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A comparative study on the tissue distributions of rhubarb anthraquinones in normal and CCl4-injured rats orally administered rhubarb extract
Journal of Ethnopharmacology 137 (2011) 1492–1497
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A comparative study on the tissue distributions of rhubarb anthraquinones in normal and CCl4 -injured rats orally administered rhubarb extract Fang Fang a,b,1 , Jia-bo Wang a,1 , Yan-ling Zhao a , Cheng Jin a , Wei-jun Kong a,b , Hai-ping Zhao c , Hong-juan Wang a , Xiao-he Xiao a,∗ a
China Military Institute of Chinese Materia Medica, 302 Military Hospital, Beijing 100039, PR China Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China c Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, PR China b
a r t i c l e
i n f o
Article history: Received 1 April 2011 Received in revised form 4 August 2011 Accepted 14 August 2011 Available online 30 August 2011 Keywords: Rhubarb Anthraquinone derivatives Tissue distribution LC–MS You Gu Wu Yun
a b s t r a c t Aim of the study: The present study comparatively investigated the tissue distributions of rhubarb anthraquinone derivatives (AQs) to examine whether they undergo different uptakes in normal or CCl4 induced liver-damaged rats, to explore possible reasons for the different toxicities of AQs in pathological model rats and normal rats at the tissue distribution level. Materials and methods: The total rhubarb extract (14.49 g kg−1 of body weight per day based on the quantity of crude material) was administrated orally to normal and model rats for 12 weeks. The concentrations of free AQs in tissues were quantitated by liquid chromatography–tandem mass spectrometry (LC–MS). After drug withdrawal for 4 weeks, tissue distributions were again determined. Results: The five free AQs—aloe-emodin, rhein, emodin, chrysophanol and physcion—were detected in the liver, kidney and spleen, while only rhein, aloe-emodin and emodin reached the quantitative limit. The tissue distributions of rhein (p < 0.001), aloe-emodin (p < 0.001) and emodin (p < 0.05) in normal rats were higher than those in model rats with rhein > aloe-emodin > emodin in kidney and spleen tissues and aloe-emodin > rhein > emodin in liver tissues. Free AQs were not detected in the tissues after drug withdrawal for 4 weeks. Conclusions: These results suggest that the tissue toxicity of AQs in normal animals is higher than that in pathological model animals with little accumulative toxicity of rhubarb. The results are concordant with the traditional Chinese theory of You Gu Wu Yun recorded first in Su Wen, a classical Chinese medical treatise. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Rhubarb (Radix et rhizoma rhei, Dahuang in Chinese) has been used in traditional Chinese medicine (TCM) for more than 2000 years and was first documented in “The Shen Nong Ben Cao Jing”, the earliest book on materia medica in the world. Rhubarb was named general with beneficial effects on constipation (Jong et al., 2010), hepatitis (Cui et al., 2010), cholecystitis (Jiao and Liu, 1982), diabetic nephropathy (DN) (Gu et al., 2003) and chronic renal failure (CRF) (Xiao et al., 2002). Rhubarb contains five free anthraquinone derivatives (AQs), which account for the main medicinal properties of rhubarb and have similar chemical structures, as shown in Fig. 1. These AQs have been documented to have numerous therapeutic
∗ Corresponding author at: 100#, the 4th West Ring Road, 302 Military Hospital, Beijing 100039, PR China. Tel.: +86 10 66933322; fax: +86 10 63879915. E-mail address: pharm [email protected] (X.-h. Xiao). 1 These authors contributed equally to this work. 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.08.028
benefits, including inducing purgation (Sun, 1992), inhibiting bacterial growth (Wang et al., 2010), protecting the liver (Huang et al., 1997; Zhao et al., 2009) and treating CRF (Wang et al., 2009a) and jaundice (Ho, 1996). However, recent studies have reported that AQs are cytotoxic to the liver and kidney (Wojcikowski et al., 2006) and have mutagenic and carcinogenic effects in vitro. A NIH 2001 panel conducted a two-year experimental study on the toxic effects of emodin in animals (National Toxicology Program (NTP), 2001). They showed that emodin has no carcinogenic effects and leads to obvious pathological changes of the renal tubule. Recent risk assessments of rhubarb in animals also concluded that AQs cause injury to the liver and kidney (Wang et al., 2007a, 2009a, 2011; Xing et al., 2011). The discrepancies among these studies have raised doubts concerning the medicinal use of rhubarb. Therefore, the protective or destructive effects of rhubarb should be assessed scientifically; the results of such studies will be of great significance to clinical diagnosis and treatment. We previously determined that rhubarb-induced injury to the liver or kidney is more pronounced in normal rats than in
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(20060525, Beijing Beihua Fine Chemicals Co. Ltd.), and olive oil (F20080219, Sinopharm Chemical Reagent Beijing Co. Ltd.) were of analytical grade.UPLC analyses of free AQs in rhubarb total extract were performed using a Waters Acquity System equipped with a binary solvent delivery pump, an auto sampler and a photodiode array detector. The UPLC–ESI-MS/MS analyses for tissue distribution of AQs were performed using an Acquity UPLC–MS/MS System (Waters Corp., Milford, MA, USA) with a Waters tandem quadrupole mass spectrometer (Milford, MA, USA) equipped with an electrospray source. An Acquity BEH UPLC C18 column (100 mm × 2.1 mm, 1.7 m) was used for the separation. The signal acquisition, peak integration and concentration determination were performed using ChemStation software (MassLynx 4.1) supplied by Waters Technologies. Other instruments used included a solid-phase extraction column with an OASIS MAX cartridge 3 cc (60 mg) (Waters, USA), a Waters extraction manifold system (Waters, USA), an ALl04 electronic balance, a low-speed centrifuge, and a manually adjustable pipette gun. 2.2. Animals
Fig. 1. Chemical structures of aloe-emodin, rhein, emodin, chrysophanol and physcion.
pathological rat models. This finding suggests that the medicinal effects of rhubarb are dependent on the pathological status (Wang et al., 2009a). In general, the protective or toxic effects of a drug on target organs are related to the concentration of the drug in the respective tissues. Therefore, the differing effects of rhubarb are possibly due to accumulation of the drug in the organs leading to different pathological conditions. To date, there have not been any studies of the tissue distribution of AQs in healthy rats compared to pathological rat models. In the present study, we determined the concentrations of free AQs in different tissues and evaluated their toxic effects on the liver or kidney in normal rats and in rats with CCl4 -induced liver damage. Possible explanations for the differences in free AQ tissue distribution are explored preliminarily.
