Pliarttiacology & Toxicology 1992, 71, 383-387

Picroliv and its Components Kutkoside and Picroside I Protect Liver Against Galactosamine-Induced

Damage in Rats Yogesh Dwivedi, Ran Rastogi, N. K. Garg and B. N. Dhawan

ICMR Centre for Advanced Pharmacological Research on Traditional Remedies, Central Drug Research Institute, Lucknow-226OOl, India

(Received December 13, 1991; Accepted June 25, 1992)

Abstract: D-Galactosamine (800 mgikg, intraperitoneally) caused significant decrease in the activities of 5'-nucleotidase, glucose-6-phosphatase and cytochrome P450 and increase in activities of y-glutamyl transpeptidase, succinate dehydrogen- ase, acid phosphatase and acid ribonuclease in liver after 24 hr. The levels of RNA, protein and glycogen decreased while total lipids, phospholipids. cholesterol and lipid peroxides increased. It also increased the serum levels of transaminases, alkaline phosphatase and bilirubin while protein concentration decreased significantly. Oral administration of Picroliv (12 mglkgiday for 7 days), a standardised iridoid glycoside fraction of Picrorhiza kurroa, significantly prevented the biochemical changes in liver and serum of galactosamine-toxicated rats. Kutkoside (12 mgikgiday for 7 days) also protected against changes in most of the hepatic and serum constituents studied. Another iridoid glycoside from Picroliv, Picroside I . at the same dose level could only prevent toxicant-induced changes in acid phosphatase, phospholipids and lipid peroxides in liver and alkaline phosphatase in serum. Mixture of Picroside I and Kutkoside in the ratio of 1:l.S at 12 mgikg dose elicited lesser response than Picroliv.

D-Galactosamine produces hepatocellular injury which closely resembles histological changes observed in liver dur- ing acute viral hepatitis (Keppler rt al. 1968). Since the damage is reversible it serves as a good model for evaluation of hepatoprotective agents. Picrorhiza kurroa has been used for treatment of liver disorders in Indian traditional systems of medicine. The ethanolic extract of P kurroa has been shown to possess hepatoprotective activity against carbon tetrachloride- and galactosamine-induced liver damage in rats (Pandey & Chaturvedi 1969; Ansari et al. 1988). Picroliv is a standardised fraction isolated from roots and rhizomes of P kurroa. It contains two major iridoid g1ycosides:Picros- ide I and Kutkoside in ratio of 1:1.5 along with several other uncharacterized glycosides as minor constituents. He- patoprotective efficacy of Picroliv against carbon tetra- chloride (Dwivedi et ul. 1990), paracetamol (Dwivedi et al. 1991a). thioacetamide (Dwivedi et al. I991 b), mono- crotaline (Dwivedi et al. 1991~) in rats and Plasmodium brrghei infection in mastomys (Chander et uI. 1990) has earlier been reported. In this communication we report pro- tection by Picroliv, Kutkoside and Picroside I of several biochemical changes induced in liver and serum of rats 24 hr after administration of galactosamine.

Materials and Methods Adult male albino rats (120+ 10 g, Sprague Dawley) were divided into 6 groups of 6 rats each. The animals of group I received normal saline by gavage for 7 days and by intraperitoneal route on the 6th day and served as normal controls. The rats of group I1 received normal saline for 7 days and an intraperitoneal injection of 800 mgikg D-galactosamine hydrochloride (dissolved in normal saline) on the 6th day. This group served as intoxicated control. Group

111, IV, V and VI received aqueous solutions of Picroliv, Kutkoside, Picroside I and a mixture of Picroside I and Kutkoside (in ratio of 1:1.5 w/w) respectively by gavage at a dose of 12 mgikglday for 7 days. The rats were fasted overnight. Twenty-four hr after galactosa- mine injection, blood was collected from the retro-orbital plexus. Serum was obtained by centrifugation of the blood. The rats were sacrificed by cervical dislocation. Liver was quickly removed, washed with 145 mM sodium chloride and 10% ( w i v ) liver homo- genate was prepared in 154 mM potassium chloride.

Enzyme assays. The activities of 5'-nucleotidase EC 3.1.3.5 (Aron- son & Touster 1974), y-glutamyl transpeptidase EC 2.3.2.2 (Boel- sterli & Zbinden 1979), glucose 6-phosphatase EC 1.3.3.9 (Hubsch- er & West 1965), succinate dehydrogenase EC 1.3.99.1 (Slater & Bonner 1952), acid phosphatase EC 3.1.3.2 (Wright et al. 1972) and acid ribonuclease EC 3.1.4.22 (de Duve et al. 1955) were assayed directly in liver homogenates. The contents of cytochrome P450 and cytochrome b, were estimated in microsomal fractions of liver (prepared as described by Schneider & Hogeboom 1950) according to the method of Omura & Sat0 (1964). The activities of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transam- inase (GPT) were determined by the procedure of Reitman & Frank- el (1957). while serum alkaline phosphatase was measured according to Bessey et al. (1946).

DNA and RNA in liver were extracted by the procedures of Schneider (1945) and Schneider & Klug (1946). respectively. DNA in the TCA extract was estimated as described by Dische (1930) while RNA was estimated by orcinol reaction (Mejbaum 1939). Total lipids were extracted from liver homogenate as described by Folch et al. (1957). Phospholipids and cholesterol were estimated in the extracted lipids according to Wagner el al. (1962) and Zlatkis et al. (1953), respectively. Lipid peroxides were estimated by the method of Ohkawa et al. (1979). Glycogen was extracted from liver (Good et al. 1933) and estimated according to Montgomery (1957). Total proteins in liver and serum were estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard. The contents of bilirubin and albumin in serum were measured according to Jendrassik & Grof (1938) and Stavric-Hirosho ef al. (1974), respectively.

