Planta Med. 57(1991) 25
Picroliv Affords Protection Against Thioacetamide-Induced Hepatic Damage in Rats1 Yogesh Dwivedi2, Ravi Rastogi2. Sri Kant Sharma2, Narendra Kumar Garg2, and Bhola Nath Dhawan23 1 c• D. 2
R. I. Communication No. 4556
ICMR Centre for Advanced Pharmacological Research on Traditional Remedies, Central Drug Research Institute,
Lucknow-226 001, India Address for correspondence Received: August 21, 1989
rachloride-induced liver damage in rats (10) and hepatic
Thioacetamide (100mg/kg), when ad-
damage produced by Plasmodium berghei infection in mastomys (11). In this communication we report the protective effect of oral feeding of Picroliv on the biochemical lesions
ministered to normal rats, caused a significant increase in the activities of 5' -nucleotidase and y-glutamyl transpeptidase and a decrease in the activities of glucose 6phosphatase and succinate dehydrogenase enzymes in the liver. DNA, RNA, and proteins were increased while the cytochrome P450 in the micnosomal fraction and the glycogen content in the liver were decreased significantly. Elevations in the activities of GOT, GPT, and alkaline phosphatase and bilirubin content in serum were also observed. Picroliv, a standardised glycoside fraction of Picrorhiza kurroa, in doses of 12.5 and 25 mg/kg pre-
vented most of the biochemical changes induced by thioacetamide in liver and serum. The hepatoprotective activity of Picroliv was comparable with that of silyma-
nfl, a known hepatoprotective agent obtained from seeds of Silybuin marianum.
Key words
Picrorhiza kurrooa, thioacetamide, silymann, hepatotoxicity, hepatoprotection, Picroliv
Introduction Thioacetamide has been reported to cause inhibition of the respiratory metabolism of the liver due to the uncontrolled entry of Ca ions into the hepatocytes resulting in inhibition of oxidative phosphorylation (1). Early metabolic disturbances caused by thioacetamide include considerable increases in the ribonucleic acid and the pro-
tein content of the nuclear fraction of hepatocytes (2). Thioacetamide has widely been used to produce varying grades of liver damage in rats including nodular cirrhosis (3), liver cell proliferation, production of psuedolobules (4), and parenchymal cell necrosis (5).
Earlier publications from several laboratories including our own have reported that the ethanolic extract of Picrorhiza kurroa (6, 7, 8, 9) as well as its stand-
ardized fraction Picroliv, which contains at least a 60% mixture of the iridoid glycosides, picroside I and kut-
associated with damage to plasma membrane, mitochondna, microsomes, lysosomes, and nucleus of liver cells and changes in serum parameters produced by thioacetamide in rats.
Materials and Methods Adult male rats (body wt. 130 15 g, SpragueDawley strain) inbred in the CDRI animal house were used. The animals were fed ad lib standard pellet diet (Lipton, Bombay) and had free access to water. The rats were given single, subcutaneous injections of thioacetamide (100 mg/kg) as 2% (w/v) solution. Aqueous solutions of Picroliv (12.5 mg/ml) and suspensions of silymarin (2% w/v, gum accacia in water, 20 mg/ml), a known hepatoprotective agent, were prepared. Picroliv (12.5 and 25 mg/kg) and silymarin (20 mg/kg) were administered every day by intubation for seven days, i.e. five days before, one day along with, and one day after
thioacetamide administration. Control groups of normal and thioacetamide-treated rats were not given the hepatoprotective agents. At the end of this experimental regimen, the rats were fasted overnight and sacrificed by decapitation, 48 hours after the thioacetamide injection. Blood was collected from the retro-orbital plexus before decapitation and kept for 30 mm to obtain serum. The liver was excised out, washed with chilled normal saline, and 10% (w/v) liver homogenates were prepared in ice-cold 1.15% potassium chloride solution.
