology 107, 308–311. Yokozawa, T., Suzuki, N., Okuda, I., Oura, H., Nishioka, I., 1985. Changes in the urinary constituents in rats with chronic renal failure during oral administration of rhubarb extract. Chemical and Pharmaceutical Bulletin 33, 4508–4514.
J. Wang et al. / Journal of Ethnopharmacology 124 (2009) 18–25 Yokozawa, T., Zheng, P.D., Oura, H., Koizumi, F., 1986. Animal model of adenineinduced chronic renal failure in rats. Nephron 44, 230–234. Zhan, Y.T., Wei, H.S., Wang, Z.R., Huang, X., Xu, Q.F., Li, D.G., Lu, H.M., 2001. Effects of emodin on hepatic fibrosis in rats. Chinese Journal of Hepatology 9, 235– 239.
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Zhang, J.H., Yao, X.D., Song, Y., 1993. The long-term effect of retarding chronic renal failure progression with rhubarb and catopril. Journal of Chinese Nephrology 9, 197–201. Zhou, Y.P., Jiang, J.L., 1991. Review on the pharmacological researches of rhubarb. Pharmacology and Clinics of Chinese Materia Medica 7, 41–43.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Assessment of the renal protection and hepatotoxicity of rhubarb extract in rats Jiabo Wang, Yanling Zhao, Xiaohe Xiao ∗ , Huifang Li, Haiping Zhao, Ping Zhang, Cheng Jin China Military Institute of Chinese Materia Medica, 302 Military Hospital, Beijing 100039, PR China
a r t i c l e
i n f o
Article history: Received 5 November 2008 Received in revised form 7 April 2009 Accepted 8 April 2009 Available online 17 April 2009 Keywords: Chronic renal failure (CRF) Rhubarb Renal protection Toxicity Anthraquinones High performance liquid chromatography (HPLC)
a b s t r a c t Aim of the study: Rhubarb is well used to treat chronic renal failure (CRF) in China and Japan, but recent studies reported that the anthraquinone derivatives contained in rhubarb had nephrotoxicity. In this investigation an attempt was made to assess the value and toxic potential of rhubarb to treat CRF. Materials and methods: Histopathologic and biochemical tests combined with toxicokinetic analysis were performed to investigate the nephrotoxic potential and protective effect of rhubarb extract. Results: In normal rat groups, no death was observed and no renal lesion was found after repetitive administration of rhubarb for 3 weeks. The survival rate, pathologic conditions and biochemical indexes of CRF rats treated with rhubarb at two dosages were all improved and significant amelioration was found in the low dosage group compared to the untreated CRF group. Rhein was the mainly absorbable anthraquinone derivative into systemic circulation after oral administration and the area under curve of rhein in CRF groups was lower than that in normal groups at same dosage. Conclusions: After 3 weeks of administration of rhubarb extract, there was evidence of protective effect to CRF rats, while incidences of hepatotoxicity with minimal to mild hyaline droplets were also observed in normal rats. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Anthraquinones (AQs) are the active components present in the root and rhizoma of rhubarb, which have wide pharmacological effects and also some toxic concerns on genetic toxicity (Mueller et al., 1998a,b) and nephrotoxicity (Yan et al., 2006). Despite the positive in vitro results of genetic toxicity, results from in vivo studies in rodents showed lack of mutagenic activity (Mengs et al., 1997; National Toxicology Program, 2001). However, the nephrotoxicity of AQs has been revealed through both in vitro and in vivo studies. Two in vitro studies reported that emodin, one of the anthraquinones contained in rhubarb, could induce lesion on human proximal tubular epithelial cell line HK-2 cells (Wang et al., 2007a) and repress the proliferation of rat kidney glomerular mesangial cells (MC) through inhibition on c-myc mRNA expression (Liu et al., 1996). In 2001, the National Toxicology Program reported that exposure of rats to emodin resulted in an increase in incidence of renal tubule hyaline droplets and severities of renal tubule pigmentation in both male and female animals. On the other hand, rhubarb has been well used in China and Japan to treat chronic renal failure (CRF) and reveals desirable prospects in clinic (Mitsuma et al., 1984, 1987; Huang, 1993; Li and Wang, 2005). And there were few clinical reports in China about the increase of renal risk of rhubarb in treatment of
