Evaluation of the liver protection and toxicity of Da-Huang-Zhe-Chong pill in rats

Abstract

Context: Da-Huang-Zhe-Chong pill (DHZCP), a classical traditional Chinese formula, consists of 12 crude drugs which have been widely used with significant therapeutic effects. Some drugs in this formula have toxicities that might result in some adverse effects of DHZCP.

Objective: The liver protection and toxicity of DHZCP were first evaluated against chronic carbon tetrachloride (CCl₄)-induced liver injury in rats.

Materials and methods: The rats were treated by intraperitoneal injection of 10% CCl₄ for 12 weeks. At the end of week 4, DHZCP at doses of 44 g/kg (high-dose group) and 22 g/kg (low-dose group) was intragastrically administered to CCl₄-treated rats, once a day for four weeks. At the end of weeks 8 and 12, the general status of the rats, histopathology of liver, serum alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) levels were observed or determined and recorded. By correspondence analysis (CA) on biochemical markers and liver histopathological score (HS), the “dose-time-response” relationship of DHZCP on the hepatic injury rats was evaluated.

Results: The results showed that DHZCP exhibited a significant protective effect on liver injury by reversing the biochemical parameters and histopathological changes. However, this hepatoprotective effect may be weakened, or even be transferred to toxicity with the increase of the administration dose (44 g·kg⁻¹·d⁻¹) and time (more than 2 months) of this formula. These results were consistent with the histopathological observation and the serum levels.

Discussion and conclusion: Administration of proper dose and time of DHZCP could well play its hepatoprotective effect and even treat hepatitis, but the safety on liver should be considered when large-dose (44 g·kg⁻¹·d⁻¹) DHZCP is used for long time (more than 2 months). We suggest that the administration dose and time of DHZCP in clinical use should not be increased and prolonged, and simultaneously liver function should be regularly monitored.

Keywords::

• Chronic liver injury
• protection effect
• toxicity
• “dose-time-response” relationship
• correspondence analysis

Introduction

Liver diseases are a serious health problem. For the lack of reliable liver protective drugs in allopathic medical practices, traditional Chinese drugs (TCDs) play an important role in the management of these liver diseases. Numerous medicinal TCDs and their formula are used for these diseases in ethnomedical practices and in traditional medicine system in China. However, some TCDs and their formula, such as Radix aconiti, Radix puerariae, Rhizoma coptidis, Niu-Huang-Jie-Du pill, Xiao-Cai-Hu broth and Shen-Mai injection, etc., can result in liver injury (Dai et al., 2004; Lu & Peng, 2005). This is a handicap on the development of TCDs in treating liver diseases. Therefore, there is an urgent need to search for hepatoprotective drugs with high efficacy and safety.

Da-Huang-Zhe-Chong Pill (DHZCP) is a classical traditional Chinese formula comprising 12 crude drugs: Radix et rhizoma rhei (Da-Huang), Eupolyphaga seu steleophaga (Tu-Bie-Chong), Hirudo (Shui-Zhi), Gradfly (Meng-Chong), Grub (Qi-Cao), Resina toxicodendri (Gan-Qi), Semen persicae (Tao-Ren), Semen armeniacae amarum (Ku-Xing-Ren), Radix scutellariae (Huang-Qin), Radix rehmanniae (Di-Huang), Raidix paeoniae alba (Bai-Shao) and Radix glycyrrhizae (Gan-Cao) (China Pharmacopoeia Committee, 2005). This formula was first recorded in Jin-Gui-Yao-Lue (Synopsis of Golden Chamber) with widely pharmacodynamic actions for treating diseases like blood stasis, hepatic cirrhosis, cyclomastopathy, chronic active hepatitis (CAH) for nearly 2000 years (Chen & Tan, 2006; Cao et al., 2006). Since the 1980s, many reports have confirmed the significant therapeutic effects of DHZCP for treatment of chronic hepatitis B and cirrhosis (Jiao et al., 1999; Sun, 1998; Pan et al., 2004, 2005). But, Gradfly, Grub and Resina toxicodendri in this formula have certain toxicities (China Pharmacopoeia Committee, 2005), and also, rhubarb can cause liver and renal toxicity along with carcinogenicity for its active components of anthraquinones (Cao et al., 2006; Wang et al., 2009 a,b). The inapposite or indiscriminate use of large amounts of DHZCP by some people may result in adverse side effects, even become life-threatening. So, the major objective to evaluate the hepatoprotective and toxic effects of DHZCP is important for its clinical use.