Male and female Sprague Dawley (SD) rats 6–8 weeks of age and weighing 180 ± 30 g were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (License No. SYXK 2007-004). The animals were separated by gender and were given unlimited access to food and water in an environmentally controlled breeding room (temperature 22 ± 2 ◦ C, humidity 60–80%). The breeding room was illuminated by artificial light with a 12-h light/12-h dark cycle every day; the room was disinfected regularly. 2.3. Preparation of extracts A total of 100 kg of rhubarb was added to 600 L of 90% ethanol, heated and extracted 3 times for 1 h each. The residual rhubarb after extraction was added to 1000 L of water and heated and extracted for 1 h (Wang et al., 2011). The filtrate was merged and spray-dried, and the final extract yield was 29.3%. The samples were stored at 4 ◦ C for later experiments. 2.4. Animal experiments and sample collection
2. Materials and methods 2.1. Materials and instruments Rhubarb is the peeled and dried root of Rheum palmatum L., Rheum tanguticum Maxim. ex Balf. or Rheum officinale Baill. (Polygonaceae family) described in the Chinese Pharmacopoeia (PPRC, 2010). Rhubarb is also officially listed in the European and Japanese Pharmacopoeia (European Pharmacopoeia, 2001; Japanese Pharmacopoeia, 2006). Rheum palmatum was specifically examined in these studies because it is documented in all three pharmacopoeias mentioned above and is more commonly used than other species. The dried root and rhizoma of Rheum palmatum L. of the Polygonaceae family were collected in Lixian County, Gansu province of China, and were classified by Professor Xiaohe Xiao, a taxonomist at the PLA Institute of Traditional Chinese Material Medica. Standard chemicals (aloe-emodin, emodin, rhein, chrysophanol, physcion and 1,8-dihydroxy anthraquinone) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol was HPLC grade (Fisher, USA). Formic acid was of analytical grade purity and was purchased from Beijing Chemical Regents Inc., PRC (Beijing, China). High-purity water was produced by the Milli-Q water purification system (Millipore, Bedford, MA, USA). Carbon tetrachloride
In this study, a CCl4 -induced liver injury model was used because it has been well researched and demonstrates good reproducibility and high reliability. The mechanism by which CCl4 induces liver injury involves the formation of free radicals and the subsequent peroxidation chain reaction, which leads to a significant and chronic injury of hepatic cells (Jian et al., 2008). The experiment encompassed a total of 16 weeks. First, 54 rats were randomized into three groups of 18 animals each (9 males and 9 females). One group (the model group) was injected intraperitoneally with CCl4 oil (containing 1 portion CCl4 and 9 portions olive oil, 5 mL/kg) two times per week for a total of 12 weeks to induce chronic liver injury. The others received physiologic levels of saline. The medication group was administered the RE intragastrically once every day from the 4th week after modeling to the end of the 16th week. The control and model groups were administered physiologic levels of saline intragastrically. The dosage of total RE was 14.69 g herbs kg−1 of body weight per day and is equivalent to 4.9 times (converted with body surface area) (Wang et al., 2011) the upper dosage limit for humans as described in the Chinese Pharmacopoeia (0.5 g kg−1 ) (PPRC, 2010). Food and water were available to the animals ad libitum. 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
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Table 1 Amounts of AQs in rhubarb extract.
AQs AQGs
Aloe-emodin (%)
Rhein (%)
Emodin (%)
Chrysophanol (%)
Physcion (%)
0.204 ± 0.001 0.364 ± 0.002
0.267 ± 0.001 0.168 ± 0.001
0.954 ± 0.001 0.467 ± 0.003
0.329 ± 0.001 0.10S ± 0.001
0.455 ± 0.002 0.443 ± 0.003
AQs: anthraquinone derivatives; AQGs: anthraciuinone glycosides.
guidelines approved by the Ministry of Science and Technology of China. The tissues of liver, spleen and kidney were preserved at −80 ◦ C and thawed before use. 2.5. Quantitative determination 2.5.1. Assay of free AQs in the total rhubarb extract Preparation of standard solutions: Stock solutions of the five rhubarb anthraquinones (emodin, rhein, physcion, chrysophanol and aloe-emodin) were prepared at a concentration of 40 g mL−1 in methanol. All the solutions were stored in dark glass bottles at 4 ◦ C and were stable for at least 1 month. Working solutions were freshly prepared by diluting suitable amounts of the above solutions with methanol before injection. Sample preparation: Appropriate amounts of test samples were precisely weighed and then extracted with 25 mL of methanol by refluxing for 60 min. The extracted solution was prepared by the method of weight relief, in which there was compensation for the weight lost in the extraction procedure. After filtering, 5 mL of filtrate was transferred into a flask and then evaporated to dryness. Subsequently, 10 mL of 2 M HCl and 20 mL of chloroform were added to dissolve the residue, and the solution was incubated in 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 with methanol and transferred into a 50-mL volumetric flask. The solution was filtered through a 0.22-m filter before injection (Wang et al., 2008). Chromatographic conditions: The mobile phase was a methanol-0.1% formic acid water solution (70:30, v/v) at a flow rate of 0.3 mL min−1 . A single wavelength of 254 nm was employed for detection of AQs. The final injection volume was 5 L. The column temperature was maintained at 30 ◦ C (Wang et al., 2008). 2.5.2. Assay of free AQs in tissues Standard solutions were prepared as described in Section 2.5.1. The concentration of IS was 30 g mL−1 (1,8-dihydroxy anthraquinone) prepared in methanol. Sample preparation: In this study, aliquots of 0.2-g rat tissue samples and 500 L IS (30 g mL−1 ) were placed into 10-mL centrifuge tubes, homogenized and vortex-mixed. After being centrifuged for 15 min (10,000 rpm), 500 L of supernatant was transferred into an OASIS MAX cartridge 3 cc (60 mg) for further purification. The SPE column was conditioned with 1 mL of MeOH and then 1 mL of water, eluted with 3 mL of 5% ammonium water, then with 3 mL of methanol, and, finally, with 1 mL of 12% formic acid–isopropanol solution. A 500-L aliquot of the last eluted solution was collected to be injected into the UPLC–ESI-MS for analysis. The SPE procedure was performed on a Waters extraction manifold system. Chromatographic conditions: In addition to the UPLC conditions described in Section 2.5.1, the UPLC–ESI-MS/MS was conducted using high-purity nitrogen (N2 ) to assist nebulization. The quadrupole mass spectrometer equipped with an ESI source was set with a desolvation gas (N2 ) flow of 600 L min−1 , nebulizer pressure of 600 kPa, desolvation gas temperature of 450 ◦ C,