Lipid peroxides, protein, and total lipids in the liver homogenates and phospholipids and cholesterol from the extracted liver lipids were estimated as described earlier (12). 10% Trichloroacetic acid was added to the liver homogenates and the precipitate was extracted with ethanol (95%, v/v). The resulting pellet was used for estimations of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) according to Dische (13) and Mejbaum (14), respectively. Glycogen was extracted from liver tissue as described by Good et al. (15) and estimated by the method of Montgomery (16).
Succinate dehydrogenase, glucose 6-phos-
phatase, acid phosphatase, and acid ribonuclease were assayed in liver homogenates by the procedure described ealier (17). Acid
phosphatase and acid ribonuclease were estimated in liver homogenates without submitting them to freezing and thawing. This indicated the magnitude of rupture of lysosomes/leakage of
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
koside in a ratio of 1: 1.5, protect against carbon tetAbstract
Planta Med. 57(1991)
Yogesh Dwivedi at at
these enzymes in situ as a result of damage to the integrity of the
hepatic lysosomes by thioacetamide. 5'-Nucleotidase and yglutamyl transpeptidase were assayed by the methods of Aronson et al. (18) and Boelsterli et al. (19), respectively. Superoxide dismutase was measured in the post-mitochondrial fraction according to Kakkar et al. (20). Cytochrome P450 and cytochrome b5 were estimated in the microsomal fraction using the procedure of Omura and Sato (21). Bilirubin (22) and albumin (23) were estimated in
serum by standard methods. Glutamate oxaloacetate trans-
mutase and the amount oflipid peroxides, total lipids, phospholipids, and cholesterol also did not exhibit any appreciable changes after thioacetamide administration.
Thioacetamide caused a significant eleva-
tion in the activities of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and alkaline phosphatase and the content of bilirubin in serum (Fig. 1). No change was observed in serum total protein and albumin.
aminase and glutamate pyruvate transaminase were assayed in serum according to Reitman and Frankel (24) and alkaline phos-
Hepatoprotection by Picroliv
phatase by the method of Bessay et al. (25).
The results in Table 1 indicate that oral feeding of Picroliv (12.5 and 25mg/kg) significantly re-
Results and Discussion Changes induced by thioacetainide
duced changes in the activities of succinate dehydrogenase,
Thioacetamide affected several of the hepatic and serum parameters used in this study. The results in Table 1 show that it caused significant increases in the ac-
tivities of 5'-nucleotidase and y-glutamyl transpeptidase, marker enzymes of the plasma membrane of liver cells and indices of transport accross the membrane, and decreases in the activities of succinate dehydrogenase and glucose 6phosphatase (indicating mitochondrial and microsomal damage). The activity of free lysosomal acid ribonuclease was increased significantly (74%) but the other lysosomal enzyme, acid phosphatase was not affected. Deoxyribonucleic acid (nuclear damage), ribonucleic acid (microsomal damage), and protein content of the liver were increased by 73, 35, and 27%, respectively, associatedwith a decrease in glycogen (52 %). Cytochrome P45 activity in the liver micro-
somes decreased significantly but there was no change in the content of cytochrome b5. The activity of superoxide dis-
acid ribonuclease, and glucose 6-phosphatase and increases in deoxyribonucleic acid produced by thioacetamide; however, increases in the activities of 5'-nucleotidase and y-glutamyl transpeptidase were significantly
prevented only at the 25mg/kg dose. The changes in the microsomal content of cytochrome P45 as well as the contents of glycogen and protein in the liver were not modified by Picroliv treatment.
The elevated levels of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and alkaline phosphatase in serum were restored towards their respective normal values with both the doses of Picroliv (Fig. 1). In the case of glutamate pyruvate transaminase, however, protection appeared to be higher at the lower dose of Picroliv. Picroliv was ineffective in preventing the in-
crease in serum bilirubin caused by thioacetamide.