In the present study, we evaluated the liver protection and toxicity of different doses of DHZCP by intragastrically administering it to CCl₄-induced liver injury rats. By examining the general status of rats, histopathology of liver, biochemical parameters including serum ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase) and TBIL (total bilirubin) levels, combined with correspondence analysis, the hepatoprotective effect and toxicity of DHZCP on liver injury rats were investigated. Further, the “dose-time-response” relationship of it on liver injury rats was discussed to promote safe use of this kind of formula.

Materials and methods

Materials

Da-Huang-Zhe-Chong Pill (DHZCP) was prepared according to Chiniese Pharmacopoeia (China Pharmacopoeia Committee, 2005). Analytical-grade CCl₄ was purchased from Beihua Fine Chemicals Co., Ltd. (Beijing, China) and dissolved in olive oil to a final concentration of 10% before use. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) kits were purchased from Nanjing Jiancheng Bioengineering Institute (China). Other chemicals and reagents were obtained from local sources and were all analytical grades.

Animals

Eighty, male and female Sprague-Dawley rats, 6–8 weeks old and weighing 150–180 g were obtained from the Vital River Lab Animal Technology Co., Ltd. (Beijing, China) (License No. SCXK 2003–0003). These animals were housed under controlled conditions before the commencement of the experiments. The conditions were as follows: light (12 h light/dark cycle, lights on at 7:00 am), controlled temperature (20 ± 2°C), humidity (60–70%), draughty, free access to food and water. Animal welfare and experimental procedures were carried out strictly in accordance with the institutional animal care guidelines approved by the Ministry of Science and Technology of China and the related ethical regulations.

Experimental procedure

All the animals were randomly divided into four groups: normal group (N, n = 20), CCl₄ model group (M, n = 20), CCl₄ + high- dose of DHZCP (44 g/kg, H, n = 20) group and CCl₄ + low -dose of DHZCP (22 g/kg, L, n = 20) group. The rats [except the normal group, which received an equal volume of Isotonic Saline Solution (ISS)] were intraperitoneally injected with a solution of 10% CCl₄ (5 mL/kg body weight, in olive oil, 1:10, v/v) for 12 weeks to induce chronic liver injury. All the rats in H and L groups were treated intragastrically with 20 mL/kg of 44 g/kg and 22 g/kg of DHZCP, respectively, once a day from the beginning of week 4 to the end of week 12, while the rats in N and M groups received an equal volume of water. At the end of week 8 (when the rats had received CCl₄ for 2 months and DHZCP for 1 month) and week 12 (when the rats had received CCl₄ for 3 months and DHZCP for 2 months), the rats had free access to water, but not food for 12 h, they were then sacrificed. Blood was taken from the femoral vein to prepare serum for biochemical assay and the liver was obtained for histopathological observations.

General observation

The general status of the rats, including mental status, weight change, food intake, etc, was first visually observed and recorded (Suresh et al., 2008; Wang et al., 2009 a,c).

Biochemical assays

The blood sample (5.0 mL) was centrifuged at 3000 × g for 10 min to separate serum. ALT, AST, ALP and TBIL activities were determined with a commercial kit (Roche) by Roche Modular Autoanalyzer.

Histopathological studies

Portions of tissues from the same lobe of liver in each animal were immediately fixed in 10% formaldehyde, embedded in paraffin, cut into 4–5 μm-thick sections, stained with hematoxylin-eosin (H & E), and observed under a light microscope. Histological damage was expressed according to the “semiquantitative grading” standard (Liu et al., 2001) using the following score system: 0, absent; 1, few; 2, mild; 3, severe, the average value were then calculated and expressed as mean ± S.D.

Statistical analysis

Data were expressed as mean ± S.D. Significant difference between groups was analyzed by Student’s t-test using SAS 8.0 software (SAS, USA). The Mann–Whitney rank sum test was used for the degree of histopathological liver injury. P < 0.05 was considered to be statistically significant.

Correspondence analysis (CA)

CA (Park et al., 2007; Pusha et al., 2009; Kong et al., 2009) is an important multivariate statistical method for studying the relationship between investigated factor and multi-variables. Based on the idea of principal component analysis (PCA), CA can compress these multi-variables to two principal component (PC) variables Z1 and Z2, which have the main contributions to the total data set. Then, the PC variable aggregations [Z1, Z2] of every valid point are calculated and depicted in the form of 2D-projection. Thus, the internal change regularity of investigated factor can be directly represented. In this study, the “dose-time-response” relationships of the liver protection and toxicity of DHZCP on hepatic injury rats were investigated by CA on the biochemical markers (serum ALT, AST, ALP and TBIL levels) and liver histopathological scores (HS) using SAS 8.0 software (SAS, USA).