∗ Corresponding author at: 100#, the 4th Ring Road, 302 Military Hospital, Beijing 100039, PR China. Tel.: +86 10 66933322; fax: +86 10 63879915. E-mail addresses: [email protected], pharm [email protected] (X. Xiao). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.04.018
nephritic diseases. The possibility of an association between concurrent renal protection and lesion with the use of rhubarb to treat CRF needs to be assessed. Former studies have focused on the safety of the components contained in rhubarb, such as emodin, but a single component could not represent the whole of the rhubarb and the decision could not be applied to such phytomedicines processed with crude materials. Furthermore, such evaluations were only performed on normal animals and the differences between normal and morbid animals were not considered. The harms of a drug to renal failure animals might be obviously distinct to normal ones. It is, therefore, necessary to assess the value and toxic potential of rhubarb to treat CRF based on comparative investigation on both normal and CRF animals by histopathologic and biochemical tests. Besides, toxicokinetic studies were performed because the changes of a drug’s side effects from normal animals to morbid ones might be correlated with the alteration of its metabolism. Some experimental animal models of chronic renal failure, such as 5/6 nephrectomy/bipolar resection and adenine-induced renal failure models, were well documented (Yokozawa et al., 1986; Terzi et al., 1992). In this study, adenine-induced CRF model was selected because of the convenience of operation and high achievement ratio compared to nephrectomy and other chemical inducement (Lacour et al., 2005), and adenine was administered intragastricly at 250 mg kg−1 of body weight per day for 2 weeks instead of using an adenine content in diet of 0.75%. Rhubarb is the peeled and dried root of Rheum palmatum L., Rheum tanguticum Maxim. ex Balf. or Rheum officinale
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Baill. (family: Polygonaceae) stipulated in Chinese Pharmacopoeia (PPRC, 2005). Rhubarb is also officially listed in European and Japanese Pharmacopoeia (European Pharmacopoeia, 2001; Japanese Pharmacopoeia, 2006). Rheum palmatum was researched in this paper for it is stipulated in all of the three pharmacopoeias mentioned above and is more commonly used than other species. There are various kinds of constituents contained in rhubarb, which can be classified as anthraquinones, dianthrones, stilbens, anthocyanins, flavonoids, polyphenols, organic acids, and chromones (Kashiwada et al., 1989). Among them, anthraquinone derivatives, including emodin, chrysophanol, rhein, aloe-emodin, physcion, and their glucosides, are accepted as important active components with various pharmacological effects such as purgation, antibacterial, anti-inflammation, antitumor activity and nephric protection (Yokozawa et al., 1985; Dictionary of Traditional Chinese Medicine, 1997). The blood concentrations of these five AQs were determined to describe the pharmacokinetic course of rhubarb. 2. Materials and methods 2.1. Plant material and chemicals The dried root and rhizoma of Rheum palmatum were collected in Lixian county of Gansu province of China in November of 2006 and were identified by Professor Xiaohe Xiao, a taxonomist at PLA Institute of Traditional Chinese Material Medica. A voucher specimen (Rh200611W) was deposited in the institute. Rhubarb was grinded and decocted twice with water for 30 min each. The resulting extracts were decanted, filtered and evaporated to dryness under reduced pressure. This dried extract was re-dissolved and dispersed in warm water and then cooled for intragastric administration to rats. Aloe-emodin, rhein, emodin, chrysophanol and physcion were also supplied by National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) and the chemical structure of these anthraquinone derivatives are shown in Fig. 1. Methanol (HPLC grade, Fisher USA), aether, hydrochloric acid and phosphoric acid (AR, Beijing Chemical Regents Inc., PR China) were used for the mobile phase of chromatographic analysis and blood sample preparation. Adenine (>98%, Bioszume Inc., Beijing) was used to copy the chronic renal failure on rats. 2.2. Animals and study design Male and female Sprague–Dawley (S.D.) rats (License No.: SCXK2000-0010) of approximately 140 g were obtained from NICPBP. Just prior to the experiment, 120 Sprague–Dawley (S.D.)
