paracetamol belongs to the class of medications called analgesic ( pain reliever ) and antipyretic ( fever reducer ) and is widely used as an over - the - counter ( otc ) pain and fever medication to relieve mild to moderate pain from head , muscle , menstrual period , cold , sore throat , tooth , reactions to vaccination ( shot ) , in osteoarthritis ( arthritis caused by the breakdown of the lining of the joints ) and to reduce fever .
paracetamol is known to cause liver and renal injury in humans and experimental animals when administered in higher and repeated doses .
the incidences associated with liver injury include abdominal pain , nausea , vomiting , low blood sugar , low blood ph , easy bleeding , oliguria ( decreased output of urine ) and hepatic encephalopathy .
the excess use of paracetamol leads to liver and renal failure , and even causes death .
the mechanism of paracetamol toxicity in the liver is well described , but in the kidney it is less clearly understood .
paracetamol is primarily metabolized and excreted as paracetamol  glucuronide and paracetamol  so4 in the hepatocytes .
when paracetamol is taken at a higher dose ( larger amounts of paracetamol are bioactivated ) , it produces a reactive metabolite n - acetyl - p - benzoquinone imine ( napqi ) , generated by several hepatic cytochrome p-450 isoenzymes .
glutathione ( gsh ) can reduce napqi , recycling to paracetamol , or conjugate the napqi forming paracetamol 
generation of the reactive oxygen species has appeared as an early event that is preceded by intracellular gsh depletion .
depletion of gsh level permits the protein thiolarylation by napqi that may compromise the protein function and contribute to paracetamol , leading to hepatocellular damage .
paracetamol - induced renal insufficiency depends on the drug , dose , duration of pharmacological effect and the health of the patient .
overdose of paracetamol causes various adverse effects in the kidney , which may partly be due to the vasoconstriction resulting in inhibition of renal prostaglandin - mediated vasodilatation , decreasing renal blood flow and finally decreasing the glomerular filtration rate .
oxidative stress , with decline of gsh and lipid peroxidation , has also been suggested as a mechanism by which paracetamol could induce renal injury .
triphala is the combination ( equal proportion 1:1:1 ) of dried fruits ( phala ) of amalaki ( embelica officinalis ) , bibhitaki ( terminalia bellirica ) and haritaki ( terminalia chebula ) . as per ayurveda , in the indian system of medicine
, it is commonly used to balance the three doshas ( humors ) in the body , viz .
traditionally , the triphala formulation has been used as a laxative in chronic constipation , as a detoxifying agent of the colon , and as a rejuvenator of the body . according to recent researches
, the triphala formulation also shows antidiabetic and hepatoprotective activities and plays an important role in blood pressure control and balances cholesterol .
triphala is an important medicine in rasayana therapy that preserves and promotes health , immunity and longevity .
the individual plants of triphala have been reported to be a rich source of vitamin c , ellagic acid , gallic acid , chebulinic acid , bellericanin , -sitosterol and flavanoids , which possess several other health benefits as well .
these herbs are also reported for their anti - inflammatory , anti - mutagenic , antioxidant , cytoprotective , gastroprotective , anti - cancer activity and anti - bacterial effects .
renal toxicity has not yet been reported till date to the best of our knowledge .
this study was , therefore , undertaken to examine the protective effect of triphala against paracetamol- induced hepatic 
potassium dihydrogen phosphate , dibasic monohydrogen phosphate , formaldehyde , paraffin wax , xylene and biochemical kits were purchased from merck chemicals private ltd .
dried fruits of amalaki ( emblica officinalis ) , bibhitaki ( terminalia bellirica ) and haritaki ( terminalia chebula ) were collected from the farm of csir - cimap , lucknow and authenticated by an ayurvedic physician at the hmpd , csir - cimap lucknow , india .
a voucher specimen of amalaki ( emblica officinalis ) ( hmp101 ) , bibhitaki ( terminalia bellirica ) ( hmp102 ) and haritaki ( terminalia chebula ) ( hmp103 ) was deposited in the departmental herbarium of the csir - cimap , lucknow , up , india . dried fruits of amalaki , bibhitaki and haritaki
the dried powder of all the three fruits was mixed in a defined ratio of 1:1:1 and the mixture was extracted with distilled water by using a cold maceration process .
one hundred grams of the above mixture was taken and soaked overnight in 400 ml of distilled water .
