This work was about Andrographis peniculata (Burm. F). Ab initio , the LD 50 test showed non-toxicity at the highest administered dose of 5000 mg/kg in rats. Male albino Wistar rats were treated with daily single doses of ethanol extracts (100/200/500 mg/kg) of Andrographis peniculata ( A.p. ) for 14 days with intermittent administration, i.p. , of CCl 4 every four days. Liver and kidney TBARS concentrations showed lower values with increased doses of extract administration. In both cases, “CCl 4 A.p. 500 mg/kg” values compared to “CCl 4 Silymarin ” group, while “CCl 4 A.p. 100 mg/kg” showed no significant difference from “CCl 4 only” group. The “NC” (normal control) however, presented the least concentration of 66.17 ± 2.74 and 38.04 ± 4.34 nmol/mg protein, respectively. Total and Indirect bilirubin concentrations indicated decreased values with increasing doses, such that respectively, the lowest values of 1.18 ± 0.47 and 0.98 ± 0.31 mg/dl in the “CCl 4 A.p. 500 mg/kg” group were observed. There was no significant difference among all the various groups except the “NC” which showed the least value. Urea and creatinine levels were significantly higher (p ≤ 0.05) in the “CCl 4 only” group than all others. Liver function parameters, viz., AST and ALT indicated significantly higher values in the “CCl 4 only” group, compared to all others (p ≤ 0.05). Values obtained for “CCl 4 A.p. (500 mg/kg)” were comparable to the “NC” and “CCl 4 Silymarin ” groups.
Free radicals are organic molecules responsible for aging, tissue damage, and possibly so many diseases. These molecules, are very unstable, therefore they look to bond with other molecules, destroying their health and further continuing the damage processes. Antioxidants present in many foods are molecules that prevent free radicals from harming healthy tissues.
Generally, free radicals attack the nearest stable molecule, “stealing” its electron. When the “attacked” molecule loses its electron, it becomes a free radical itself, beginning a chain reaction. Once the process is started, it can cascade, finally resulting in the disruption of a living cell. Some free radicals arise normally during metabolism. Sometimes the body’s immune system purposefully creates them to neutralize viruses and bacteria. However, environmental factors such as pollution, radiation, cigarette smoke and herbicides can also spawn free radicals. Normally, the body can handle free radicals but if antioxidants are unavailable, or if the free radical production becomes excessive, damage can occur. Of particular importance is that free radical damage accumulates with age.
Antioxidants work to protect lipids from peroxidation by radicals. Antioxidants are effective because they are willing to give up their own electrons to free radicals. When a free radical gains the electron from an antioxidant, it no longer needs to attack the cell and the chain reaction of oxidation is broken [
Andrographis paniculata (Burm. F.) Nees, commonly known as the king of bitters, it is an herbaceous plant belonging to the Acanthaceae family and is found throughout tropical and subtropical Asia, Southeast Asia, and India. It grows abundantly in south eastern Asia, including India, SriLanka, Pakistan, Java, Malaysia, and Indonesia, while it is cultivated extensively in India, China, and Thailand [
Liver and kidney disorders are some of the world’s major health problems [
Petroleum ether, ethanol, carbon tetrachloride (CCl4), silymarin were obtained from Sigma Chemicals Limited, USA. Assay kits for bilirubin, total protein, total cholesterol, triglyceride, creatinine, urea, AST and ALT were obtained from RANDOX Laboratories Ltd., Ardmore, Diamond Road, Crumlin, Co. Antrim, United Kingdom.
Spectrophotometer (6405 UV/VIS Jenway laboratory equipment, Japan); Soxhlet extractor (500 ml capacity, Tech-Lab scientific sdn. bath model EB-6.SKU); Centrifug (Labofuge 300 Heraeus, UK); Tissue homogenizer (Eberbach lab tools-Model 7000); rotary evaporator and oral cannula.
Plant leaves were obtained from cultivated fields of Bingham University, Nigeria. The plant was identified at National Institute for Pharmaceuticals and Research Development (NIPRD) Abuja by comparison with the voucher specimen number NIPRD/H/3720.
