Hepatoprotective and curative activities of aqueous extract of decoction containing 10 Chinese medicinal herbs (HPE-XA-08) were evaluated in Sprague–Dawley albino rats with liver damage induced by thioacetamide (TAA). These activities were assessed by investigating the liver enzymes level and also histopathology investigation. Increases in alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) levels were observed in rats with cirrhotic liver. No significant alterations of the liver enzymes were observed following treatment with HPE-XA-08. Histopathology examination of rats treated with HPE-XA-08 at 250 mg/kg body weight, however, exhibited moderate liver protective effects. Reduced extracellular matrix (ECM) proteins within the hepatocytes were noted in comparison to the cirrhotic liver. The curative effects of HPE-XA-08 were observed with marked decrease in the level of ALP (more than 3x) and level of GGT (more than 2x) in cirrhotic rat treated with 600 mg/kg body weight HPE-XA-08 in comparison to cirrhotic rat treated with just water diluent. Reversion of cirrhotic liver to normal liver condition in rats treated with HPE-XA-08 was observed. Results from the present study suggest that HPE-XA-08 treatment assisted in the protection from liver cirrhosis and improved the recovery of cirrhotic liver.
Chronic liver disease is the ninth leading cause of mortality in Western and developing countries [
To date, there is no specific treatment for liver cirrhosis. Sufferers are treated to reduce the complications due to the damaged liver from exacerbating. The treatments are often expensive especially for those in developing countries where there is high rate of liver cirrhosis in the population. Due to these factors, treatment using ethnobotanical approach has gained popularity as an alternative cost-effective approach [
The present study aims to evaluate the preventive and potential curative effects of a decoction containing 10 Chinese medicinal herbs, HPE-XA-08, in which a number of the herbs have been reported to be useful in supporting liver functions and help to ameliorate liver fibrosis/cancer [
Aqueous extract of HPE-XA-08 containing mixture of 10 Chinese medicinal herbs, comprising fructus
Male Sprague–Dawley (SD) rats aged between 7 and 10 weeks were kept in the University Malaya animal breeding house. Animals were fed with standard pellet diet and water ad libitum at 20–25°C. All animal handlings and protocols were in accordance with the institutional guidelines for laboratory animals (Ethic Reference Number PM/27/08/2011/MAA(R)).
Preventive effects for liver cirrhosis conferred by HPE-XA-08 were evaluated using the SD rats. Rats were assigned to 4 groups, namely, Group 1P: control group 1 (normal saline + H2O), Group 2P: control group 2 (normal saline + HPE-XA-08), Group 3P: TAA control group (TAA+ H2O), and Group 4P: treated group (TAA + HPE-XA-08 treatment; pretreatment with HPE-XA-08, 300 mg/kg). Rats from Groups 1P and 2P were injected intraperitoneally (ip) with normal saline while Groups 3P and 4P were injected with thioacetamide (TAA) at a dose of 200 mg/kg twice weekly for 12 weeks. HPE-XA-08 was administered orally at 300 mg/kg via a stomach tube to rats in Groups 2P and 4P daily, up to 12 weeks, while Group 1P and Group 3P were fed with distilled water. For the evaluation of HPE-XA-08 treatment on TAA-induced liver cirrhosis, 3 rat groups were assigned, namely, Group 1T: normal control group, Group 2T: cirrhosis control group, and Group 3T: treatment group (posttreatment with HPE-XA-08, 600 mg/kg). Liver cirrhosis was induced in rats of Groups 2T and 3T by inoculating TAA at 200 mg/kg intraperitoneally, twice weekly, for 12 weeks. Rats of Group 1T that served as normal control were inoculated with normal saline. After 12 weeks, the TAA was stopped and the rats from Group 3T were treated with 600 mg/kg HPE-XA-08 as described above while rats of Groups 1T and 2T were treated with distilled water of the equivalent volume. Blood was collected from each rat before and after the treatment. The treatment was performed for 30 days.
Blood of each rat was collected via tail vein before the treatment and at the end of the treatment regime. The blood was collected in tube containing EDTA and analyzed for the liver function enzymes serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) in the Clinical Diagnostic Laboratory (CDL) at University Malaya Medical Centre (UMMC). Immediately after the final blood collection, the rats were euthanized and the organs were harvested for histological investigation.
Liver samples harvested from the rats were washed with the normal saline and immediately fixed in 10% buffered neutral formalin for 48 hours. Samples were then embedded in paraffin wax. Sections of 5-micron thickness were prepared, processed in alcohol-xylene series, and stained with alum-haematoxylin and eosin, prior to histopathological examination.
Results were presented as mean ± SEM of six animals in each group. The data were subjected to one-way ANOVA followed by Bonferroni’s posttest.
The effects of HPE-XA-08 on serum ALT, AST, ALP, and GGT and bilirubin activities in rats from all treatment groups were shown in Table
Effect of HPE-XA-08 on blood biochemicals related to liver damage (ALT, AST, ALP, and GGT) for observation of protective activity.