capillary voltage of 3.5 kV and the negative ion mode. The ESI-MS was performed in the multiple reaction monitoring (MRM) mode using the target ions [M−H]− at m/z 269 > daughter ions at m/z 240 for aloe-emodin, m/z 283 > 211 for rhein, m/z 269 > 225 for emodin, m/z 253 > 225 for chrysophanol, m/z 283 > 240 for physcion and m/z 239 > 211 for IS. Ultra-high-purity helium (He) was used as the collision gas at a flow rate of 0.25 mL min−1 (Fang et al., 2011). 2.6. Statistical analysis The experimental values were expressed as mean ± standard deviation (S.D.). All data were processed with the statistical software Windows SAS 8.0 (SAS, USA). The groups were analyzed using factor analysis, and the significance probability was ˛ = 0.05. 3. Results 3.1. Distributions of AQs in rhubarb extract and tissues The yield of total RE was 29.3%. The contents of free AQs in the total extract are listed in Table 1. The 5 free AQs—aloeemodin, rhein, emodin, chrysophanol and physcion—were detected in liver, kidney and spleen, but only rhein, aloe-emodin and emodin reached the quantitative limit. The tissue distributions of aloeemodin, rhein and emodin in the normal rats were all higher than those in the model rats with rhein > aloe-emodin > emodin in kidney and spleen tissues and aloe-emodin > rhein > emodin in liver tissues (Table 2). AQs were not detected in control animals and methanol control group, which suggests that there is no interference from control or methanol. After 4 weeks of discontinued administration as indicated, AQs were not detected in tissues. 3.2. Tissue distribution of aloe-emodin After successive administration for 12 weeks, the tissue distributions of aloe-emodin were significantly different between the normal and model groups (p < 0.0001). The concentrations of aloeemodin in the spleen and kidney tissues of normal rats were significantly higher than in the model rats (p < 0.01) (Tables 2 and 3). The concentration of aloe-emodin in the liver tissue of normal rats was also higher than in model animals (Table 2). 3.3. Tissue distribution of rhein The results show a statistically significant difference in distribution of rhein (p < 0.0001). The distribution of rhein between the two different groups of rats was significantly different (p < 0.0001), and the distribution of rhein among the tissues was also significantly different (p < 0.0001) (Table 4). The concentration of rhein in the spleen and kidney of normal rats was significantly higher than in the same tissues of the pathological model rats (p < 0.01) (Tables 2 and 4). The concentration of rhein in the liver of normal rats was also higher than in model rats, but these differences are not statistically significant. In addition, the distribution of rhein was not statistically significant in the tissues of model rats, but it was significant in the normal animals (p < 0.01). These data indicate
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Table 2 Amounts of free anthraquinones in rat tissue after oral administration for 12 weeks. Animals
Tissue
Aloe-emodin (g/g)
Rhein (g/g)
Emodin (g/g)
Control
Liver Kidney Spleen
1.13 ± 0.31** 1.11 ± 0.66** 1.94 ± 1.2**
0.43 ± 0.11 1.59 ± 0.74 2.68 ± 1.49
0.22 ± 0.07** 0.07 ± 0.01** 0.14 ± 0.09**
Mod
Liver Kidney Spleen
0.71 ± 0.16** 0.39 ± 0.19** 0.56 ± 0.24**
0.30 ± 0.76 0.68 ± 0.23 0.87 ± 0.13
0.14 ± 0.04** 0.05 ± 0.01** 0.08 ± 0.02**
**
p < 0.01 compared to rhein.
Table 3 Statistical results of tissue distribution of aloe-emodin in different animal groups. Source
DF
F value
Pr > F
Model Group Tissue Group × tissue
5 1 2 2
6.92 28.03 3.01 2.77
0.0002 <0.0001 0.0642 0.0784
Group
Tissue
Liver-m
Spleen-m
Model
Liver-m Spleen-m Kidney-m
0.5378 0.1270
0.4873
Liver-c Spleen-c Kidney-c
0.0999 0.0003 0.1184
0.0495 0.0002 0.0584
Control
Kidney-m
Liver-c
Spleen-c
0.0051 <0.0001 0.0064
0.0189 0.9373
0.0161
Liver-m, spleen-m, kidney-m represent CCl4 -injured model rats; liver-c, spleen-c, kidney-c represent control rats.
that the pharmacokinetics of AQs in normal animals and pathological animals may be different, which would cause the tissue concentrations of AQs in normal rats to be higher than those in the pathological model rats. Further experiments are necessary to determine an exact mechanism responsible for these observations (Table 4).
statistical details which scarcely published data did so. The significance of the whole statistic model constructed was reflected in the model item in Tables 3–5. Further statistic items (such as group and tissue) may actually make statistic sense only when the statistic model constructed have statistical significance (p < 0.05). The group item reflect the statistical difference between the normal rats and the model rats, and the tissue item show the differences among the visceras. In this study, we compared the different tissue distributions of rhubarb anthraquinone derivatives (AQs) in normal and CCl4 injured rats orally administered RE. The results indicated that after drug withdrawal for 4 weeks, AQs were no longer detected in tissues. These data suggest that this dosage of rhubarb may not cause accumulative toxicity. In addition, the tissue concentrations of all the AQs in normal rats were higher than those in model rats with rhein > aloe-emodin > emodin in kidney and spleen tissues and aloe-emodin > rhein > emodin in liver tissues (Table 2). Tissue distribution and excretion of aloe-emodin occurs quickly in rats (Jiang et al., 2003; Fuchikami et al., 2006), but the elimination half-life of aloe-emodin is shorter than that of rhein and emodin (Han, 1999).