Table 1 Effect of Picroliv and silymann en hepatic biochemi cal parameters altered by thioacetamide. Parameter
control
Thioacetamide
Picroliv+Thioacetamide 12.5mg/kg 25mg/kg
Silymarin
(20mg/kg)+ Thioacetamide
Enzyme (units/ 100mg liver protein) 5l.Nucleotidasea
3.00
0.09
5.14
0.53
5.23
0.28
(71) .y.Glutamyltranspeptidaseb
47.31
3.07
90.00
11.87
94.00
9.06
(90)
Succinatedehydrogenasec Acid ribonucleasec
0.99 4.16
0.08
0.62
0.25
(37) 7.25 0.68
0.05
(74)
Glucose 6phosphatase'
9.73
1.00
4.91
0.38
(49) Cytochrome P4554
24.90
2.08
5.56
0.50
0.98 0.09" [97] 0.64' 6.36 [29] 7.45 0.89" [53] 5.81
0.84
4.46 0.36' [32] 65.36 1.93" [58] 0.83 0.16' [57] 5.87 0.36" [44] 5.50 0.42' [12] 5.69 1.04
3.92 0.30" [57] 89.20 15.07
0.15"
0.86 165]
4.19 0.15" [99]
0.30"
7.89
[62] 4.40
1.79
3.41
0.31"
(77)
Other constituents (mg/g liver) DNA
2.34
0.17
4.05
0.13
(73) RNA
3.72
0.23
5.04
0.30
(35) Glycogen
Protein
62.70 135.08
3.59 12.60
30.19
7.18
(52) 171.25 7.36 (27)
3.60 0.17" [26] 4.54 0.35' [38] 33.38 12.56 174.43
6.75
3.11 0.08" [55] 4.06 0.30" [74] 29.93 5.04 185.38
5.89
[37]
0.34"
4.39
[49] 36.90
12.08
139.96 11.45" [85]
Enzyme units: a. moles of pi released/mm; b. .ug of p-nitroaniline released/mm; c. LOD/min; d. nnoles/100 mg microsomal protein. Values are mean S.D. from 8 animals (control and thioacetamide-treated( or 6 animals (thioacetamide-treated given Picroliv or silymarin(. All the changes in thioacetanidetreated groups were significant (P
Picroliv Affords Protection Against Thioacetamide-Induced Hepatic Damage in Rats1 Yogesh Dwivedi2, Ravi Rastogi2. Sri Kant Sharma2, Narendra Kumar Garg2, and Bhola Nath Dhawan23 1 c• D. 2
R. I. Communication No. 4556
ICMR Centre for Advanced Pharmacological Research on Traditional Remedies, Central Drug Research Institute,
Lucknow-226 001, India Address for correspondence Received: August 21, 1989
rachloride-induced liver damage in rats (10) and hepatic
Thioacetamide (100mg/kg), when ad-
damage produced by Plasmodium berghei infection in mastomys (11). In this communication we report the protective effect of oral feeding of Picroliv on the biochemical lesions
ministered to normal rats, caused a significant increase in the activities of 5' -nucleotidase and y-glutamyl transpeptidase and a decrease in the activities of glucose 6phosphatase and succinate dehydrogenase enzymes in the liver. DNA, RNA, and proteins were increased while the cytochrome P450 in the micnosomal fraction and the glycogen content in the liver were decreased significantly. Elevations in the activities of GOT, GPT, and alkaline phosphatase and bilirubin content in serum were also observed. Picroliv, a standardised glycoside fraction of Picrorhiza kurroa, in doses of 12.5 and 25 mg/kg pre-
vented most of the biochemical changes induced by thioacetamide in liver and serum. The hepatoprotective activity of Picroliv was comparable with that of silyma-
nfl, a known hepatoprotective agent obtained from seeds of Silybuin marianum.
Key words
Picrorhiza kurrooa, thioacetamide, silymann, hepatotoxicity, hepatoprotection, Picroliv
Introduction Thioacetamide has been reported to cause inhibition of the respiratory metabolism of the liver due to the uncontrolled entry of Ca ions into the hepatocytes resulting in inhibition of oxidative phosphorylation (1). Early metabolic disturbances caused by thioacetamide include considerable increases in the ribonucleic acid and the pro-
tein content of the nuclear fraction of hepatocytes (2). Thioacetamide has widely been used to produce varying grades of liver damage in rats including nodular cirrhosis (3), liver cell proliferation, production of psuedolobules (4), and parenchymal cell necrosis (5).