the extract was filtered using a filter paper ( whatman filter paper grade 1 ) and then the extract was concentrated in a rotary evaporator ( buchi r-210 , switzerland ) and freeze - dried under vacuum for 50 h using the lobconco freeze drying system ( 42c to 47c temperature with 0.340 mbar pressure ) .
completely dried material was collected and stored in a closed air - tight container at room temperature until use .
swiss albino mice ( weight 2025 g ) were maintained in an air conditioned room ( 22  3c ) with relative humidity of 50  5% and 12-h light/12-h dark cycle and acclimatized for 1 week prior to the study .
they were fed with a certified pellet rodent diet ( dayal industries , lucknow , india ) and water was provided ad libitum .
the institutional animal ethics committee of csir - cimap approved the protocol of the experiment ( ah-2012 - 10 ) .
the guidelines for animal care were followed as recommended by the committee for the purpose of control and supervision of experiments on animals ( cpcsea ) , government of india .
group 1 : ( vehicle control ) animals received distilled water for 7 days and served as normal controlsgroup 2 : ( paracetamol ) animals in this group received ( only the paracetamol challenge ) distilled watergroup 3 : ( liv-52 ) positive control group , pre - treated with liv-52 at 5.2 ml / kg body weight ( bw ) for 7 daysgroup 4 and group 5 : served as the treatment groups and pre - treated with triphala extract at 100 mg / kg and 300 mg / kg body weight for 7 days . 
 
group 1 : ( vehicle control ) animals received distilled water for 7 days and served as normal controls group 2 : ( paracetamol ) animals in this group received ( only the paracetamol challenge ) distilled water group 3 : ( liv-52 ) positive control group , pre - treated with liv-52 at 5.2 ml / kg body weight ( bw ) for 7 days group 4 and group 5 : served as the treatment groups and pre - treated with triphala extract at 100 mg / kg and 300 mg / kg body weight for 7 days . on the 8 day , all the groups except group 1 were given an intraperitoneal injection of paracetamol at 500 mg / kg bw .
after 2.5 h of paracetamol administration , 500 l of blood was collected from the retro - orbital plexus of the mice using non - heparinised hematocrit capillaries . the serum was separated and stored at -20c for experimental analysis .
the collected blood samples were used for the analysis of different biochemical parameters , viz .
aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , alkaline phosphatase ( alp ) , bilirubin , creatinine , urea and uric acid , by using standard diagnostic kits as per the manufacturer 's protocol ( labkit from merck chemicals private ltd . , india ) .
after blood collection , animals were sacrificed and their liver was dissected out and washed with phosphate buffer ( ph 7.4 ) for complete removal of blood stains and clot .
after washing , tissue was fixed in 10% formalin and embedded in molten paraffin wax followed by cutting of sections with microtome .
deparaffinized sections were then stained with hematoxylin and eosin and observed microscopically for histopathological changes .
all the statistical analyses were performed with the help of graph pad instat software ( version 3.0 ) .
difference between means was calculated by one - way analysis of variance ( anova ) followed by the student  newman 
potassium dihydrogen phosphate , dibasic monohydrogen phosphate , formaldehyde , paraffin wax , xylene and biochemical kits were purchased from merck chemicals private ltd .
dried fruits of amalaki ( emblica officinalis ) , bibhitaki ( terminalia bellirica ) and haritaki ( terminalia chebula ) were collected from the farm of csir - cimap , lucknow and authenticated by an ayurvedic physician at the hmpd , csir - cimap lucknow , india .
a voucher specimen of amalaki ( emblica officinalis ) ( hmp101 ) , bibhitaki ( terminalia bellirica ) ( hmp102 ) and haritaki ( terminalia chebula ) ( hmp103 ) was deposited in the departmental herbarium of the csir - cimap , lucknow , up , india .
dried fruits of amalaki , bibhitaki and haritaki were collected and powdered using a mill .
the dried powder of all the three fruits was mixed in a defined ratio of 1:1:1 and the mixture was extracted with distilled water by using a cold maceration process .
one hundred grams of the above mixture was taken and soaked overnight in 400 ml of distilled water .
the extract was filtered using a filter paper ( whatman filter paper grade 1 ) and then the extract was concentrated in a rotary evaporator ( buchi r-210 , switzerland ) and freeze - dried under vacuum for 50 h using the lobconco freeze drying system ( 42c to 47c temperature with 0.340 mbar pressure ) .
completely dried material was collected and stored in a closed air - tight container at room temperature until use .