Apparently healthy male albino Wistar rats weighing between 160 - 220 g obtained from the animal house of College of Medicine, Bingham University Nasarawa state, Nigeria were used for the study. They were fed ad libithum with commercial rat feed pellets obtained from vital feed depot Gizzard Plaza, Mararaba, Nasarawa state Nigeria, and allowed clean drinking water. Rats were divided into six groups of five rats per group and treated as shown below:
Rats were treated over a period of 14 days and sacrificed under chloroform anesthesia by decapitation the next day after being fasted overnight. CCl4 only group was administered i.p. with 0.012 ml/kg CCl4 every 4 days. Standard (Silymarin) and extract groups were orally administered with 0.0025 ml/kg Silymarin or A.P. (100, 200 and 500 mg/kg) daily. However, every successive 4th day from commencement of experiment, 0.012 ml/kg CCl4 was administered 1hr after Silymarin or A.p. administration. NC group received no treatment.
Blood samples for determination of sero-biochemical parameters were immediately collected. At necropsy, all rats were examined to identify lesions, and the specimen of the liver and kidney were quickly removed and fixed in 10% formol-saline for histopathology studies.
This was carried out by collecting fresh plants and drying under shade then pulverized using mortar and pestle. This was followed by de-fatting with petroleum ether. Upon drying, powders were extracted with ethanol then concentrated with rotary evaporator at 60˚C. The pasty extracts were evaporated further over water bath at 45˚C then transferred to desiccators containing activated silica gel until fully dry, then quantified [
This was done by allowing collected blood to stand for 1 hour to clot then centrifuged at 1000 × g for 15 min to separate the serum which was collected and stored in a freezer. The homogenates of liver and kidney portions were removed, blotted and weighed then processed for the relevant assays. These procedures have been previously described [
LD50 was determined using method described elsewhere [
Finally, histological examination was further carried out after the animals were sacrificed by cervical decapitation. The liver tissues were excised, trimmed of fat and other connective tissues then, blotted dry and weighed on a balance. Sections of the liver from the autopsy samples were stored in 10% formalin and transferred to the Department of Pathology, Ahmadu Bello University teaching hospital Zaria for assessment. Slides were prepared, observed under the light microscope and photomicrographs were obtained.
Data were presented as mean ± SD of six determinations. The significance of difference was evaluated by ANOVA and Duncan Multiple Range test, with confidence limit set at 0.05 (95%). SPSS version 16.0 and Microsoft excel (Windows 7) were used as analytical tools.
As seen in
There were significant increase (p ≤ 0.05) in TBARS, creatinine and urea concentrations in the kidney and liver of the “CCl4 only” administered group compared to the NC and all the other groups except the “CCl4 + A.p. 100 mg/kg” group (
As depicted in
In
Treatment Group | % change in body weight | % change in liver/ body weight ratio | % change in kidney/body weight ratio |
---|---|---|---|
Normal Control (NC) | 4.30 ± 1.45b | 3.43 ± 0.15b | 0.34 ± 0.07b |
CCl4 only | −6.05 ± 1.60a | 2.59 ± 0.39ac | 0.27 ± 0.07c |
CCl4 + silymarin | −3.29 ± 1.48c | 2.92 ± 0.94c | 0.26 ± 0.12c |
CCl4 + A.p. 100 mg/kg | −2.18 ± 0.09cd | 2.63 ± 0.74c | 0.23 ± 0.08a |
CCl4 + A.p. 200 mg/kg | −2.68 ± 0.25c | 3.37 ± 0.32b | 0.32 ± 0.02b |
CCl4 + A.p. 500 mg/kg | −1.68 ± 0.39d | 3.51 ± 0.11b | 0.36 ± 0.04b |
Values are expressed as mean ± SD, (n = 5). No significant difference between values bearing same alphabets (p ≤ 0.05).
