EFFECT OF CORN SILK EXTRACT ON ACETAMINOPHEN INDUCED RENAL DAMAGE IN MICE.
Objective: To evaluate the protective role of Corn Silk extract on Acetaminophen induced nephrotoxicity in albino mice.
Study Design: Laboratory based randomized controlled trials.
Place and Duration of Study: The study was carried out in experimental research laboratory University of Health Sciences and Anatomy department, Lahore. The study duration was one year from February 2012 to February 2013.
Material and Methods: Twenty seven male albino mice, 6-8 weeks old weighing 30 5 gm, were used; these animals were randomly divided into three groups having nine mice in each group. Group A served as control and was given 16.6ml/kg normal saline intraperitoneally on first day of experiment and was sacrificed on 10th day of the experiment. Group B was treated with acetaminophen 600 mg/kg dissolved in 16.6 ml of normal saline intraperitoneally on 1st day of experiment and was sacrificed after 48 hours. Group C was given acetaminophen at a dose of 600 mg/kg intraperitoneally on first day of experiment and then corn silk extract was given by oral route at a dose of 400 mg/kg for next 8 days. The animals were sacrificed on 10th day of the experiment, the kidneys were removed; 3mm three tissue pieces were fixed in 10% formaline; processed and stained with HandE for histological study.
Results: It was observed on microscopic examination that Corn silk extract reduced deleterious effects of acetaminophen on tubules of kidney as evidenced by reduction of tubular vacuolation and necrosis, absence of protein casts, vascular congestion and inflammation.
Conclusion: It is concluded from current results that corn silk extract protects acetaminophen induced nephrotoxicity.
Keywords: Acetaminophen, Corn silk extract, Mice, Nephrotoxicity.
Acetaminophen, commonly known as Paracetamol, is one of the widely used over the counter drug with potent antipyretic and analgesic effects. It is the drug of choice for management of pain, fever and osteoarthritis1.
Acetaminophen inhibits prostaglandin synthesis by inhibiting cycloxygenases and exerts its antipyretic and analgesic effects2. It passes through first phase of metabolism in liver. A small fraction is oxidized by cytochrome P- 450 system resulting in production of highly reactive toxic metabolite, N- acetyl-p-benzo-quinine imine (NAPQI), which normally reacts with sulfhydryl groups in glutathione and is thereby rendered harmless3.
Different products are used to treat acetaminophen induced nephrotoxicity such as Garlic oil4, Propylthiouracil5, Harungana madagascariensis6 and Phyllantusamarus7, these works were mainly focused on the antioxidant properties of the remedies mentioned to combat the toxic effects of acetaminophen.
Corn silk comprises the long silky threads which are the stigma and styles of the maize plant that cover the corn8.The major constituents of corn silk are amines, fixed oils, saponins, pigments, resin, flavonoids, alkaloids, tannins, chlorogenic acid, phytosterols, allantoin, vitamin C, E and K9. Corn silk is traditionally used as herbal medicine which has been used in different parts of the world for the management of odema, gout, inflammatory conditions of bladder, renal stones, nephritis, diabetes mallitus and prostatitis10. It is a mild stimulant, diuretic and demulcent. It is useful in acute and chronic cystitis, in bladder irritation of uric acid and in gonorrhea. Recent studies have indicated that corn silk can reduce clotting time and blood pressure11.
Flavonoids from corn silk (FCS) have been investigated and confirmed to possess various pharmacological activities such as antihypertensive, anti-inflammatory, anti- oxidative and anti- diabetic12. Nephroprotective effect of corn silk extract alongside Gentamicin induced nephrotoxicity has also been reported13.
Damage to the kidney by acetaminophen is reported to be due to oxidative stress produced by free oxygen radicals and on promise that corn silk extract possesses strong anti oxidant properties, it is presumed that it could serve as a protective agent for acetaminophen induced nephrotoxicity. The present study were therefore designed accordingly.
MATERIAL AND METHODS
These laboratory based randomized controlled trials were conducted in experimental research laboratory, anatomy department of University of Health Sciences Lahore from Feb 2012 to Feb 2013.
Dose of acetaminophen was based on previous studies14 i.e., single dose of 600mg/kg intraperitoneally. Acetaminophen was obtained from Merck Pharmaceuticals in a powdered form. Solution of acetaminophen was prepared by dissolving 600 mg of acetaminophen in 16.6ml of 0.9% normal saline; thus each ml of solution would contain 36 mg of acetaminophen.
Corn silk extract was prepared at PCSIR Laboratory, Lahore. 100 gm of corn silk powder was swamped in 1000 ml of 80% methanol for 72 hours and the extract was made by a rotating evaporator. Prepared extract was collected, weighed and stored in refrigerator till use13.
Dose of corn silk extract was calculated 400 mg/kg/day according to formula for dose translation13.