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Fig. 1. Structures of rhein, emodin, aloe-emodin, chrysophanol and physcion.
rats were randomized into six groups of 20 animals each. The groups shown in Table 1 were differentiated based on animal model and dosage. Three groups were treated with adenine (250 mg/kg of body weight per day) for 2 weeks to induce the chronic renal failure experimentally and the others received physiologic saline. The dosages of rhubarb were 3 and 20 g/kg of body weight per day od (counted on the quantity of crude material) and were equivalent to 6 and 40 times of the upper dose of human stipulated in Chinese Pharmacopoeia (0.5 g/kg), respectively. The extract of rhubarb was administered intragastricly. The dosage was set at high level to uncover any potential toxicity in order to investigate the renal risk of rhubarb rigorously. Feed and water were available ad libitum. The experiment lasted for 3 weeks. At the 7th, 14th and 21st experimental day, blood samples were collected after administration at eight time points (10, 20, 30, 50, 90, 150, 360 and 1440 min) for toxicokinetic analysis. Five AQs in specimens were determined by high performance liquid chromatography (HPLC). Necropsies were performed on all animals. Animals sacrificed midway were dissected in time for histologic examination. All experiments using rodents were performed in accordance with the applicable guidelines and regulations. All rats received humane care in compliance with the institutional animal care guidelines approved by the Ministry of Science and Technology of China. 2.3. Histopathologic examination and biochemical analysis Necropsies were performed on all study animals. Tissues for histopathologic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of approximately 5 m, and stained with hematoxylin and eosin. Biochemical analysis was performed on all studied animals. Biochemical parameters, serum urea nitrogen (SUN), creatinine (CREA), alanine aminotransferase (ALT), aspartate aminotrans-
Table 1 Survival and incidences of histopathologic lesions on kidney and liver in different groups. Animal model
Dosagea (g kg−1 )
Survivalb
N NL NH
Normal
0 3 20
20c 20 20
0d (0e ) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
CRF CRFL CRFH
CRF
0 3 20
6 16** 9
20 (3.3) 13 (2.2** ) 15 (2.5** )
20 (2.4) 14 (1.9* ) 14 (2.0)
Group
Renal tubule hyaline droplet
Renal tubule pigmentation
Glomeruli necrosis 0 (0) 0 (0) 0 (0) 20 (2) 10 (1.2** ) 14 (1.4* )
Hepatic hyaline droplets 0 (0) 8 (1.5** ) 15 (2** ) 15 (2.5) 15 (2.4) 14 (2.6)
N, normal rats group without treatment; NL, normal rats groups treated by rhubarb in low dosage (3 g kg−1 ); NH, normal rats groups treated by rhubarb in high dosage (20 g kg−1 ); CRF, CRF rats group without treatment; CRFL, CRF rats group treated by rhubarb in low dosage (3 g kg−1 ); CRFH, CRF rats group treated by rhubarb in high dosage (20 g kg−1 ). a Dosage was counted with the quantity of crude material. b Animals initially in the study of every group are 20. c Number of survived animals. d Number of animals with lesion. e Average severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked. * p < 0.05, comparison between untreated and rhubarb-treated groups. ** p < 0.01, comparison between untreated and rhubarb-treated groups.
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ferase (AST), total protein (TP), albumin (ALB), calcium (CA), inorganic phosphate (P), total cholesterol (CHOL), alkaline phosphatase and total bile acids, were determined using the Hitachi clinical analyzer 7020 (Hitachi High-Technologies Corporation, Japan). 2.4. Specimen preparation for determination of anthraquinones For the determination of the aqueous extract of rhubarb, 0.15 g of the extract was dissolved in 10 ml of 2 M HCl and 20 ml of chloroform was added and the solution was kept on a water bath for 1 h. The hydrolyzed solution was extracted with 10 ml of chloroform four times and the combined extract was then evaporated to dryness. The residue was dissolved and transferred into a 50 ml volumetric flask by methanol. The solution was filtered through a 0.45 m filter before analysis. Blood specimens of about 0.3 ml were taken from the retroorbital sinus into heparinized polypropylene tubes. The blood was centrifuged (3000 × g, 10 min) and 100 l plasma was removed in a 1.5 ml tube. Then 10 l 3 M HCl water-solution and 10 l 120 g ml−1 danthron in methanol were added into the tube and stirred for 30 s. Aether of 1 ml was added and stirred for 2 min before centrifugation (10,000 × g, 10 min). The upper clear liquid of 800 l in the tube was transferred into a plain tube and was evaporated gently (60 ◦ C, stream of nitrogen) to dryness. The dry residue was reconstituted in 100 l of the mobile phase for the fluorescence analysis.