swiss albino mice ( weight 2025 g ) were maintained in an air conditioned room ( 22  3c ) with relative humidity of 50  5% and 12-h light/12-h dark cycle and acclimatized for 1 week prior to the study .
they were fed with a certified pellet rodent diet ( dayal industries , lucknow , india ) and water was provided ad libitum .
the institutional animal ethics committee of csir - cimap approved the protocol of the experiment ( ah-2012 - 10 ) .
the guidelines for animal care were followed as recommended by the committee for the purpose of control and supervision of experiments on animals ( cpcsea ) , government of india .
group 1 : ( vehicle control ) animals received distilled water for 7 days and served as normal controlsgroup 2 : ( paracetamol ) animals in this group received ( only the paracetamol challenge ) distilled watergroup 3 : ( liv-52 ) positive control group , pre - treated with liv-52 at 5.2 ml / kg body weight ( bw ) for 7 daysgroup 4 and group 5 : served as the treatment groups and pre - treated with triphala extract at 100 mg / kg and 300 mg / kg body weight for 7 days . 
 
group 1 : ( vehicle control ) animals received distilled water for 7 days and served as normal controls group 2 : ( paracetamol ) animals in this group received ( only the paracetamol challenge ) distilled water group 3 : ( liv-52 ) positive control group , pre - treated with liv-52 at 5.2 ml / kg body weight ( bw ) for 7 days group 4 and group 5 : served as the treatment groups and pre - treated with triphala extract at 100 mg / kg and 300 mg / kg body weight for 7 days . on the 8 day , all the groups except group 1 were given an intraperitoneal injection of paracetamol at 500 mg / kg bw .
after 2.5 h of paracetamol administration , 500 l of blood was collected from the retro - orbital plexus of the mice using non - heparinised hematocrit capillaries . the serum was separated and stored at -20c for experimental analysis .
the collected blood samples were used for the analysis of different biochemical parameters , viz .
aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , alkaline phosphatase ( alp ) , bilirubin , creatinine , urea and uric acid , by using standard diagnostic kits as per the manufacturer 's protocol ( labkit from merck chemicals private ltd . , india ) .
after blood collection , animals were sacrificed and their liver was dissected out and washed with phosphate buffer ( ph 7.4 ) for complete removal of blood stains and clot .
after washing , tissue was fixed in 10% formalin and embedded in molten paraffin wax followed by cutting of sections with microtome .
deparaffinized sections were then stained with hematoxylin and eosin and observed microscopically for histopathological changes .
all the statistical analyses were performed with the help of graph pad instat software ( version 3.0 ) .
difference between means was calculated by one - way analysis of variance ( anova ) followed by the student  newman 
intraperitonial ( ip ) administration of paracetamol in swissalbino mice ( group 2 ) at the dose of 500 mg / kg bw produced a significant ( p < 0.001 ) increase in the serum levels of ast , alt , alp and bilirubin , while pre - treatment with the liv-52 syrup ( 5.2 ml / kg body weight ) ( group 3 ) and triphala extract at the rate of 100 mg / kg and 300 mg / kg bw ( groups 3 and 4 ) significantly ( p < 0.01 ) prevented the elevation in these liver enzymes [ table 1 , figure 1 ] .
similarly , the serum levels of creatinine , urea and uric acid were increased after paracetamol administration , which were significantly ( p < 0.001 ) different from the vehicle control group .
pre - treatment with the triphala extract at the above - mentioned dose and liv-52 syrup effectively ( p < 0.01 ) prevented the increase in the serum levels of these renal function tests [ table 1 , figure 1 ] .
the effect of triphala extract on liver function tests ( ast , alt , alp and bilirubin ) and renal function tests ( creatinine , urea and uric acid ) shows its effect in a dose - dependent manner .
the triphala extract exhibited more protective effects at 300 mg / kg bw than 100 mg / kg bw [ table 1 and figure 1 ] .
effect of triphala rasayana on liver and kidney bio - markers for assessing hepatic - renal toxicity in swiss albino mice effect of triphala extract on different groups of mice against paracetamol - induced hepatic 
the effect of pre - treatment with triphala extract at 100 mg / kg and 300 mg / kg body weight dose on ast , alt , alp , bilirubin , creatinine , urea and uric acid of mice in paracetamol - induced liver and renal toxicity . * p < 0.05 , * * p < 0.01 , * * * p <
0.001 , paracetamol compared with the vehicle control group , p < 0.05 , p < 0.01 , p < 0.001 , liv-52 and treatment groups 4 and 5 compared with the paracetamol group ( n = 6 mice / group ) histopathological observation of liver tissues showed that there were no abnormal morphological changes in the liver specimens of the control group [ figure 2a ] .