3.4. Tissue distribution of emodin There was a significant difference (p < 0.05) in the tissue concentration of emodin between the normal and CCl4 -induced injury rats. However, there was not a significant difference between the two animal groups. Therefore, we did not further explore significant differences between the tissues in the same group (p < 0.01) (Table 5). Overall, the tissue concentration of emodin in normal rats was higher than in the model rats (Table 2). 4. Discussion Whether the statistic method is rational concerns the reliability of the research results. We attempted to convey the complete Table 4 Statistical results of tissue distribution of rhein in different animal groups. Source
DF
F value
Pr > F
Model Group Tissue Group × tissue
5 1 2 2
13.77 28.03 3.01 2.77
<0.0001 <0.0001 <0.0001 0.0018
Group
Tissue
Mod
Liver-m Spleen-m Kidney-m
0.0662 0.1588
0.4997
Liver-c Spleen-c Kidney-c
0.7421 <0.0001 <0.0001
0.1532 <0.0001 0.0227
Control
Liver-m
Spleen-m
Kidney-m
Liver-c
Spleen-c
0.3450 <0.0001 0.0015
<0.0001 0.0015
0.0038
Liver-m, spleen-m, kidney-m represent CCl4 -injured model rats; liver-c, spleen-c, kidney-c represent control rats.
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Table 5 Statistical results of tissue distribution of emodin in different animal groups. Source
DF
F value
Pr > F
Model Group Tissue Group × tissue
5 1 2 2
3.00 2.04 6.07 0.55
0.0250 0.1626 0.0058 0.5803
Group
Tissue
Liver-m
Spleen-m
Mod
Liver-m Spleen-m Kidney-m
0.0399 0.0233
0.8986
Liver-c Spleen-c Kidney-c
0.4640 0.7997 0.0557
0.0134 0.1704 0.8080
Control
Kidney-m
Liver-c
Spleen-c
0.0072 0.1627 0.8852
0.4332 0.0182
0.2245
Liver-m, spleen-m, kidney-m represent CCL-4-injured model rats; liver-c, spleen-c, Iddney-c represent control rats.
Rhein and emodin have a lower clearance rate (Han, 1999). Aloeemodin converts into rhein easily in rats (Krumbiegel and Schulz, 1993). Therefore, aloe-emodin may have a lower tissue distribution. However, aloe-emodin is more liposoluble than emodin and rhein, which may be the cause for the higher tissue concentration of aloe-emodin when administered continuously for 12 weeks. In previous studies, intragastric administration of free or conjunct anthraquinones caused similar levels of the chemicals in the serum of rat, with the concentration of rhein being the highest (Fuchikami et al., 2006; Shia et al., 2009). These results are in accordance with the findings of our study. Absorbency of rhein into the organism is higher than that of the other AQs (Ma et al., 2005; Zhu et al., 2005). Rhein and its main metabolites are mainly excreted through the liver and kidney. Bile excretion and enterohepatic circulation of rhein and its metabolites in rats increase the concentration and time (Bachmann and Schlatter, 1981; Ma et al., 2005). Rhein and emodin have longer elimination half-lives and lower clearance rates (Han, 1999). Aloe-emodin and emodin are oxidized and converted to rhein, but to date there is no evidence to show that rhein reduces to aloe-emodin or emodin (Krumbiegel and Schulz, 1993). For the above-mentioned reasons, rhein may accumulate in tissues, and these results suggest that rhein may be a major hazardous component of rhubarb. To date, research on the toxicity of rhein has been mainly focused on the liver and kidney (Wang et al., 2009a,b). However, in this study, the concentration of rhein in the spleen was significantly higher than in the liver and kidney. Further studies are necessary to determine the affinity of rhein for the spleen as compared to the liver and kidney and the effects of rhubarb on spleen toxicity. In this study, we observed that the tissue concentrations of rhein, aloe-emodin and emodin in normal rats were all higher than those in model rats. It is known that there is a relationship between the dose of a drug and the side effects or toxicities. Therefore, the toxic effects are directly correlated with the drug concentrations in the target tissues. The results from this study suggest that the toxic effects of AQs on normal tissues are greater than on pathological model tissues. In addition to the tissue distributions of AQs, we also measured other indexes, such as the organ coefficient of the liver, spleen and kidney, liver enzymology, serum ALT, AST, BIL, HA and a pathological examination of tissues. The results indicate that rhubarb was mainly toxic to normal rats but had therapeutic effects on CCl4 -induced injury rats when administrated orally at the same dose (data to be published in another article). In this study, we analyzed the pharmacological and toxicological differences of various concentrations of rhubarb in tissues. Further experiments are necessary to determine the relationship between the tissue concentration of AQs and the effects or toxicities on cells in vitro. AQs have been shown to have toxic effects on the liver and kidney. It is noteworthy that the published studies have analyzed the
effects of AQs on the normal state. However, therapeutics are commonly used for diagnosis, treatment or prevention of a disease, and therefore, most patients are in a pathological state. Because the condition of the organ is different in a normal or pathological situation, medicines may have different toxicities in these pathological settings. The findings that the concentrations of AQs in normal rat tissues were higher than in pathological model rat tissues are concordant with the ancient traditional Chinese medicine (TCM) theory of You Gu Wu Yun. This TCM theory has been an important guideline in the treatments of diseases and disorders for more than 2000 years. It emphasizes the combination of disease and syndrome differentiation with the purpose of more positive effects and less toxicity to patients (Sun and Yu, 2009). The principle of TCM is summarized as follows: a drug will reveal its therapeutic effect when it is prescribed for the correct indication; otherwise, it could lead to harmful effects for the patient or if administered to a healthy person as the result of misdiagnosis. Our results indicate that AQs cause greater tissue toxicity to normal animals than to pathological animals, which provides an opportunity to decode the ancient TCM theory and elucidate its medical implications with the use of modern biochemical tools. We hope that this study will provide helpful information for the evaluation of the adverse and toxic effects of rhubarb at the tissue-concentration level.