anthraquinone:danthron). The correlation coefficient (r) was also expressed. Accuracy was calculated as the percentage found on the standard curve, and the precision of the method, expressed as the relative standard deviation (R.S.D. = 100 S.D./mean), was also evaluated. 2.6. Statistics Statistical analysis was performed using an unpaired t-test. When variances were not homogeneous a Welch’s correction was employed. p-Values lower than 0.05 were considered significant. Values are expressed as means ± standard error (S.E.). 3. Results 3.1. Common changes All rhubarb-treated rats had a dosage-related body color change pattern from yellow to red, which appeared earlier in the study as the exposure concentration increased. Red to brown urine and reddish-brown staining of the anal area were noted for all the groups treated with rhubarb. Marked and durative diarrhea accompanied with emaciation and inactivity was observed in all CRF and normal rats administered 20 g/kg of rhubarb. The average urine pH value of CRF rats was about 8.5, while the value of normal ones was in the range of 7.0–7.5.
2.5. Chromatography and calibration
3.2. Survival and histopathologic analysis
Chromatographic analyses were performed using an Agilent 1100 chromatograph. The chromatographic system consisted of a solvent degasser, a quaternary gradient pump before column, fluorescence detector (FLD), diode array detector (DAD) and a data station with analytical software Chemstation 8.03 (Agilent, Inc., USA). The method to analyze the anthraquinones in the aqueous extract of rhubarb was well established (PPRC, 2005) and it was used in this study. The mobile phase was a mixture of methanol–0.1% phosphoric acid in water (85:15, v:v). The flow rate was 1 ml min−1 . The injection volume was 10 l while the diode array detector (ultraviolet detector) was set at 254 nm. The chromatographic separation was performed on a Kromasil® C18 analytical column (250 mm × 4.6 mm i.d., 5 m) and maintained at 25 ◦ C. The standard solution of five AQs (16 g/ml of each) was prepared in methanol. Since the plasma concentrations of anthraquinones absorbed into the body were very low, a sensitive and rapid method with fluorescence analysis was developed and calibrated in this study. The chromatographic separation was performed on a Phenix® C18 analytical column (150 mm × 4.6 mm i.d., 5 m) and maintained at 40 ◦ C. Mobile phase consisting of a mixture of methanol–0.1% phosphoric acid in water (80:20, v:v) with the flow rate of 2 ml/min was employed. Fluorescence detection was performed with excitation wavelength at 256 nm and emission wavelength at 520 nm. For the calibration of fluorescence analysis, a mixed standard stock solution (1 mg ml−1 of five AQs each in methanol) was prepared. A calibration series of mixed standard solution were diluted as 10, 30, 50, 100, 200, and 300 g ml−1 (six samples for each concentration), all containing 10 g ml−1 danthron as internal standard. Transfer 10 l above mixed standard solutions into drug-free rat plasma (100 l) and following the sample preparation procedure was performed as described above. Six-level calibration series with six analyses at each concentration level were measured. The linearity of the calibration curve was tested and evaluated (y = kx + b, where x is the concentration ratio of an anthraquinone to danthron (internal standard) and y is the corresponding peak-area ratio
A summary of survival and histopathologic results for different animal groups is shown in Table 1. Typical histopathologic section photos are shown in Fig. 2. In normal groups, no death was observed and no renal lesion was found after repetitive administration of rhubarb for 3 weeks. However, minimal to mild hepatic lesion was observed in rhubarb-treated normal rats and it was dose dependent. In CRF groups, evident renal lesions were observed after modeling in histopathologic examination, principally involving the proximal tubules, some distal tubules and glomeruli, characterized by moderate to crucial necrosis of tubules, slight necrosis of glomeruli and lots of light brown uroliths (possibly 2,8dihydroxyadenine). Rats without treatment died successively and only 6/20 animals survived at the end of 3 weeks. The survival of two rhubarb-treated CRF groups both increased and a significant enhancement was found in the low dosage group compared to the untreated CRF group. The symptoms of renal and hepatic lesions found in rhubarb-treated CRF groups were similar to those in untreated group, as well as the extent of some renal lesions was significantly lessened. Based on those results, it could be concluded that there was no explicit evidence of renal toxicity in normal rats and there was evidence of a treatment-related renal protection of rhubarb to CRF rats. The phenomenon that the curative effect of rhubarb to chronic renal failure was not improved with dosage increase from 3 to 20 g/kg might be related to the marked and durative diarrhea, which led to electrolyte imbalance and consequently worsened renal disorder. Another phenomenon of mild erosion at gastric mucosa that was only observed in CRF rats might relate to the occurrence of the pharmacokinetic change of rhubarb and it would be discussed below. The phenomenon that a definite dosage-related increase of hepatic lesion of rhubarb was found in normal groups while a marginal increase of hepatic lesion was observed with dosage enhancement in CRF groups over the background led by adenine, might relate to the alteration of pharmacokinetics and hepatic metabolism induced by adenine and need be investigated further.