Treatment Group | TBARS (nmol/mg protein) | Total protein (nmol/mg) | Creatinine (nmol/mg protein) | Urea (mg/dl) | |
---|---|---|---|---|---|
Liver | Kidney | ||||
Normal Control | 66.17 ± 2.74a | 38.04 ± 4.34ac | 36.40 ± 2.61d | 1.8 ± 0.62c | 41.46 ± 6.92d |
CCl4 only | 94.79 ± 1.16b | 46.96 ± 4.47b | 27.36 ± 3.17ac | 3.64 ± 0.36b | 50.61 ± 7.47b |
CCl4 + silymarin | 74.62 ± 2.69c | 41.98 ± 4.60c | 29.98 ± 1.85c | 2.88 ± 0.72d | 34.55 ± 6.91e |
CCl4 + A.p. 100 mg/kg | 93.40 ± 1.31b | 45.55 ± 3.91b | 44.07 ± 1.60b | 3.01 ± 0.91bd | 41.47 ± 9.76d |
CCl4 + A.p. 200 mg/kg | 86.59 ± 2.03d | 41.29 ± 4.51c | 39.68 ± 2.66d | 1.68 ± 0.55c | 20.70 ± 6.60ac |
CCl4 + A.p. 500 mg/kg | 75.99 ± 1.46c | 39.42 ± 3.00c | 31.91 ± 1.41c | 1.32 ± 0.42a | 18.24 ± 3.98a |
Values are expressed as mean ± SD, (n = 5). No significant difference between values bearing same superscripts (p ≤ 0.05).
Treatment Group | Concentration of liver enzymes (u/l) | Bilirubin concentration (mg/dl) | |||
---|---|---|---|---|---|
AST | ALT | Direct | Indirect | Total | |
Normal Control | 16.49 ± 1.98c | 136.96 ± 9.92c | 0.12 ± 0.07a | 2.47 ± 0.39d | 2.58 ± 0.34c |
CCl4 only | 26.06 ± 1.14b | 158.53 ± 4.70b | 0.24 ± 0.02b | 3.11 ± 0.56c | 3.34 ± 0.54b |
CCl4 + silymarin | 17.71 ± 3.65c | 142.53 ± 4.87c | 0.20 ± 0.02d | 2.24 ± 0.54d | 2.44 ± 0.56c |
CCl4 + A.p. 100 mg/kg | 18.00 ± 2.71c | 151.28 ± 8.84c | 0.23 ± 0.01c | 3.35 ± 0.25bc | 3.59 ± 0.24b |
CCl4 + A.p. 200 mg/kg | 17.36 ± 1.97c | 147.46 ± 6.48c | 0.24 ± 0.01bc | 2.81 ± 0.97cd | 3.04 ± 0.96bc |
CCl4 + A.p. 500 mg/kg | 16.41 ± 1.85ac | 128.49 ± 3.05ac | 0.20 ± 0.02d | 0.98 ± 0.31a | 1.18 ± 0.47a |
Values are expressed as mean ± SD, (n = 5). No significant difference between values bearing same alphabets (p ≤ 0.05).
Treatment Group | Lipid profile | |||
---|---|---|---|---|
HDL (mg/dl) | LDL (mg/dl) | TG (mg/dl) | TC (U/I) | |
Normal Control | 38.82 ± 0.79c | 36.67 ± 1.51c | 75.22 ± 0.16a | 41.72 ± 0.31d |
CCl4 only | 27.11 ± 1.12b | 66.97 ± 0.47a | 157.42 ± 0.08d | 97.11 ± 0.51a |
CCl4 + silymarin | 37.51 ± 1.05c | 38.78 ± 0.28c | 82.08 ± 0.10b | 32.24 ± 0.14d |
CCl4 + A.p. 100 mg/kg | 33.08 ± 2.51c | 44.21 ± 0.84b | 112.22 ± 0.20c | 53.35 ± 0.21c |
CCl4 + A.p. 200 mg/kg | 36.36 ± 0.98c | 39.46 ± 1.40c | 93.43 ± 0.08b | 42.18 ± 0.17d |
CCl4 + A.p. 500 mg/kg | 40.43 ± 1.45c | 35.01 ± 1.05c | 78.02 ± 0.12a | 36.97 ± 0.64d |
Values are expressed as mean ± SD, (n = 5). No significant difference between values bearing same alphabets in the same column (p ≤ 0.05).
than the “NC” and all other groups except in HDL concentration where the least concentration was recorded.
Statistically significant increases in LDL, TG and TC among the “CCl4 only” rats group indicated hyperlipidemic and hypercholesterolemic effects of the hepatotoxin, while the decreased concentration values with increased doses of the extract showed the antihyperlipidemic and antihypercholesterolemic effects of A.p. extract in a dose-dependent manner. On the contrary, an observed increase in HDL among all the rat groups treated with A.p. after CCl4 administrations was equally indicative of the antihyperlipidemic effect of the plant extracts. HDL concentration in the “CCl4 only” group was significantly low due to the effect of CCl4.