Earlier publications from several laboratories including our own have reported that the ethanolic extract of Picrorhiza kurroa (6, 7, 8, 9) as well as its stand-
ardized fraction Picroliv, which contains at least a 60% mixture of the iridoid glycosides, picroside I and kut-
associated with damage to plasma membrane, mitochondna, microsomes, lysosomes, and nucleus of liver cells and changes in serum parameters produced by thioacetamide in rats.
Materials and Methods Adult male rats (body wt. 130 15 g, SpragueDawley strain) inbred in the CDRI animal house were used. The animals were fed ad lib standard pellet diet (Lipton, Bombay) and had free access to water. The rats were given single, subcutaneous injections of thioacetamide (100 mg/kg) as 2% (w/v) solution. Aqueous solutions of Picroliv (12.5 mg/ml) and suspensions of silymarin (2% w/v, gum accacia in water, 20 mg/ml), a known hepatoprotective agent, were prepared. Picroliv (12.5 and 25 mg/kg) and silymarin (20 mg/kg) were administered every day by intubation for seven days, i.e. five days before, one day along with, and one day after
thioacetamide administration. Control groups of normal and thioacetamide-treated rats were not given the hepatoprotective agents. At the end of this experimental regimen, the rats were fasted overnight and sacrificed by decapitation, 48 hours after the thioacetamide injection. Blood was collected from the retro-orbital plexus before decapitation and kept for 30 mm to obtain serum. The liver was excised out, washed with chilled normal saline, and 10% (w/v) liver homogenates were prepared in ice-cold 1.15% potassium chloride solution.
Lipid peroxides, protein, and total lipids in the liver homogenates and phospholipids and cholesterol from the extracted liver lipids were estimated as described earlier (12). 10% Trichloroacetic acid was added to the liver homogenates and the precipitate was extracted with ethanol (95%, v/v). The resulting pellet was used for estimations of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) according to Dische (13) and Mejbaum (14), respectively. Glycogen was extracted from liver tissue as described by Good et al. (15) and estimated by the method of Montgomery (16).
Succinate dehydrogenase, glucose 6-phos-
phatase, acid phosphatase, and acid ribonuclease were assayed in liver homogenates by the procedure described ealier (17). Acid
phosphatase and acid ribonuclease were estimated in liver homogenates without submitting them to freezing and thawing. This indicated the magnitude of rupture of lysosomes/leakage of
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
koside in a ratio of 1: 1.5, protect against carbon tetAbstract
Planta Med. 57(1991)
Yogesh Dwivedi at at
these enzymes in situ as a result of damage to the integrity of the
hepatic lysosomes by thioacetamide. 5'-Nucleotidase and yglutamyl transpeptidase were assayed by the methods of Aronson et al. (18) and Boelsterli et al. (19), respectively. Superoxide dismutase was measured in the post-mitochondrial fraction according to Kakkar et al. (20). Cytochrome P450 and cytochrome b5 were estimated in the microsomal fraction using the procedure of Omura and Sato (21). Bilirubin (22) and albumin (23) were estimated in
serum by standard methods. Glutamate oxaloacetate trans-
mutase and the amount oflipid peroxides, total lipids, phospholipids, and cholesterol also did not exhibit any appreciable changes after thioacetamide administration.
Thioacetamide caused a significant eleva-
tion in the activities of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and alkaline phosphatase and the content of bilirubin in serum (Fig. 1). No change was observed in serum total protein and albumin.
aminase and glutamate pyruvate transaminase were assayed in serum according to Reitman and Frankel (24) and alkaline phos-
Hepatoprotection by Picroliv
phatase by the method of Bessay et al. (25).