Results

General status

Rats in the normal (N) group and CCl₄ + DHZCP groups appeared healthy with good mental status and increased weight, while the CCl₄ (M) group showed decreased activity, less food intake, loose stool, piloerection, dark fur, and retarded weight gain or even weight loss. The animals in CCl₄ + high- dose of DHZCP (H) group and CCl₄ + low- dose of DHZCP (L) group had little mortality (Table 1), slight hepatocyte steatosis, liver fibrosis, cholestasis or small bile duct hyperplasia as compared with those of the CCl₄ model (M) group.

Table 1.  Histopathological changes in the liver of rats of the four groups.

Time	Group	Dose (g/kg)	n	Survival	Microscopic observation of damage in liver
					Number of rats in four scores				Average value of scores
					0	1	2	3
Week 8	N		10	10	10	0	0	0	0
	M		10	9	0	2	6	1	4 ± 1.53^ΔΔ
	H	44	10	10	0	6	4	0	3.14 ± 1.21*
	L	22	10	10	0	7	3	0	2.71 ± 1.11**
Week 12	N		10	10	10	0	0	0	0
	M		10	6	0	0	2	4	S.4 ± 2.07^ΔΔ
	H	44	10	8	0	3	4	1	4.33 ± 1.51**
	L	22	10	9	0	6	3	0	3.17 ± 1.47**^▴

Histological damage was expressed using the following score system: 0, absent; 1, few; 2, mild; 3, severe. Values represent the mean ± S.D. The Student’s t-test was performed on the raw data.

^Δp < 0.05, ^ΔΔp < 0.01 compared with the normal group.

*p<0.05, **p<0.01 compared with the CCl₄ model group.

▴p<0.05 compared with the CCl4 + high-dose of DHZCP group.

Activities of DHZCP on serum ALT, AST, ALP and TBIL levels

The serum activities of ALT, AST, ALP and TBIL were used as biochemical markers for the chronic hepatic damage and the contents of these four markers in the four groups at different times are shown in Figure 1. At the end of week 8, compared to N group, the levels of ALT, AST and ALP in the M group were significantly elevated (p<F0.01) (ALT: from 71.60 ± 34.95 U/L to 426.90 ± 102.35 U/L, AST: from 288.50 ± 42.71 U/L to 826.50 ± 88.85 U/L, ALP: from 75.50 ± 33.52 IU/L to 281.60 ± 94.99 IU/L), and TBIL was also increased (p<0.05) from 2.70 ± 1.56 µmoL/L to 5.62 ± 3.95 µmoL/L, illustrating that the hepatocyte was damaged along with cholestasis. DHZCP administration with low and high doses (H and L groups) all significantly lowered transaminase level and cholestasis in rats compared with the corresponding levels in M group. The serum ALT level in low-dose group was higher (p<0.05) than the corresponding value in high-dose group, showing that high- dose of DHZCP had significant enzyme-lowering and liver-protecting effects on the CCl₄-induced liver injury rats. At the end of week 12: the levels of ALT (1665.80 ± 37.71 U/L), AST (1629.20 ± 39.67 U/L), ALP (251.60 ± 26.40 U/L) and TBIL (2.34 ± 0.44 U/L) in M group were significantly elevated (p<0.01) from the corresponding values in N group (ALT: 166.29 ± 29.91 U/L, AST: 807.57 ± 59.567 U/L, ALP: 85.29 ± 28.14 U/L, TBIL: 2.56 ± 0.85 U/L), illustrating the notable hepatocyte damage. DHZCP administration in the H and L group could significantly lower transaminase level, protect liver and normalize gall bladder to cure jaundice. The rats treated with low dose of DHZCP showed a significant decrease in AST, ALT and ALP levels (p<0.01), and in TBIL level (p<0.05) compared with the corresponding values of rats treated with high- dose of DHZCP.

Figure 1.  Activities of DHZCP on serum ALT, AST, TBIL and ALP levels in 4 groups of rats. N, normal group; M, model group, H, high-dose group, L, low-dose group. A–D: serum levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), alkaline phosphatase (ALP) and total bilirubin (TBIL) in 4 groups at weeks 8 and 12, respectively. Data are expressed as mean ± S.D. of 20 rats for each group. ^△p < 0.05, ^△△p < 0.01 compared with the normal group. *p < 0.05, **p < 0.01 compared with the CCl₄ model group. ^▴p < 0.05, ^▴▴p < 0.01 compared with the CCl₄ + high-dose of DHZCP group.