Hisptopathological slides have shown the protective capabilities of A.p. ( Plate 1(F) ) especially at 500 mg/kg comparable to silymarin ( Plate 1(C) ) even though at a lower dose of 200 mg/kg ( Plate 1(E) ) where there were evidences of liver tissue regeneration. Furthermore, ( Plate 2(B) ) indicated a similar pattern with respect to the protective effect of A.p. against kidney tissue damage as a result of CCl4 administrations. Regeneration of cells of the damaged tissue seemed to be in a dose-dependent manner ( Plates 2(D)-(F) ).
Herbal medicines derived from plant extracts are gaining greater attention due to their effectiveness as chemo-therapeutic agents. As such there has been increased utilization of these herbs in treating a wide variety of diseases and the use of herbal medicines have continued to expand rapidly across the world. In fact, vast numbers of people now solely rely on herbal medicines or herbal products for their primary health care requirements
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The present study was undertaken to demonstrate the free radical scavenging abilities of ethanolic leaf extracts of Andrographis peniculata against induced liver damage by CCl4 and the toxic effects of similar doses in rats. CCl4 is one of the most widely used toxicants for experimental induction of liver damage in laboratory animals [
Plate 1. Photomicrographs of liver sections of CCl4-induced hepatotoxicity in rats pre-administered with varying doses Andrographis peniculata ethanol leaf extracts ((H) & (E) stain 250×), showing (A) Untreated, (B) Treated with CCl4 only, (C)-(F) Treated with: Silymarin, 100, 200 and 500 mg/kg of extract after CCl4 administration, respectively.
Plate 2. Photomicrographs of kidney sections of CCl4-induced hepatotoxicity in rats pre-administered with varying doses Andrographis peniculata ethanol leaf extracts ((H) & (E) stain ×250), showing (A) Untreated, (B) Treated with CCl4 only, (C)-(F) Treated with: Silymarin, 100, 200 and 500 mg/kg of extract after CCl4 administration, respectively.
trichloromethyl free radical (▪CCl3). Trichloromethyl free radical when combined with cellular lipids and proteins in the presence of oxygen form trichloromethylperoxyl radical, which attacks lipids on the membrane of endoplasmic reticulum faster than a trichloromethyl free radical. Thus, trichloromethylperoxyl free radical elicits lipid peroxidation, eventual cell necrosis and finally resulting to cell death [
Intra-peritoneal administration of CCl4 caused significant liver damage as evidenced by altered biochemical parameters in the findings here. There was a significant reduction in body weight of the “CCl4 only” group as compared to the various extract treated groups, silymarin treated group and the normal control group. CCl4 significantly (p < 0.05) increased serum levels of ALT, AST, urea, creatinine, total, direct and indirect bilirubin levels, and also lipidemic parameters, namely, low density lipoprotein, triglycerides and total cholesterol. Treatment with the ethanolic leaf extract of Andrographis peniculata attenuated the elevated enzyme levels towards normal ranges. The hepatoprotective and nephroprotective efficacies of the extract were comparable to that of the standard drug-silymarin, used in the experiment. These effects against CCl4 intoxication was further confirmed by histopathological examinations. The liver samples of “CCl4 only” administered rats showed cell vacuolation, necrosis and degeneration of nuclei and bile capillaries. However, in the extract-treated groups, mild degenerative changes and marked recovery from necrosis and degeneration of bile capillaries in the liver samples were evident. Also, kidney samples showed necrosis in the “CCl4 only” administered rats whereas, these tissues were preserved with well regenerated glomerulus and tubules as a result of the A.p. extracts treatments.
The results of this study have clearly demonstrated the antiperoxidative, hepatoprotective and nephroprotective properties of Andrographis peniculata which were likely as a result of the free radical scavenging properties of the extract, equally shown to be safe even at very high dose.
Godwin O. Adejo,Joseph M. Gnimintakpa,Olufunsho D. Olowoniyi,Paulinus Obinna Matthew, (2016) Andrographis peniculata: Capabilities against Free Radicals, Lipid Peroxidation, Hepatotoxicity, and Nephrotoxicity. Open Access Library Journal,03,1-9. doi: 10.4236/oalib.1102541