the liver samples of the paracetamol group showed necrosis , phagocyte filtration and fat degeneration [ figure 2b ] .
the liver tissues in the groups pre - treated with liv-52 syrup and triphala extract protects the liver cells , central vein and portal vein from paracetamol toxicity and maintains the normal architecture of the liver tissues [ figure 2c  e ] .
control group ( a ) mice showing normal histological architecture and chords of hepatocytes are radiating .
toxin ( paracetamol)-induced group ( b ) showing centri - zonal necrotic areas and degenerated liver tissues .
liv-52 ( 5.2 ml / kg body weight ) and triphala   extract at 100 mg / kg and 300 mg / kg body weight ( c , d and e ) showing protection of hepatocytes with mild changes and pyknotic nuclei around the central vein indicating necrotic changes
intraperitonial ( ip ) administration of paracetamol in swissalbino mice ( group 2 ) at the dose of 500 mg / kg bw produced a significant ( p < 0.001 ) increase in the serum levels of ast , alt , alp and bilirubin , while pre - treatment with the liv-52 syrup ( 5.2 ml / kg body weight ) ( group 3 ) and triphala extract at the rate of 100 mg / kg and 300 mg / kg bw ( groups 3 and 4 ) significantly ( p < 0.01 ) prevented the elevation in these liver enzymes [ table 1 , figure 1 ] .
similarly , the serum levels of creatinine , urea and uric acid were increased after paracetamol administration , which were significantly ( p < 0.001 ) different from the vehicle control group .
pre - treatment with the triphala extract at the above - mentioned dose and liv-52 syrup effectively ( p < 0.01 ) prevented the increase in the serum levels of these renal function tests [ table 1 , figure 1 ] .
the effect of triphala extract on liver function tests ( ast , alt , alp and bilirubin ) and renal function tests ( creatinine , urea and uric acid ) shows its effect in a dose - dependent manner .
the triphala extract exhibited more protective effects at 300 mg / kg bw than 100 mg / kg bw [ table 1 and figure 1 ] .
effect of triphala rasayana on liver and kidney bio - markers for assessing hepatic - renal toxicity in swiss albino mice effect of triphala extract on different groups of mice against paracetamol - induced hepatic 
the effect of pre - treatment with triphala extract at 100 mg / kg and 300 mg / kg body weight dose on ast , alt , alp , bilirubin , creatinine , urea and uric acid of mice in paracetamol - induced liver and renal toxicity . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 , paracetamol compared with the vehicle control group , p
< 0.05 , p < 0.01 , p < 0.001 , liv-52 and treatment groups 4 and 5 compared with the paracetamol group ( n = 6 mice / group )
histopathological observation of liver tissues showed that there were no abnormal morphological changes in the liver specimens of the control group [ figure 2a ] .
the liver samples of the paracetamol group showed necrosis , phagocyte filtration and fat degeneration [ figure 2b ] .
the liver tissues in the groups pre - treated with liv-52 syrup and triphala extract protects the liver cells , central vein and portal vein from paracetamol toxicity and maintains the normal architecture of the liver tissues [ figure 2c  e ] .
control group ( a ) mice showing normal histological architecture and chords of hepatocytes are radiating .
toxin ( paracetamol)-induced group ( b ) showing centri - zonal necrotic areas and degenerated liver tissues .
liv-52 ( 5.2 ml / kg body weight ) and triphala   extract at 100 mg / kg and 300 mg / kg body weight ( c , d and e ) showing protection of hepatocytes with mild changes and pyknotic nuclei around the central vein indicating necrotic changes
the liver is a multipurpose organ in the body related with regulation of various metabolic pathways .
therefore , damage of the liver tissues caused by a hepatotoxic agent is a serious consequence .
paracetamol is the most commonly used analgesic and antipyretic drug known to possess a hepatotoxic effect in humans as well as in animals at high doses .
it is generally observed that drug- or chemical - induced liver and kidney injuries are associated with the overproduction of free radicals .
parallel to this , renal insufficiency occurs in around 12% of patients with paracetamol overdose .
thus , prevention and treatment of liver injury , together with renal protection , can be a good line of treatment in liver diseases .
herbal medicines have been used since antiquity and have played a pivotal role in the prevention and treatment of various diseases .