Histopathological observation

The histological features, as shown in Figure 2A and Table 1, indicated a normal liver lobular architecture and cell structure of the livers in the control animals (group N). There were no pathological changes in healthy livers of the normal group. At the end of week 8, in the CCl₄-induced (M) group, liver pathological changes appeared characterized by swelling, hydropic degeneration, hepatocytes steatosis, small bile duct proliferation and cholestasis (Figure 2B), reflecting early biochemical and pathological changes in CCl₄-induced liver injury. Histopathologcial changes induced by CCl₄ were remarkably improved by high and low- dose of DHZCP (H and L groups) (Figures 2 C, D ), showing obvious difference from the M group except the hepatocytes steatosis, which is similar to the M group. The histopathological scores (HS) of liver pathological changes (Table 1) were significantly low after DHZCP administration (p<0.05 for H group and p<0.01 for L group compared with the M group), but there were no notable differences between the H and L group. At the end of week 12, in M group, liver pathological changes (Figures 2E) were much more severe than those at the end of week 8, which could well reflect the biochemical and pathological changes in CCl₄-induced liver injury. These histopathologcial changes were remarkably improved by DHZCP of high and low- dose (H and L groups) (Figures 2F, G). The HS of liver pathological changes (Table 1) in L group were significantly low, compared with the scores in H group (p<0.05). This shows that low- dose of DHZCP in L group could much more significantly improve the liver histopathologcial changes than high-dose DHZCP in H group, which, on the other hand, illustrated that DHZCP at high level would aggravate the liver histopathologcial damage by showing toxicity to the injured liver. The safety would be considered by successive administration of DHZCP at the dose of 44 g·kg⁻¹·d⁻¹ (H group) on the chronic liver injury rats for 2 months. The therapeutic action of DHZCP on CCl₄-induced liver injury will be weakened or will be transferred to toxicity with the increase in administration dose and time of this formula. For the investigation of the “dose-time-response” relationships of DHZCP on hepatic injury rats, an important statistical method, CA, was introduced in the next part.

Figure 2.  Light microscopic analysis (HE stain, × 200) of rat liver sections of normal rats and CCl₄ treatment with or without DHZCP administration. (A) normal group; (B) CCl₄-induced (M) group at the end of week 8; (C) high- dose of DHZCP at the end of week 8; (D) low- dose of DHZCP at the end of week 8; (E) CCl₄-induced (M) group at the end of week 12; (F) high- dose of DHZCP at the end of week 12; (G) low-dose of DHZCP at the end of week 8.

CA for the “dose-time-response” relationship of DHZCP

The “dose-time-response” relationship of DHZCP on the hepatic injury rats were investigated by CA on the biochemical markers (serum ALT, AST, ALP and TBIL levels) and liver histopathological scores (HS) in each group and shown in Figure 3. Each point, which was obtained by CA on the four markers and HS, was depicted in the rectangular coordinate system with Z1 as horizontal axis and Z2 as longitudinal axis to represent each investigated group. The point of each group was clearly distributed in this rectangular coordinate system based on the point of N8 (the normal group at the end of week 8) as zero. N8, M8, H8 and L8 represented the normal group, model group, high- dose of DHZCP group and low- dose of DHZCP group at the end of week 8 and N12, M12, H12 and L12 represented the normal group, model group, high- dose of DHZCP group and low- dose of DHZCP group at the end of week 12. The four groups at the end of week 8 were shown with solid rhombus, and the other four groups at the end of week 12 with solid triangle. The euclidean distance between each group was calculated and shown in Table 2.

Table 2.  Euclidean distance between each group.

Group	N8	M8	H8	L8	N12	M12	H12	L12
N8	—a
M8	0.2656	—
H8	0.3848	0.3961	—
L8	0.2668	0.1750	0.2217	—
N12	0.3178	—	—	—
M12	—	0.4988	—	—	0.6879	—
H12	—	—	0.7898	—	0.3684	0.3313	—
L12	—	—	—	0.5242	0.0858	0.6027	0.2870	—

^aThe euclidean distance between the same group, between the different groups at different time were not calculated and compared.

N8, M8, H8 and L8 represented the normal group, model group, high dose of DHZCP group and low dose of DHZCP group at the end of week 8.

N12, M12, H12 and L12 represented the normal group, model group, high dose of DHZCP group and low dose of DHZCP group at the end of week 12.