Stock solution of the extract was prepared by dissolving 2000 mg of extract in 50 ml of distilled water; thus 40 mg of extract was present in one ml of solution.
Twenty seven male albino mice, 6-8 weeks old, weighing 30 5gm were procured from Veterinary Research Institute, Lahore. Only healthy animals were included, weighed and housed in steel cages of appropriate size. Animals were kept under controlled environment having room temperature of 22 3C, humidity (50% 10) and light and dark cycles of 12 hours each. Animals were fed on standard rodent diet and tap water ad libitum. They were allowed to acclimatize for a period of one week before starting the experiment. The animals were divided into three groups using random number table, each groups having nine mice.
The mice of group A were given 16.6 ml/kg of 0.9% normal saline solution intraperitoneally on first day of experiment and were sacrificed under anesthesia on the 10th day. The mice of groups B and C were given Acetaminophen at a dose of 600 mg/kg dissolved in 16.6 ml normal saline intraperitoneally, on first day of experiment; Group B mice were sacrificed after 48 hours while the Group C mice were given 400 mg/kg of corn silk extract orally which was dissolved in 10ml of distilled water, for next 8 days and were sacrificed on the 10th day.
All the animals were dissected and sacrificed under chloroform anesthesia. The kidneys were removed and sliced into small pieces (3mm), fixed in 10% neutral formaline for 48 hours, dehydrated in rising grades of alcohol, washed in xylene and embedded in molten paraffin wax of melting point 560C-580C. Paraffin blocks were prepared, solidified, refrigerated and fixed in the chuk of rotator microtome. 5m thick sections were obtained and stained with haematoxylin and eosin (HandE) in a usual way and examined under light microscope.
The tubules were recorded as normal (N), mild tubular necrosis (+), moderate (++) and severe (+++). The damage was considered as mild (+), when the tubules were sparsely damaged, involving less the quarter of the total number of proximal tubules (less than 25%); moderate (++), when involvement of the tubules was from 26-50%, and severe (+++) when more than half of the tubules showed necrosis and desqumation (greater than 50%). Intraluminal protein casts were also similarly graded15.
In group B, Acetaminophen exposure for hours showed degenerative changes
Table-1: Shows comparison of histological parameters between the groups.
###Group A Group B###Group C###p-
Degree of Tubular Necrosis###Absent###9 (100%)###0 (0.0%)###6 (66.7%)
###Mild less than 25%###0 (0.0%) 2 (22.2%)###3(33.3%)
###Moderate 26-50% 0 (0.0%) 7 (77.8%)###0 (0.0%)
###less than 0.001
Degree of Tubular Vacuolation###Absent###9 (100%)###0 (0.0%)###2 (22.2%)
###Mild less than 25%###0 (0.0%)###6 (66.7%)###7 (77.8%)
###Moderate 26-50%###0 (0.0%)###3 (33.3%)###0 (0.0%)
###less than 0.001
Degree of Tubular Casts###Absent###9 (100%)###0 (0.0%)###9 (100%)
###Mild less than 25%###0 (0.0%)###0 (0.0%)###0 (0.0%)
###Moderate 26-50%###0 (0.0%)###7 (77.8%)###0 (0.0%)
###Severe greater than 50%###0 (0.0%)###2 (22.2%)###0 (0.0%)
###less than 0.001
Degree###of###Interstitial Absent###9 (100%)###1(11.1%)###9 (55.6%)
Inflammation###Mild less than 25%###0 (0.0%)###1(11.1%)###0 (0.0%)
###Moderate 26-50%###0 (0.0%)###5 (55.6%)###0 (0.0%)
###Severe greater than 50%###0 (0.0%)###2 (22.2%)###0 (0.0%)###less than 0.001
Percentage###of###Vascular###Absent###8 (88.9%)###0 (0.0%)###9 (100%)
congestion###Mildless than 25%###1(11.1%)###2 (22.2%)###0 (0.0%)
###Moderate 26-50%###0 (0.0%)###5 (55.6%)###0 (0.0%)
###Severe greater than 50%###0 (0.0%)###2 (22.2%)###0 (0.0%)###less than 0.001
###Data had been analyzed by SPSS (statistical###mutually###in###proximal###convoluted###tubules
Data had been analyzed by SPSS (statistical package for social Sciences) version 18.0. Descriptive statistics were used to describe the results. To compare the morphological features between the groups, Chi-square/Fisher Exact test was applied. A p-value of less than 0.05 was considered as significant.
Examination of the cortex of the kidneys from group A showed renal corpuscles which looked like rounded structures containing glomeruli, surrounded by double layered Bowman's capsule lined by squamous epithelial cells with flattened nuclei. The cortical tubules mainly consisted of proximal and distal convoluted tubules together with collecting tubules. Proximal convoluted tubules were lined by simple cuboidal epithelium with prominent brush border and distal convoluted tubules were lined by simple cuboidal epithelium with relatively larger lumen.