Group | ALT (IU/L) | AST (IU/L) | ALP (IU/L) | GGT (IU/L) |
---|---|---|---|---|
Normal saline + H2O (1P) | 80.00 ± 4.175 | 237.3 ± 12.84 | 173.6 ± 7.807 | 2.286 ± 0.4206 |
Normal saline + HPE-XA-08 (2P)# | 76.00 ± 4.416 | 223.3 ± 11.16 | 170.3 ± 23.68 | 3.750 ± 0.4787 |
TAA + H2O (3P) | 88.50 ± 12.75 | 215.0 ± 13.64 | 375.0 ± 10.41 | 37.50 ± 1.857 |
TAA + HPE-XA-08 (4P)+ | 69.40 ± 7.756 | 222.4 ± 5.715 | 277.0 ± 4.722 | 30.80 ± 3.980 |
#Levels of enzymes between 1P and 2P were compared to show the effects of HPE-XA-08 on the level of liver enzymes but did not show statistically significant value (
+Levels of enzymes between 3P and 4P were compared to show the effects of HPE-XA-08 on preventing TAA-induced liver cirrhosis but did not show statistically significant value (
Histological examination of liver section of rats from the control groups (Groups 1P and 2P) showed that the liver was dark red in color, with smooth homogenous surface texture (Figures
Macroscopic and histologic analysis of liver harvested from rats. Liver samples were harvested from rats inoculated with normal saline and treated with water (a), inoculated with normal saline and treated with HPE-XA-08 (c), inoculated with TAA (200 mg/kg) twice weekly and treated with water (e), and inoculated with TAA and treated with HPE-XA-08 (g). The sections from harvested liver were stained with hematoxylin-eosin, respectively (b, d, f, h).
The possibility for HPE-XA-08 to reverse liver cirrhosis in rats was also evaluated. All rats were inoculated with TAA to induce liver cirrhosis, except for rat group 1T which was inoculated with normal saline to serve as control group (normal rat). Following 12 weeks of induction of liver cirrhosis with TAA, the rats were either treated with distilled water (Group 2T) or with 600 mg/kg HPE-XA-08 for 30 days (Group 3T). The hepatic biochemical activities in normal and cirrhotic rats were evaluated before and after the treatment regime. The hepatic enzyme levels of ALT, AST, ALP, and GGT were measured (Table
Blood enzymes related to liver damage (ALT, AST, ALP, and GGT) for observation of curative activity conferred by HPE-XA-08.
Liver enzyme | Normal rat (Group 1T) | Cirrhotic rat | ||||
---|---|---|---|---|---|---|
Treated with distilled water (Group 2T) | Treated with 600 mg/kg HPE-XA-08 (Group 3T) | |||||
Concentration of liver enzyme (before treatment; IU/L) | Concentration of liver enzyme (after treatment; IU/L) | Concentration of liver enzyme (before treatment; IU/L) | Concentration of liver enzyme (after treatment; IU/L) | Concentration of liver enzyme (before treatment; IU/L) | Concentration of liver enzyme (after treatment; IU/L) | |
Alanine aminotransferase (ALT) | 77 ± 1.528 | 64 ± 0.318 | 62 ± 0.636 | 75 ± 0.577 | 63 ± 1.202 | 69 ± 0.441 |
Aspartate aminotransferase (AST) | 203 ± 3.512 | 184 ± 1.856 | 180 ± 1.155 | 175 ± 0.667 | 179 ± 0.882 | 158 ± 0.882 |
Alkaline phosphatase (ALP) | 198 ± 1.856 | 96 ± 1.155 | 368 ± 1.155 | 126 ± 0.667 | 368 ± 0.882 | 102 ± 2.333 |
G-Glutamyl transferase (GGT) | 2 ± 0.333 | 1 ± 0.067 | 38 ± 0.333 | 25 ± 0.577 | 39 ± 0.296 | 15 ± 0.441 |
Macroscopic analysis of cirrhotic liver following treatment regime. Liver cirrhosis was induced by TAA for 12 weeks. After 12 weeks, the TAA was discontinued and rats were treated with either water (b) or HPE-XA-08 (c). Normal rat was included as negative control and treated with water (a).
Hepatic cirrhosis, a consequence of hepatic fibrosis, is characterized by exaggerated production of extracellular matrix properties (ECM) [
In the present study, liver damage is manifested by increases in serum ALP, GGT, and bilirubin levels. TAA-induced liver cirrhosis, however, was usually not characterized by high serum ALT and AST, unlike those intoxicated by CCl4 and paracetamol [
Liver fibrosis and cirrhosis were earlier thought of as irreversible processes. However, recent clinical and experimental evidences suggest that the process can be reversed [
Most of the previous studies on the antihepatofibrotic were performed on single plant/herb extract. In this study, the hepatocellular injury and fibrosis in rats were treated orally with decoction consisting of 10 medicinal Chinese herbs, named HPE-XA-08. Other than the 5 plants/herbs within HPE-XA-08 that have been mentioned above, the remaining 4 plants/herbs in the decoction,
Alanine aminotransferase
Alkaline phosphatase
Aspartate aminotransferase
Extracellular matrix
Gamma-glutamyl transferase
Water
Intraperitoneal
Standard error of mean
Thioacetamide.
Tong-Hye Lim is the herbalist in Herbitec (M) Sdn Bhd. Sazaly AbuBakar is the Scientific Consultant for Herbitec (M) Sdn Bhd. Nor Aziyah Mat-Rahim and Nur-Asyura Nor-Amdan have no conflict of interests.
This study received grant support from Herbitec (M) Sdn Bhd. The authors also thank Herbitec (M) Sdn Bhd for providing continuous supply of HPE-XA-08 extract and Professor Dr. Mahmood Ameen Abdulla, Department of Molecular Medicine, Faculty of Medicine, University Malaya (UM), for his guidance and assistance with the animal study and histology.