Figure 3.  CA plot of principal components 1 and 2 (Z1, Z2) for the distribution of the investigated group. This plot was obtained by correspondence analysis (CA) using SAS 8.0 software on the biochemical markers and liver histopathological scores in each group. It could reflect the “dose-time-efficacy” relationships of DHZCP on hepatic injury rats. N8, M8, H8 and L8, which represented the normal group, model group, high- dose of DHZCP group and low- dose of DHZCP group at the end of week 8, were shown with solid triangles. N12, M12, H12 and L12, which represented the normal group, model group, high-dose of DHZCP group and low- dose of DHZCP group at the end of week 12, were shown with solid rhombuses.

From Figure 3 and Table 2, the “dose-time-response” relationships of DHZCP on hepatic injury rats could be found: (1) the distance of M12 and N12 (0.6879) was larger than that of M8 and N8 (0.2656), showing that CCl₄ could successfully induce the liver injury of rats and this injury would be aggravated by increasing the time of its use on rats; (2) at the end of week 8 (DHZCP administration for 1 month), the points L8 and M8 were distributed in the second quadrant and the distance between them was 0.1750, while H8 was alone distributed in the first quadrant and the distance of H8 and M8 was 0.3916, illustrating that high- dose (44 g·kg⁻¹·d⁻¹, which was 220-fold to the maximum dose 12 g·60 kg⁻¹·d⁻¹ in China Pharmacopoeia) of DHZCP had good therapeutic action on CCl₄-induced liver injury of rats and this action was stronger than that of low- dose (22 g·kg⁻¹·d⁻¹) of DHZCP; (3) at the end of week 12 (DHZCP administration for 2 month), the points N12, H12 and L12 were distributed in the third quadrant and M12 was in the second quadrant. The distance of L12 and M12 (0.6027) was larger than that of H12 and M12 (0.3313), and the distance of L12 and N12 (0.0858) was the smallest. These illustrated that low and high- dose of DHZCP could significantly improve the CCl₄-induced liver injury of rats at the end of week 12, and this improvement of low-dose (22 g·kg⁻¹·d⁻¹, which was 110-fold to the maximum dose 12 g·60 kg⁻¹·d⁻¹ in China Pharmacopoeia) DHZCP on this injury was better than that of high- dose of DHZCP; (4) the distance of H8 and H12 (0.7898) was the biggest among the distance of other groups, showing that the therapeutic action of high-dose DHZCP on CCl₄-induced liver injury of rats will be weakened, even be transferred to toxicity with the increase of the administration dose and time of this formula. These results were consistent with the histopathological observations and the serum levels.

Discussion

Although TCDs have been attracting more attention in recent years because of its complementary therapeutic effects to western medicines, and its capability to deal with many essential problems that have not yet been solved by conventional medicinal practices, their adverse effects or toxicity of them should not be ignored (Tai et al., 1993; De Smet, 2004; Jordan et al., 2010). These adverse effects or toxicities may result from many factors including inherent toxicity, contamination with toxic materials, improper administration and large-dose or longtime use, etc. Discussion on the safety assessment of TCDs has got increased (Jordan et al., 2010). As a classical traditional Chinese formula, DHZCP has got quick development for treating liver diseases. However, there are few recent reports to show on the toxic and adverse effects of this formula.

In the present study, the hepatoprotective effect and toxicity of DHZCP on CCl₄-induced liver injury rats were investigated based on examining the general status of the rats, histopathology of liver, biochemical parameters levels, and further, the “dose-time-response” relationship of it on liver injury was discussed combined with correspondence analysis. The results showed that DHZCP could improve the liver lesion of rats induced by CCl₄. Administration of proper dose and time of DHZCP could well play its hepatoprotective effect and even treat hepatitis, but the safety on liver should be considered when a large- dose (44 g·kg⁻¹·d⁻¹) of DHZCP was used for a long time (more than 2 months). So, the safety should be considered in the long-term and large-dose use of DHZCP. Further research elucidating the action mechanism of these effects (protective effect and toxicity) will give an insight into the usefulness of this prescription on the chronic CCl₄-induced liver injury. We suggest that the administration dose and time of DHZCP in clinical use should not be increased and prolonged, and simultaneously the liver function should be regularly monitored.

The study of “dose-time-response” relationship provided some useful references for the rational use of DHZCP and other medicinal TCDs and their formula clinically.

Acknowledgement

We are grateful to the support of National Basic Research Program of China (973 project) (No. 2007CB512607), National Nature Science Foundation (No. 30973947) and the National Key Technologies R&D Program of China (No. 2009ZX10005-017).

Declaration of interest

The authors have declared no conflict of interest.