Mutually in proximal convoluted tubules (PCTs) and distal convoluted tubules (DCTs); tubular necrosis with fractional loss of brush border of proximal convoluted tubules was observed. Vacuoles were seen in the cells of both proximal and distal convoluted tubules with presence of cellular debris in their lumens. Basement membrane of the tubules was intact in all the animals of group B. Vascular congestion and inflammation were evident in all animals and so were protein casts in the lumen of DCT, ascending thick limb of loop of Henle and collecting ducts (Fig-1).
In group C there was no evidence of vascular congestion, inflammation and protein casts but some of the tubules exhibited desquamation of epithelial cells. Some tubules also showed areas of patchy necrosis with loss of brush border and vacuolation implying thereby that tubular injury was not in fact fully reversed (Fig-2).
Significant difference was observed between groups in percentages of tubular necrosis (pless than 0.001), tubular vacuolation (p less than 0.001), tubular casts (pless than 0.001), interstitial inflammation (pless than 0.001) and vascular congestion (p less than 0.001) between the groups (Table-1).
In the current study it was observed that there was statistically significant tubular necrosis and vacuolation of epithelial cell in the animals of group B when compared with the animals of groups A and C. This showed that toxic dose of acetaminophen was responsible for tubular damage especially in proximal convoluted tubules after 48 hours. Roberts16 in 2008 stated that cytoplasmic granules are the first to show the evidence of degeneration process which is followed by the vacuolation. Results of current study is in accord with those reported earlier by Refaat and Mady17 who observed proximal tubular distortion and vacuolization after single intraperitoneal dose of 1000mg/kg of acetaminophen in rats.
In present study, loss of brush border was observed in animals of group B. This loss of brush border was statistically significant among the groups. This result is in accord with the Khorsandi and Orazizadeh18 who observed loss of brush border in proximal convoluted tubules after single oral dose of 500 mg/kg of acetaminophen in mouse. Brush border did not completely recover in group C as four animals of this group showed mild brush border loss. This result is comparable to that reported by Sepehri et al13 who stated that brush border did not completely repair by methanolic extract of corn silk when it was given along with gentamicin intraperitoneal injections.
Current study showed that luminal cast was present in distal convoluted tubules of group B. Gulnaz et al4 also reported many protein casts in distal convoluted tubules and in thick ascending limb of loop of Henle after single intraperitoneal dose of 1000 mg/kg acetaminophen. There was no protein cast in animals of group C of current study which showed that treatment with corn silk extract has improved the deleterious effects of acetaminophen on renal tubules. Present study also showed that there was marked interstitial vascular congestion and inflammation (lymphocytic infiltration) in the group treated with acetaminophen. Li et al14 also reported widespread renal interstitial congestion after single intraperitoneal dose of 600 mg/kg of acetaminophen to mouse. In current study, interstitial congestion and inflammation was not seen in mice of groups A and C. Extracts of corn silk and corn silk have antioxidant activities.
This antioxidant activity is stated to provide protection from the injury produced by free radicals19. Corn silk extract has recovered the interstitial congestion and inflammation in group C. The exact mechanism is unknown but it is probably because antioxidant causes vasodilatation hence it may reduce the toxicant induced congestion and renal injury20.
Antioxidant, antinflammatory and free- radical scavenger activity of corn silk explains its nephroprotective role12. Antioxidants present in corn silk extract are volatile oxygen containing chemicals, such as R-terpineol, eugenol and citronellol21. The free radical scavenging ability of these extracts is due to the existence of phenolic compounds for example eugenol, carvacrol and thymol, along with some polyphenols and avonoids22.
Present study revealed that corn silk extract administration ameliorated histopathological lesions induced by acetaminophen, vascular congestion and inflammation to almost normal, reduced deleterious effects of acetaminophen on tubules and caused noticeably decreased vacuolation. This indicates that considerable protection is provided by corn silk extract to acetaminophen nephrotoxicity.
On basis of our observations that administration of a single dose of 600mg/kg body weight of acetaminophen to mice resulted in tubular necrosis, cast formation, inflammation and congestion which disappeared upon treating the mice with corn silk extract with a dose of 400 mg/kg body weight for next 8 days, clearly indicating that corn silk protects renal injury induced by acetaminophen. Further investigations are required to explore the mechanism of action of corn silk extract and may possibly have a significant impact on future clinical treatments of patients with renal malfunction.
I would like to thank the Vice Chancellor of University of health Sciences Lahore for his moral help and providing funds for the research. I am also grateful to the Laboratory staff for their unhesitating help to support my work.
CONFLICT OF INTEREST
This study has no conflict of interest to declare by any author.
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|Publication:||Pakistan Armed Forces Medical Journal|
|Article Type:||Clinical report|
|Date:||Jun 30, 2015|
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