The results in Table 1 indicate that oral feeding of Picroliv (12.5 and 25mg/kg) significantly re-
Results and Discussion Changes induced by thioacetainide
duced changes in the activities of succinate dehydrogenase,
Thioacetamide affected several of the hepatic and serum parameters used in this study. The results in Table 1 show that it caused significant increases in the ac-
tivities of 5'-nucleotidase and y-glutamyl transpeptidase, marker enzymes of the plasma membrane of liver cells and indices of transport accross the membrane, and decreases in the activities of succinate dehydrogenase and glucose 6phosphatase (indicating mitochondrial and microsomal damage). The activity of free lysosomal acid ribonuclease was increased significantly (74%) but the other lysosomal enzyme, acid phosphatase was not affected. Deoxyribonucleic acid (nuclear damage), ribonucleic acid (microsomal damage), and protein content of the liver were increased by 73, 35, and 27%, respectively, associatedwith a decrease in glycogen (52 %). Cytochrome P45 activity in the liver micro-
somes decreased significantly but there was no change in the content of cytochrome b5. The activity of superoxide dis-
acid ribonuclease, and glucose 6-phosphatase and increases in deoxyribonucleic acid produced by thioacetamide; however, increases in the activities of 5'-nucleotidase and y-glutamyl transpeptidase were significantly
prevented only at the 25mg/kg dose. The changes in the microsomal content of cytochrome P45 as well as the contents of glycogen and protein in the liver were not modified by Picroliv treatment.
The elevated levels of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and alkaline phosphatase in serum were restored towards their respective normal values with both the doses of Picroliv (Fig. 1). In the case of glutamate pyruvate transaminase, however, protection appeared to be higher at the lower dose of Picroliv. Picroliv was ineffective in preventing the in-
crease in serum bilirubin caused by thioacetamide.
Table 1 Effect of Picroliv and silymann en hepatic biochemi cal parameters altered by thioacetamide. Parameter
control
Thioacetamide
Picroliv+Thioacetamide 12.5mg/kg 25mg/kg
Silymarin
(20mg/kg)+ Thioacetamide
Enzyme (units/ 100mg liver protein) 5l.Nucleotidasea
3.00
0.09
5.14
0.53
5.23
0.28
(71) .y.Glutamyltranspeptidaseb
47.31
3.07
90.00
11.87
94.00
9.06
(90)
Succinatedehydrogenasec Acid ribonucleasec
0.99 4.16
0.08
0.62
0.25
(37) 7.25 0.68
0.05
(74)
Glucose 6phosphatase'
9.73
1.00
4.91
0.38
(49) Cytochrome P4554
24.90
2.08
5.56
0.50
0.98 0.09" [97] 0.64' 6.36 [29] 7.45 0.89" [53] 5.81
0.84
4.46 0.36' [32] 65.36 1.93" [58] 0.83 0.16' [57] 5.87 0.36" [44] 5.50 0.42' [12] 5.69 1.04
3.92 0.30" [57] 89.20 15.07
0.15"
0.86 165]
4.19 0.15" [99]
0.30"
7.89
[62] 4.40
1.79
3.41
0.31"
(77)
Other constituents (mg/g liver) DNA
2.34
0.17
4.05
0.13
(73) RNA
3.72
0.23
5.04
0.30
(35) Glycogen
Protein
62.70 135.08
3.59 12.60
30.19
7.18
(52) 171.25 7.36 (27)
3.60 0.17" [26] 4.54 0.35' [38] 33.38 12.56 174.43
6.75
3.11 0.08" [55] 4.06 0.30" [74] 29.93 5.04 185.38
5.89
[37]
0.34"
4.39
[49] 36.90
12.08
139.96 11.45" [85]
Enzyme units: a. moles of pi released/mm; b. .ug of p-nitroaniline released/mm; c. LOD/min; d. nnoles/100 mg microsomal protein. Values are mean S.D. from 8 animals (control and thioacetamide-treated( or 6 animals (thioacetamide-treated given Picroliv or silymarin(. All the changes in thioacetanidetreated groups were significant (P