Acknowledgments This work was financially supported by the Key Technologies R&D Program of China (no. 2009ZX09502-022) and the National Natural Science Foundation of China (nos. 30625042 and 30973947).
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A comparative study on the tissue distributions of rhubarb anthraquinones in normal and CCl4 -injured rats orally administered rhubarb extract Fang Fang a,b,1 , Jia-bo Wang a,1 , Yan-ling Zhao a , Cheng Jin a , Wei-jun Kong a,b , Hai-ping Zhao c , Hong-juan Wang a , Xiao-he Xiao a,∗ a
China Military Institute of Chinese Materia Medica, 302 Military Hospital, Beijing 100039, PR China Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China c Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, PR China b
a r t i c l e
i n f o
Article history: Received 1 April 2011 Received in revised form 4 August 2011 Accepted 14 August 2011 Available online 30 August 2011 Keywords: Rhubarb Anthraquinone derivatives Tissue distribution LC–MS You Gu Wu Yun
a b s t r a c t Aim of the study: The present study comparatively investigated the tissue distributions of rhubarb anthraquinone derivatives (AQs) to examine whether they undergo different uptakes in normal or CCl4 induced liver-damaged rats, to explore possible reasons for the different toxicities of AQs in pathological model rats and normal rats at the tissue distribution level. Materials and methods: The total rhubarb extract (14.49 g kg−1 of body weight per day based on the quantity of crude material) was administrated orally to normal and model rats for 12 weeks. The concentrations of free AQs in tissues were quantitated by liquid chromatography–tandem mass spectrometry (LC–MS). After drug withdrawal for 4 weeks, tissue distributions were again determined. Results: The five free AQs—aloe-emodin, rhein, emodin, chrysophanol and physcion—were detected in the liver, kidney and spleen, while only rhein, aloe-emodin and emodin reached the quantitative limit. The tissue distributions of rhein (p < 0.001), aloe-emodin (p < 0.001) and emodin (p < 0.05) in normal rats were higher than those in model rats with rhein > aloe-emodin > emodin in kidney and spleen tissues and aloe-emodin > rhein > emodin in liver tissues. Free AQs were not detected in the tissues after drug withdrawal for 4 weeks. Conclusions: These results suggest that the tissue toxicity of AQs in normal animals is higher than that in pathological model animals with little accumulative toxicity of rhubarb. The results are concordant with the traditional Chinese theory of You Gu Wu Yun recorded first in Su Wen, a classical Chinese medical treatise. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Rhubarb (Radix et rhizoma rhei, Dahuang in Chinese) has been used in traditional Chinese medicine (TCM) for more than 2000 years and was first documented in “The Shen Nong Ben Cao Jing”, the earliest book on materia medica in the world. Rhubarb was named general with beneficial effects on constipation (Jong et al., 2010), hepatitis (Cui et al., 2010), cholecystitis (Jiao and Liu, 1982), diabetic nephropathy (DN) (Gu et al., 2003) and chronic renal failure (CRF) (Xiao et al., 2002). Rhubarb contains five free anthraquinone derivatives (AQs), which account for the main medicinal properties of rhubarb and have similar chemical structures, as shown in Fig. 1. These AQs have been documented to have numerous therapeutic
∗ Corresponding author at: 100#, the 4th West Ring Road, 302 Military Hospital, Beijing 100039, PR China. Tel.: +86 10 66933322; fax: +86 10 63879915. E-mail address: pharm [email protected] (X.-h. Xiao). 1 These authors contributed equally to this work. 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.08.028
benefits, including inducing purgation (Sun, 1992), inhibiting bacterial growth (Wang et al., 2010), protecting the liver (Huang et al., 1997; Zhao et al., 2009) and treating CRF (Wang et al., 2009a) and jaundice (Ho, 1996). However, recent studies have reported that AQs are cytotoxic to the liver and kidney (Wojcikowski et al., 2006) and have mutagenic and carcinogenic effects in vitro. A NIH 2001 panel conducted a two-year experimental study on the toxic effects of emodin in animals (National Toxicology Program (NTP), 2001). They showed that emodin has no carcinogenic effects and leads to obvious pathological changes of the renal tubule. Recent risk assessments of rhubarb in animals also concluded that AQs cause injury to the liver and kidney (Wang et al., 2007a, 2009a, 2011; Xing et al., 2011). The discrepancies among these studies have raised doubts concerning the medicinal use of rhubarb. Therefore, the protective or destructive effects of rhubarb should be assessed scientifically; the results of such studies will be of great significance to clinical diagnosis and treatment. We previously determined that rhubarb-induced injury to the liver or kidney is more pronounced in normal rats than in
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(20060525, Beijing Beihua Fine Chemicals Co. Ltd.), and olive oil (F20080219, Sinopharm Chemical Reagent Beijing Co. Ltd.) were of analytical grade.UPLC analyses of free AQs in rhubarb total extract were performed using a Waters Acquity System equipped with a binary solvent delivery pump, an auto sampler and a photodiode array detector. The UPLC–ESI-MS/MS analyses for tissue distribution of AQs were performed using an Acquity UPLC–MS/MS System (Waters Corp., Milford, MA, USA) with a Waters tandem quadrupole mass spectrometer (Milford, MA, USA) equipped with an electrospray source. An Acquity BEH UPLC C18 column (100 mm × 2.1 mm, 1.7 m) was used for the separation. The signal acquisition, peak integration and concentration determination were performed using ChemStation software (MassLynx 4.1) supplied by Waters Technologies. Other instruments used included a solid-phase extraction column with an OASIS MAX cartridge 3 cc (60 mg) (Waters, USA), a Waters extraction manifold system (Waters, USA), an ALl04 electronic balance, a low-speed centrifuge, and a manually adjustable pipette gun. 2.2. Animals
Fig. 1. Chemical structures of aloe-emodin, rhein, emodin, chrysophanol and physcion.
pathological rat models. This finding suggests that the medicinal effects of rhubarb are dependent on the pathological status (Wang et al., 2009a). In general, the protective or toxic effects of a drug on target organs are related to the concentration of the drug in the respective tissues. Therefore, the differing effects of rhubarb are possibly due to accumulation of the drug in the organs leading to different pathological conditions. To date, there have not been any studies of the tissue distribution of AQs in healthy rats compared to pathological rat models. In the present study, we determined the concentrations of free AQs in different tissues and evaluated their toxic effects on the liver or kidney in normal rats and in rats with CCl4 -induced liver damage. Possible explanations for the differences in free AQ tissue distribution are explored preliminarily.