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Fig. 2. Typical histopathologic section photos of rats in different groups. There was no evidence of lesion on both kidney (a) and liver (e) of normal rats without treatment. No histopathologic change was observed on kidney (b), but a minimal to mild lesion on hepatic tissue (f) was found in normal rats after administering rhubarb. In CRF groups, evident renal lesions were observed in both untreated (c) and treated (d) rats. The light brown uroliths (pointed by arrows) were possibly generated by adenine. Hepatic lesions were also observed in both untreated (g) and treated (h) CRF rats. Mild erosion at gastric mucosa was observed in CRF groups (i), but was not found in normal groups.
Fig. 3. Comparison on the biochemical parameters of survived rats within different groups at the end of experiment. Significance of differences from the value of normal rats without administration of rhubarb (*p < 0.05, **p < 0.01). Significance of differences from the value of CRF rats without administration of rhubarb ( p < 0.05, p < 0.01).
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3.3. Biochemical analysis Those biochemical parameters changed significantly in the study groups are shown in Fig. 3. In normal animal groups, SUN and CREA values held constant in low dosage group while these two indexes increased 29% and decreased 12%, respectively in high dosage group at the end of 3 weeks administration of rhubarb, compared to untreated group. ALT and AST values increased significantly in both rhubarb-treated normal groups. TP, ALB and serum calcium values also increased significantly in low dosage group. In CRF animal groups, significant changes of many biochemical parameters mostly listed in Fig. 3 were observed after modeling with adenine for 2 weeks. For example, SUN and CREA values increased 7 and 3 times, respectively compared to normal. In the subsequent 3 weeks of treatment, the mortality of rhubarb-treated CRF groups decreased and the survivors’ SUN and CREA values were also significantly decreased comparing to those CRF ones’ without treatment, which demonstrated the curative effect of rhubarb on CRF. Other biochemical parameters changed in untreated CRF rats showed reconverting trends in rhubarb-treated groups, however, some parameters did not recover to normal levels: SUN, CREA, ALT and AST values were significantly higher; TP and ALB values were significantly lower than normal. 3.4. Chromatography Typical HPLC analyses are shown in Fig. 4. Five AQs and internal standard could be separated from endogenous substances in rat plasma within 8 min under this chromatographic condition and the analytical procedure is accurate and precise. The calibration curves were found to be linear between 4.680 and 187.2; 4.680 and 187.2; 4.680 and 187.2; 4.680 and 187.2; 4.680 and 187.2 for rhein, aloe-emodin, emodin, chrysophanol and physcion, respectively. The precision in 1 day and 1 week of AQs determination in rat plasma samples were found to be less than 5.28% and 8.73%, respectively. The mean recovery of five AQs (based on the determination of six spiked plasma samples) were verified from 91.47 to 94.26 (max SD = 6.43%, n = 6). 3.5. Toxicokinetic analysis After intragastric administration of water extract of Rheum palmatum L., the plasma concentrations of AQs were followed through time by HPLC. Rhein could be detected in all animal groups in plasma. Aloe-emodin could be detected in high dosage group and other AQs, emodin, chrysophanol and physicion, could be detected at few sampling points. This is consistent with the literature (Lee et al., 2003). Hence, rhein was intensively investigated to evaluate the toxicokinetics of rhubarb in this study. Average concentration of rhein versus time profiles are shown in Fig. 5 and pharmacokinetic parameters are shown in Fig. 6. At same dosage, the values of AUC (area under curve), Cpeak (peak concentration), t1/2 Ka (half time of absorption) and t1/2  (half time of elimination) of CRF groups were generally lower than those of normal groups. While the dosage increased, the AUC and Cpeak values did not increase proportionally in both CRF and normal groups. This might be on account of non-linear pharmacokinetic course, or of the indigestion led by severe diarrhea induced by rhubarb itself at high dosage. Comparing to normal groups, the decline of AUC or Cpeak in CRF groups might be on account of the acceleration of elimination, showed by the shortening of t1/2  . A possible reason behind this is the urine alkalization of CRF rats, which resulted in accelerated elimination of acidic anthraquinones. And of note, urinary volume was markedly increased in CRF rat groups compared with normal rat groups, which could also accelerate the elimination