a large number of herbs and herbal formulations are used in the indian medicinal systems , viz .
ayurveda , siddha , unani and amchi , etc . , according to the world health organization ( who ) , 80% of the people throughout the world currently use herbal medicines for primary health care , as well as most of the modern plant - derived pharmaceutical medicines are used in ways that correlated directly with their traditional uses .
triphala is considered as one of the most important rasayana formulations in the indian system of medicine ( ism ) . in this current research work ,
triphala extract was screened for its possible hepato  renal protection in mice against paracetamol - induced toxicity . in the present study ,
paracetamol - induced hepatotoxicity was evidenced by the biochemical measurements and histopathological changes , while renal damage was indicated by rise in serum levels of renal markers . the increased level of serum ast , alt , alp , bilirubin , creatinine , urea and uric acid in mice treated with paracetamol indicated altered liver and renal functions due to its toxic effects .
pre - treatment of mice with triphala extract for seven consecutive days resulted in a significant protection of paracetamol - induced elevation of serum enzyme markers ( ast , alt , alp , bilirubin , creatinine , urea and uric acid ) given in two doses when compared with the paracetamol group [ figure 1 ] .
the triphala extract significantly reduced the elevated serum enzyme levels in the treatment group , which indicated the ability of triphala to preserve the normal functions of the liver and kidney .
it can be proposed that triphala inhibited the free radical generation and resultant damage to maintain the normal functions of the liver and renal cellular membrane ; [ figure 1 ] although the exact mechanism of action needs further investigation .
the hepatoprotective effect of the triphala extract was further confirmed by histopathological studies of the liver tissues , which basically supported the results obtained from the serum assays . moreover , as revealed by the histopathological study , the triphala extract could provide protection against paracetamol - mediated hepatic alterations by membrane regeneration and , therefore , it may be helpful in limiting the drug - induced membranal damage in the liver and kidney .
furthermore , in our study , the results clearly show that the triphala extract at the dose of 300 mg / kg bw offers more protection to liver tissues that were comparable to liv-52 , but in the case of 100 mg / kg bw , it offers comparatively less protection than liv-52 against paracetamol - induced hepato  renal injury .
however , a more elaborative study with special focus on the effects of triphala in maintaining gsh levels and possible measurement of naqpi using n aceyl cysteine as a positive control is still required .
in conclusion , the findings of the current study illustrated that the triphala extract was effective in eliminating the paracetamol - induced hepato 
it may be a potential lead for the protection of the liver and kidney against drug - induced toxicity .
 background : paracetamol , a widely used analgesic and antipyretic , is known to cause liver and renal injury in humans when administered in higher and repeated doses that cause acute liver injury . 
 triphala is a well - known ayurvedic rasayana formulation that is prescribed for balancing of vata , pitta and kapha . 
 traditionally , it is used for the treatment of liver and kidney diseases.objective : the present study was undertaken to examine the protective effect of triphala extract against paracetamol - induced hepato  
 renal injury in swiss albino mice.materials and methods : swiss albino mice ( weight 2025 g ) were used in this study . the mice were divided into five groups of six animals each . 
 the aqueous extract of triphala was given orally at two different doses ( 100 and 300 mg / kg body weight ) for seven consecutive days , followed by a single intraperitoneal injection of paracetamol ( 500 mg / kg body weight ) to induce hepato  
 renal toxicity . 
 serum levels of liver enzymes , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , alkaline phosphatase ( alp ) , bilirubin , creatinine , urea and uric acid were measured as indices of liver and renal injury . 
 all the statistical analyses were performed with the help of one - way analysis of variance ( anova ) followed by student  newman  
 keuls test as post hoc test . 
 results were considered statistically significant when p < 0.05.results:pre-treatment with triphala extract at 100 mg / kg and 300 mg / kg body weight exhibited a significant ( p < 0.01 ) hepatoprotective activity . 
 the protective effect of triphala extract at 300 mg / kg body weight appears more effective than 100 mg / kg body weight.conclusion:the present study gives an evidence of the protective role of triphala extract against paracetamol - induced hepato  
 renal toxicity and validates its traditional claim in the ayurveda system . 
INTRODUCTION
MATERIALS AND METHODS
Drugs and chemicals
Plant materials
Preparation of the extract
Experimental animals
Paracetamol-induced hepatorenal toxicity in mice
Biochemical analysis
Histopathological studies
Statistical analysis
RESULTS
Effects of 
Effect of 
DISCUSSION
CONCLUSION