Male and female Sprague Dawley (SD) rats 6–8 weeks of age and weighing 180 ± 30 g were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (License No. SYXK 2007-004). The animals were separated by gender and were given unlimited access to food and water in an environmentally controlled breeding room (temperature 22 ± 2 ◦ C, humidity 60–80%). The breeding room was illuminated by artificial light with a 12-h light/12-h dark cycle every day; the room was disinfected regularly. 2.3. Preparation of extracts A total of 100 kg of rhubarb was added to 600 L of 90% ethanol, heated and extracted 3 times for 1 h each. The residual rhubarb after extraction was added to 1000 L of water and heated and extracted for 1 h (Wang et al., 2011). The filtrate was merged and spray-dried, and the final extract yield was 29.3%. The samples were stored at 4 ◦ C for later experiments. 2.4. Animal experiments and sample collection
2. Materials and methods 2.1. Materials and instruments Rhubarb is the peeled and dried root of Rheum palmatum L., Rheum tanguticum Maxim. ex Balf. or Rheum officinale Baill. (Polygonaceae family) described in the Chinese Pharmacopoeia (PPRC, 2010). Rhubarb is also officially listed in the European and Japanese Pharmacopoeia (European Pharmacopoeia, 2001; Japanese Pharmacopoeia, 2006). Rheum palmatum was specifically examined in these studies because it is documented in all three pharmacopoeias mentioned above and is more commonly used than other species. The dried root and rhizoma of Rheum palmatum L. of the Polygonaceae family were collected in Lixian County, Gansu province of China, and were classified by Professor Xiaohe Xiao, a taxonomist at the PLA Institute of Traditional Chinese Material Medica. Standard chemicals (aloe-emodin, emodin, rhein, chrysophanol, physcion and 1,8-dihydroxy anthraquinone) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol was HPLC grade (Fisher, USA). Formic acid was of analytical grade purity and was purchased from Beijing Chemical Regents Inc., PRC (Beijing, China). High-purity water was produced by the Milli-Q water purification system (Millipore, Bedford, MA, USA). Carbon tetrachloride
In this study, a CCl4 -induced liver injury model was used because it has been well researched and demonstrates good reproducibility and high reliability. The mechanism by which CCl4 induces liver injury involves the formation of free radicals and the subsequent peroxidation chain reaction, which leads to a significant and chronic injury of hepatic cells (Jian et al., 2008). The experiment encompassed a total of 16 weeks. First, 54 rats were randomized into three groups of 18 animals each (9 males and 9 females). One group (the model group) was injected intraperitoneally with CCl4 oil (containing 1 portion CCl4 and 9 portions olive oil, 5 mL/kg) two times per week for a total of 12 weeks to induce chronic liver injury. The others received physiologic levels of saline. The medication group was administered the RE intragastrically once every day from the 4th week after modeling to the end of the 16th week. The control and model groups were administered physiologic levels of saline intragastrically. The dosage of total RE was 14.69 g herbs kg−1 of body weight per day and is equivalent to 4.9 times (converted with body surface area) (Wang et al., 2011) the upper dosage limit for humans as described in the Chinese Pharmacopoeia (0.5 g kg−1 ) (PPRC, 2010). Food and water were available to the animals ad libitum. 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
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Table 1 Amounts of AQs in rhubarb extract.
AQs AQGs
Aloe-emodin (%)
Rhein (%)
Emodin (%)
Chrysophanol (%)
Physcion (%)
0.204 ± 0.001 0.364 ± 0.002
0.267 ± 0.001 0.168 ± 0.001
0.954 ± 0.001 0.467 ± 0.003
0.329 ± 0.001 0.10S ± 0.001
0.455 ± 0.002 0.443 ± 0.003
AQs: anthraquinone derivatives; AQGs: anthraciuinone glycosides.
guidelines approved by the Ministry of Science and Technology of China. The tissues of liver, spleen and kidney were preserved at −80 ◦ C and thawed before use. 2.5. Quantitative determination 2.5.1. Assay of free AQs in the total rhubarb extract Preparation of standard solutions: Stock solutions of the five rhubarb anthraquinones (emodin, rhein, physcion, chrysophanol and aloe-emodin) were prepared at a concentration of 40 g mL−1 in methanol. All the solutions were stored in dark glass bottles at 4 ◦ C and were stable for at least 1 month. Working solutions were freshly prepared by diluting suitable amounts of the above solutions with methanol before injection. Sample preparation: Appropriate amounts of test samples were precisely weighed and then extracted with 25 mL of methanol by refluxing for 60 min. The extracted solution was prepared by the method of weight relief, in which there was compensation for the weight lost in the extraction procedure. After filtering, 5 mL of filtrate was transferred into a flask and then evaporated to dryness. Subsequently, 10 mL of 2 M HCl and 20 mL of chloroform were added to dissolve the residue, and the solution was incubated in 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 with methanol and transferred into a 50-mL volumetric flask. The solution was filtered through a 0.22-m filter before injection (Wang et al., 2008). Chromatographic conditions: The mobile phase was a methanol-0.1% formic acid water solution (70:30, v/v) at a flow rate of 0.3 mL min−1 . A single wavelength of 254 nm was employed for detection of AQs. The final injection volume was 5 L. The column temperature was maintained at 30 ◦ C (Wang et al., 2008). 2.5.2. Assay of free AQs in tissues Standard solutions were prepared as described in Section 2.5.1. The concentration of IS was 30 g mL−1 (1,8-dihydroxy anthraquinone) prepared in methanol. Sample preparation: In this study, aliquots of 0.2-g rat tissue samples and 500 L IS (30 g mL−1 ) were placed into 10-mL centrifuge tubes, homogenized and vortex-mixed. After being centrifuged for 15 min (10,000 rpm), 500 L of supernatant was transferred into an OASIS MAX cartridge 3 cc (60 mg) for further purification. The SPE column was conditioned with 1 mL of MeOH and then 1 mL of water, eluted with 3 mL of 5% ammonium water, then with 3 mL of methanol, and, finally, with 1 mL of 12% formic acid–isopropanol solution. A 500-L aliquot of the last eluted solution was collected to be injected into the UPLC–ESI-MS for analysis. The SPE procedure was performed on a Waters extraction manifold system. Chromatographic conditions: In addition to the UPLC conditions described in Section 2.5.1, the UPLC–ESI-MS/MS was conducted using high-purity nitrogen (N2 ) to assist nebulization. The quadrupole mass spectrometer equipped with an ESI source was set with a desolvation gas (N2 ) flow of 600 L min−1 , nebulizer pressure of 600 kPa, desolvation gas temperature of 450 ◦ C,