Fig. 4. Typical chromatograms from the aqueous extract of Rheum palmatum L. and the plasma samples. (a) A chromatogram of mixed standard solution of five AQs. (b) A representative chromatogram from the extract of rhubarb. (c) A chromatogram of plasma sample contained standards of five AQs and danthron as internal standard. (d) A representative chromatogram of a rat plasma after administrated the extract of rhubarb which demonstrated that rhein was the most massively and frequently absorbed anthraquinone. (e) A chromatogram of blank plasma sample with internal standard. (1) Aloe-emodin; (2) rhein; (3) danthron; (4) emodin; (5) chrysophanol; (6) physcion.
of anthraquinones. According to the results in histologic test, the shortening of the half time of absorption (t1/2 Ka ) in CRF groups might be on account of the mild erosion at gastric mucosa found in CRF rats, which led to acceleration of absorption.
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Fig. 5. Average plasma concentration of rhein versus time profiles after oral administration of Rheum palmatum extract. Observed data of CRF and normal rats are differentiated with filled and opened symbols, respectively. High and low dosage groups are differentiated with square and circle, respectively.
4. Discussion A recent in vitro study showed that emodin, one of the anthraquinones contained in rhubarb, could induce lesion on
human proximal tubular epithelial cell line HK-2 cells (Wang et al., 2007a). In a comprehensive feeding study (National Toxicology Program, 2001), increased incidences of hyaline droplets of renal tubule epithelial cells were observed in F344/N rats receiving over
Fig. 6. Comparison on pharmacokinetic parameters of survived rats within different groups at the different time points of experiment. Significance of differences from the value of normal rats administered low dosage of rhubarb (**p < 0.01). Significance of differences from the value of normal rats administered high dosage of rhubarb ( p < 0.01).
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17000 ppm of emodin in diet for 16 days, over 321 ppm for 14 weeks and 280 ppm for 2 years. And it was reported that the total anthraquinones extracted from rhubarb could induce swell and denaturation of renal tubule epithelial cells in Sprague–Dawley (S.D.) rats with 13 weeks of administration at dosage of 4.5 g/kg of body weight per day counted with the quantity of the extract (Yan et al., 2006). Another 6-month study showed that the critical dosage of rhubarb extract inducing above renal lesion in rats was 10 g/kg of body weight per day counted with the quantity of crude material, and such lesion could be recovered after drug withdrawal (Wang et al., 2007b). In this study, however, no renal lesion was found after repetitive administration of rhubarb to normal rats for 3 weeks at the dosage of 3 and 20 g/kg of body weight per day (counted with the quantity of crude material), and no additional rhubarb-related renal lesion was found in CRF rats over which was induced by adenine. In addition to this, there were few clinical reports in China about the increase of renal risk of rhubarb in treatment of nephritic diseases unless it was misused (Xiao, 2002b). Therefore, data suggest that the possibility of renal lesion with the use of rhubarb to treat CRF would be little when the dosage was properly controlled. Beyond the effect on kidney, an incidence of minimal to mild hyaline droplets in hepatic tissues indicated by histopathologic examination and biochemical analysis was observed in normal rats after administration of rhubarb at dosage of 3 g/kg for 3 weeks and the lesion was consequently worsened while the dosage increased. Such hepatic injury was also reported in the above-mentioned literatures: the 6-month study on rhubarb extract and the 2-year study on emodin, though it could be recovered after drug withdrawal (Wang et al., 2007b). But to adenine-induced CRF rats, the enhancement of such hepatic lesion was marginal with the increase of dosage when the lesion background induced by adenine was subtracted. In addition, the present toxicokinetic results showed that the values of AUC (area under curve, of plasma rhein) of CRF groups were significantly lower than those of normal groups at same dosage. Hence, an intuitionistic relationship between hepatic lesion and rhein, or other anthraquinones, could be included and the plasma level of rhein might be valuable to indicate the toxicity of rhubarb. However, it was reported that rhubarb and emodin could protect animals against hepatic injury induced by tetrachloromathane (Zhou and Jiang, 1991; Zhan et al., 2001). The divergence of the effect on liver of rhubarb is rarely concerned and requires further investigation. Rhubarb has been used to treat renal diseases in China for several decades and reveals desirable prospects to treat CRF (Xiao et al., 2002a; Wang et al., 2006). In a systemic review of 18 randomized or quasi-randomized trials from 15 Chinese journals, rhubarb showed a positive effect on relieving uremic symptoms, lowering serum creatinine, improving hemoglobin levels, and adjusting disturbance of lipid metabolism in 1322 patients with chronic renal disease, but the effect on reducing the number of deaths and the progression to end-stage kidney failure was not confirmed because of the small sample size (Li et al., 2004). In a prospective open-label trial involving 151 patients, the beneficial effect of rhubarb on the prevention of progression in chronic renal disease is more marked than that of captopril and their therapeutic effects are additive (Zhang et al., 1993). The combined treatment with rhubarb and an angiotensin-convertion enzyme (ACE) inhibitor seems to be quite promising (Li, 1996), but the interaction between them remains in uncertainty. In the present study, we found that the curative effect of rhubarb on CRF induced by adenine was positive in reducing the death number of rats and protecting the renal tissue. However, this curative effect was weakened by severe diarrhea, and side effect of rhubarb itself occurring at very high dosage, which would lead to electrolyte imbalance and indigestion, and consequently worsen the kidney disorder of CRF bodies and cause the decline of bioavailability, respectively. In addition, the alteration of phar-
macokinetic course in CRF rats compared to normal ones, involving the decline of plasma concentration and acceleration of elimination, indicated the decrease of exposure and shortening of half life of rhubarb in plasma. The effect of histopathologic condition on rhubarb pharmacokinetics remains uncertain. These results indicate that a well-designed clinical trial of rhubarb with long-term follow-up to treat chronic renal failure is warranted. Acknowledgement This work was financially supported by the National Key Technologies R&D Program of China (No. 2004BA721A14). References Dictionary of Traditional Chinese Medicine, 1st ed., 1997. New Medical College of Jiangsu, People’s Published of Shanghai, p. 102. European Pharmacopoeia, 4th ed., 2001. European Directorate for the Quality of Medicines, Council of Europe, Strasbourg, p. 1858. Huang, K.C., 1993. The Pharmacology of Chinese Herbs. CRC Press, Boca Raton, FL, pp. 233–234. Japanese Pharmacopoeia, 15th ed., 2006. Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Tokyo, p. 1344. Kashiwada, Y., Nonaka, G.I., Nishioka, I., 1989. Studies on Rhubarb (Rhei Rhizoma). XV. Simultaneous determination of phenolic constituents by high-performance liquid chromatography. Chemical and Pharmaceutical Bulletin 37, 999–1004. Lacour, B., Lucas, A., Auchère, D., Ruellan, N., De Serre Patey, N.M., Drueke, T.B., 2005. Chronic renal failure is associated with increased tissue deposition of lanthanum after 28-day oral administration. Kidney International 67, 1062–1069. Lee, J.H., Kim, J.M., Kim, C., 2003. Pharmacokinetic analysis of rhein in Rheum undulatum L. Journal of Ethnopharmacology 84, 5–9. Li, L., 1996. End-stage renal disease in China. Kidney International 49, 287–301. Li, X., Wang, H., 2005. Chinese herbal medicine in the treatment of chronic kidney disease. Advances in Chronic Kidney Diseases 12, 276–281. Li, Z., Qing, P., Ji, L., Su, B.H., He, L., Fan, J.M., 2004. Systematic review of rhubarb for chronic renal failure. Chinese Journal of Evidence-Based Medicine 4, 468–473. Liu, Z.H., Li, L.S., Hu, W.X., Zhou, H., 1996. Effect of emodin on c-myc proto-oncogene expression in cultured rat mesangial cells. Acta Pharmacology Sinica 17, 61–63. Mengs, U., Krumbiegel, G., Völkner, W., 1997. Lack of emodin genotoxicity in the mouse micronucleus assay. Mutation Research 393, 289–293. Mitsuma, T., Terasawa, K., Yokozawa, T., Oura, H., 1984. Rhubarb therapy in patients with chronic renal failure (Part 1). Journal of Medical and Pharmaceutical Society for Wakan-Yaku 1, 266–278. Mitsuma, T., Yokozawa, T., Oura, H., Terasawa, K., 1987. Rhubarb therapy in patients with chronic renal failure (Part 2). Japanese Journal of Nephrology 29, 195–207. Mueller, S.O., Lutz, W.K., Stopper, H., 1998a. Factors affecting the genotoxic potency ranking of natural anthraquinones in mammalian cell culture systems. Mutation Research 414, 125–129. Mueller, S.O., Stopper, H., Dekant, W., 1998b. Biotransformation of the anthraquinones emodin and chrysophanol by cytochrome P450 enzymes: bioactivation to genotoxic metabolites. Drug Metabolism and Disposition 26, 540–546. National Toxicology Program, 2001. Technical report on the toxicology and carcinogenesis studies of emodin in F344/N rats and B6C3F1 mice. Series No. 493. NIH Publication No. 01-3952. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. Pharmacopoeia of the People’s Republic of China, First Div., 2005. China Chemical Industry Press, Beijing, pp. 17–18. Terzi, F., Beaufils, H., Laouari, D., Burtin, M., Kleinknecht, C., 1992. Renal effect of antihypertensive drugs depends on sodium diet in the excision remnant kidney model. Kidney International 42, 354–363. Wang, C.F., Wu, X.D., Chen, M., Duan, W.G., Sun, L.X., Yan, M., Zhang, L.Y., 2007a. Emodin induces apoptosis through caspase 3-dependent pathway in HK-2 cells. Toxicology 231, 120–128. Wang, Q.X., Wu, C.Q., Liao, M.Y., 2007b. Study on the toxicity of rhubarb and main components contained in it. Journal of Toxicology 21, 301–302. Wang, X.H., Wang, X.Y., Xiong, M.X., Zhang, F.F., Wang, N.N., Li, J., He, D.Y., Yang, J.W., 2006. Administration of rhubarb extract attenuates renal fibrosis in mice. Chinese Journal of Nephrology 22, 360–363. Xiao, W., Deng, H.Z., Ma, Y., 2002a. Summarization of the clinical and laboratory study on the rhubarb in treating chronic renal failure. Chinese Journal of Chinese Materia Medica 27, 4–7, 25. Xiao, X.R., 2002b. The correct application of rhubarb on chronic renal failure. Liaoning Journal of Traditional Chinese Medicine 29, 311–312. Yan, M., Zhang, L.Y., Sun, L.X., Jiang, Z.Z., Xiao, X.H., 2006. Nephrotoxicity study of total rhubarb anthraquinones on Sprague Dawley rats using DNA microarrays. Journal of Ethnopharmacology 107, 308–311. Yokozawa, T., Suzuki, N., Okuda, I., Oura, H., Nishioka, I., 1985. Changes in the urinary constituents in rats with chronic renal failure during oral administration of rhubarb extract. Chemical and Pharmaceutical Bulletin 33, 4508–4514.
J. Wang et al. / Journal of Ethnopharmacology 124 (2009) 18–25 Yokozawa, T., Zheng, P.D., Oura, H., Koizumi, F., 1986. Animal model of adenineinduced chronic renal failure in rats. Nephron 44, 230–234. Zhan, Y.T., Wei, H.S., Wang, Z.R., Huang, X., Xu, Q.F., Li, D.G., Lu, H.M., 2001. Effects of emodin on hepatic fibrosis in rats. Chinese Journal of Hepatology 9, 235– 239.
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Zhang, J.H., Yao, X.D., Song, Y., 1993. The long-term effect of retarding chronic renal failure progression with rhubarb and catopril. Journal of Chinese Nephrology 9, 197–201. Zhou, Y.P., Jiang, J.L., 1991. Review on the pharmacological researches of rhubarb. Pharmacology and Clinics of Chinese Materia Medica 7, 41–43.