capillary voltage of 3.5 kV and the negative ion mode. The ESI-MS was performed in the multiple reaction monitoring (MRM) mode using the target ions [M−H]− at m/z 269 > daughter ions at m/z 240 for aloe-emodin, m/z 283 > 211 for rhein, m/z 269 > 225 for emodin, m/z 253 > 225 for chrysophanol, m/z 283 > 240 for physcion and m/z 239 > 211 for IS. Ultra-high-purity helium (He) was used as the collision gas at a flow rate of 0.25 mL min−1 (Fang et al., 2011). 2.6. Statistical analysis The experimental values were expressed as mean ± standard deviation (S.D.). All data were processed with the statistical software Windows SAS 8.0 (SAS, USA). The groups were analyzed using factor analysis, and the significance probability was ˛ = 0.05. 3. Results 3.1. Distributions of AQs in rhubarb extract and tissues The yield of total RE was 29.3%. The contents of free AQs in the total extract are listed in Table 1. The 5 free AQs—aloeemodin, rhein, emodin, chrysophanol and physcion—were detected in liver, kidney and spleen, but only rhein, aloe-emodin and emodin reached the quantitative limit. The tissue distributions of aloeemodin, rhein and emodin in the normal rats were all higher than those in the model rats with rhein > aloe-emodin > emodin in kidney and spleen tissues and aloe-emodin > rhein > emodin in liver tissues (Table 2). AQs were not detected in control animals and methanol control group, which suggests that there is no interference from control or methanol. After 4 weeks of discontinued administration as indicated, AQs were not detected in tissues. 3.2. Tissue distribution of aloe-emodin After successive administration for 12 weeks, the tissue distributions of aloe-emodin were significantly different between the normal and model groups (p < 0.0001). The concentrations of aloeemodin in the spleen and kidney tissues of normal rats were significantly higher than in the model rats (p < 0.01) (Tables 2 and 3). The concentration of aloe-emodin in the liver tissue of normal rats was also higher than in model animals (Table 2). 3.3. Tissue distribution of rhein The results show a statistically significant difference in distribution of rhein (p < 0.0001). The distribution of rhein between the two different groups of rats was significantly different (p < 0.0001), and the distribution of rhein among the tissues was also significantly different (p < 0.0001) (Table 4). The concentration of rhein in the spleen and kidney of normal rats was significantly higher than in the same tissues of the pathological model rats (p < 0.01) (Tables 2 and 4). The concentration of rhein in the liver of normal rats was also higher than in model rats, but these differences are not statistically significant. In addition, the distribution of rhein was not statistically significant in the tissues of model rats, but it was significant in the normal animals (p < 0.01). These data indicate
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Table 2 Amounts of free anthraquinones in rat tissue after oral administration for 12 weeks. Animals
Tissue
Aloe-emodin (g/g)
Rhein (g/g)
Emodin (g/g)
Control
Liver Kidney Spleen
1.13 ± 0.31** 1.11 ± 0.66** 1.94 ± 1.2**
0.43 ± 0.11 1.59 ± 0.74 2.68 ± 1.49
0.22 ± 0.07** 0.07 ± 0.01** 0.14 ± 0.09**
Mod
Liver Kidney Spleen
0.71 ± 0.16** 0.39 ± 0.19** 0.56 ± 0.24**
0.30 ± 0.76 0.68 ± 0.23 0.87 ± 0.13
0.14 ± 0.04** 0.05 ± 0.01** 0.08 ± 0.02**
**
p < 0.01 compared to rhein.
Table 3 Statistical results of tissue distribution of aloe-emodin in different animal groups. Source
DF
F value
Pr > F
Model Group Tissue Group × tissue
5 1 2 2
6.92 28.03 3.01 2.77
0.0002 <0.0001 0.0642 0.0784
Group
Tissue
Liver-m
Spleen-m
Model
Liver-m Spleen-m Kidney-m
0.5378 0.1270
0.4873
Liver-c Spleen-c Kidney-c
0.0999 0.0003 0.1184
0.0495 0.0002 0.0584
Control
Kidney-m
Liver-c
Spleen-c
0.0051 <0.0001 0.0064
0.0189 0.9373
0.0161
Liver-m, spleen-m, kidney-m represent CCl4 -injured model rats; liver-c, spleen-c, kidney-c represent control rats.
that the pharmacokinetics of AQs in normal animals and pathological animals may be different, which would cause the tissue concentrations of AQs in normal rats to be higher than those in the pathological model rats. Further experiments are necessary to determine an exact mechanism responsible for these observations (Table 4).
statistical details which scarcely published data did so. The significance of the whole statistic model constructed was reflected in the model item in Tables 3–5. Further statistic items (such as group and tissue) may actually make statistic sense only when the statistic model constructed have statistical significance (p < 0.05). The group item reflect the statistical difference between the normal rats and the model rats, and the tissue item show the differences among the visceras. In this study, we compared the different tissue distributions of rhubarb anthraquinone derivatives (AQs) in normal and CCl4 injured rats orally administered RE. The results indicated that after drug withdrawal for 4 weeks, AQs were no longer detected in tissues. These data suggest that this dosage of rhubarb may not cause accumulative toxicity. In addition, the tissue concentrations of all the AQs in normal rats were higher than those in model rats with rhein > aloe-emodin > emodin in kidney and spleen tissues and aloe-emodin > rhein > emodin in liver tissues (Table 2). Tissue distribution and excretion of aloe-emodin occurs quickly in rats (Jiang et al., 2003; Fuchikami et al., 2006), but the elimination half-life of aloe-emodin is shorter than that of rhein and emodin (Han, 1999).
3.4. Tissue distribution of emodin There was a significant difference (p < 0.05) in the tissue concentration of emodin between the normal and CCl4 -induced injury rats. However, there was not a significant difference between the two animal groups. Therefore, we did not further explore significant differences between the tissues in the same group (p < 0.01) (Table 5). Overall, the tissue concentration of emodin in normal rats was higher than in the model rats (Table 2). 4. Discussion Whether the statistic method is rational concerns the reliability of the research results. We attempted to convey the complete Table 4 Statistical results of tissue distribution of rhein in different animal groups. Source
DF
F value
Pr > F
Model Group Tissue Group × tissue
5 1 2 2
13.77 28.03 3.01 2.77
<0.0001 <0.0001 <0.0001 0.0018
Group
Tissue
Mod
Liver-m Spleen-m Kidney-m
0.0662 0.1588
0.4997
Liver-c Spleen-c Kidney-c
0.7421 <0.0001 <0.0001
0.1532 <0.0001 0.0227
Control
Liver-m
Spleen-m
Kidney-m
Liver-c
Spleen-c
0.3450 <0.0001 0.0015
<0.0001 0.0015
0.0038
Liver-m, spleen-m, kidney-m represent CCl4 -injured model rats; liver-c, spleen-c, kidney-c represent control rats.
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Table 5 Statistical results of tissue distribution of emodin in different animal groups. Source
DF
F value
Pr > F
Model Group Tissue Group × tissue
5 1 2 2
3.00 2.04 6.07 0.55
0.0250 0.1626 0.0058 0.5803
Group
Tissue
Liver-m
Spleen-m
Mod
Liver-m Spleen-m Kidney-m
0.0399 0.0233
0.8986
Liver-c Spleen-c Kidney-c
0.4640 0.7997 0.0557
0.0134 0.1704 0.8080
Control
Kidney-m
Liver-c
Spleen-c
0.0072 0.1627 0.8852
0.4332 0.0182
0.2245
Liver-m, spleen-m, kidney-m represent CCL-4-injured model rats; liver-c, spleen-c, Iddney-c represent control rats.
Rhein and emodin have a lower clearance rate (Han, 1999). Aloeemodin converts into rhein easily in rats (Krumbiegel and Schulz, 1993). Therefore, aloe-emodin may have a lower tissue distribution. However, aloe-emodin is more liposoluble than emodin and rhein, which may be the cause for the higher tissue concentration of aloe-emodin when administered continuously for 12 weeks. In previous studies, intragastric administration of free or conjunct anthraquinones caused similar levels of the chemicals in the serum of rat, with the concentration of rhein being the highest (Fuchikami et al., 2006; Shia et al., 2009). These results are in accordance with the findings of our study. Absorbency of rhein into the organism is higher than that of the other AQs (Ma et al., 2005; Zhu et al., 2005). Rhein and its main metabolites are mainly excreted through the liver and kidney. Bile excretion and enterohepatic circulation of rhein and its metabolites in rats increase the concentration and time (Bachmann and Schlatter, 1981; Ma et al., 2005). Rhein and emodin have longer elimination half-lives and lower clearance rates (Han, 1999). Aloe-emodin and emodin are oxidized and converted to rhein, but to date there is no evidence to show that rhein reduces to aloe-emodin or emodin (Krumbiegel and Schulz, 1993). For the above-mentioned reasons, rhein may accumulate in tissues, and these results suggest that rhein may be a major hazardous component of rhubarb. To date, research on the toxicity of rhein has been mainly focused on the liver and kidney (Wang et al., 2009a,b). However, in this study, the concentration of rhein in the spleen was significantly higher than in the liver and kidney. Further studies are necessary to determine the affinity of rhein for the spleen as compared to the liver and kidney and the effects of rhubarb on spleen toxicity. In this study, we observed that the tissue concentrations of rhein, aloe-emodin and emodin in normal rats were all higher than those in model rats. It is known that there is a relationship between the dose of a drug and the side effects or toxicities. Therefore, the toxic effects are directly correlated with the drug concentrations in the target tissues. The results from this study suggest that the toxic effects of AQs on normal tissues are greater than on pathological model tissues. In addition to the tissue distributions of AQs, we also measured other indexes, such as the organ coefficient of the liver, spleen and kidney, liver enzymology, serum ALT, AST, BIL, HA and a pathological examination of tissues. The results indicate that rhubarb was mainly toxic to normal rats but had therapeutic effects on CCl4 -induced injury rats when administrated orally at the same dose (data to be published in another article). In this study, we analyzed the pharmacological and toxicological differences of various concentrations of rhubarb in tissues. Further experiments are necessary to determine the relationship between the tissue concentration of AQs and the effects or toxicities on cells in vitro. AQs have been shown to have toxic effects on the liver and kidney. It is noteworthy that the published studies have analyzed the
effects of AQs on the normal state. However, therapeutics are commonly used for diagnosis, treatment or prevention of a disease, and therefore, most patients are in a pathological state. Because the condition of the organ is different in a normal or pathological situation, medicines may have different toxicities in these pathological settings. The findings that the concentrations of AQs in normal rat tissues were higher than in pathological model rat tissues are concordant with the ancient traditional Chinese medicine (TCM) theory of You Gu Wu Yun. This TCM theory has been an important guideline in the treatments of diseases and disorders for more than 2000 years. It emphasizes the combination of disease and syndrome differentiation with the purpose of more positive effects and less toxicity to patients (Sun and Yu, 2009). The principle of TCM is summarized as follows: a drug will reveal its therapeutic effect when it is prescribed for the correct indication; otherwise, it could lead to harmful effects for the patient or if administered to a healthy person as the result of misdiagnosis. Our results indicate that AQs cause greater tissue toxicity to normal animals than to pathological animals, which provides an opportunity to decode the ancient TCM theory and elucidate its medical implications with the use of modern biochemical tools. We hope that this study will provide helpful information for the evaluation of the adverse and toxic effects of rhubarb at the tissue-concentration level.
Acknowledgments This work was financially supported by the Key Technologies R&D Program of China (no. 2009ZX09502-022) and the National Natural Science Foundation of China (nos. 30625042 and 30973947).
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