384 YOGESH DWIVEDI ET A L

S/u/is/icul unulysis. Mean and standard deviations were calculated for all parameters using standard procedures. For each parameter one-way analysis of variance was applied to ascertain the signifi- cance of the difference among group means, where groups were independent. Means of the individual group were compared with the rest of the groups by least significance difference, where vari- ation ratio (F value) was found significant (Zar 1974).

Results

Biochemical changes induced by D-Galactosamine. The results given in table 1 show that administration of galactosamine to rats caused significant elevation in the activities of hepatic y-glutamyl transpeptidase, succinate de- hydrogenase, acid phosphatase and acid ribonuclease. Since the lysosomal enzymes were assayed without submitting the homogenate to freezing and thawing, the increase in activities of these enzymes indicates the degree of leakage or rupture of lysosomes. The activities of 5'-nucleotidase and glucose-6-phosphatase in liver and levels of cytochrome P450 in hepatic microsomal fraction decreased, but there was no change in the content of cytochrome b,. The results given in table 2 show that the levels of RNA, proteins and glycogen in liver decreased significantly but the level of DNA remained unchanged. The contents of total lipids, phospholipids, cholesterol and lipid peroxides in liver in- creased.

The activities of GOT, GPT, alkaline phosphatase and levels of bilirubin in serum were elevated after exposure of rats to galactosamine (fig. I) . There was a moderate decrease in total proteins (fig. 1) but not in albumin content (3.46k 0.16 g/dl).

Prevention of biochemical changes by Picroliv, Kutkoside and Picroside I. The galactosamine-induced increase in the activities of y- glutamyl transpeptidase, acid phosphatase, acid ribonu-

clease and succinate dehydrogenase in liver was significantly (59-1000/) prevented by Picroliv. The decrease in activity of 5'-nucleotidase and glucose 6-phosphatase was completely blocked, while the decrease in cytochrome P450 content was restored by about 39% (table I). Picroliv also provided significant protection against change in RNA (72o/u), pro- teins (630/), total lipids (67%), phospholipids (84%), chol- esterol (64%) and lipid peroxides (560/0), but no protection was evident in case of hepatic glycogen (table 2). The elev- ation of GOT, GPT and alkaline phosphatase in serum was decreased by 5 I , 84 and 55%, respectively in Picroliv treated animals. It also restored the increase in bilirubin and de- crease in proteins towards normal values (70 and 35'X. respectively) (fig. 1).

Kutkoside (12 mg/ kg) significantly prevented galactosa- mine-induced decrease in activities of hepatic 5'-nucleo- tidase (39%), glucose-6-phosphatase (66%) and increase in activities of acid phosphatase (48%) and acid ribonuclease (360/0), but did not significantly affect changes in y-glutamyl transpeptidase, succinate dehydrogenase and cytochrome P450 (table I). The rise in the levels of total lipids, phospho- lipids, cholesterol and lipid peroxides was also prevented significantly by Kutkoside but the effect was less marked than with Picroliv. The restoration in case of RNA and total proteins was also 6% and 21% lower than with Picroliv. Among the serum parameters, maximum restoration (52%) occurred in bilirubin followed by GPT and alkaline phos- phatase (38-39'%) but changes in GOT remained unaffected (fig. I ) . The effect on the serum parameters was also less marked.

Picroside I (12 mg/kg) was able to affect only the in- creased levels of acid phosphatase (49%) (table I ) , phospho- lipids (31%) and lipid peroxides (40%) in liver (table 2) and alkaline phosphatase (25%) in serum (fig. 1). The changes in other parameters remained unaltered.

Tuble I. Effect of Picroliv, Kutkoside, Picroside-I and mixture of Picroside-I & Kutkoside (l: l .5) on changes in hepatic enzymes of rats induced by D-galactosamine.

D-Galactosamine + Picroside I +

Control D-Galactosamine Picroliv Kutkoside Picroside I Kutkoside Enzymesa (Group 1) (Group 11) (Group 111) (Group IV) (Group V) (Group VI)

5'-Nucleotidase' 0.0340+0.0028 0.0222+0.0020** 0.0357+0.0028** 0.0268k0.0008** 0.0235+0.0035 0.0251 k0.0026* y-Glutamyl transpeptidase* 0.0029& 0.0005 0.0059~0.0001** 0.0041 +O.OOOO** 0.0061 +0.0008 0.0059k0.0006 0.0058 &0.0002 Succinate dehydrogenase' 0.0075 kO.0007 0.0123 +0.0004** 0.0072f0.0009** 0.0124+0.0008 0.0127 kO.0008 0.0132~0.0014 Acid phosphatase4 0.0533k0.0039 0.0787f0.0045** 0.0538k0.0018** 0.0664+0.0019** 0.0663~0.0010** 0.0661 *0.0027** Acid ribonuclease' 0.0490k 0.0054 0.0740~0.0009** 0.0593 &0.0032** 0.0649k0.0049* 0.0863 +0.0113 0.0675 +0.0017** Glucose-6- phosphatase' 0.0948~0.0111 0.0534k0.0030** 0.0968k0.0097** 0.0807~0.0079** 0.0537k0.0050 0.0717~0.0020** Cytochrome P4505 0.3778+0.0390 0.2266+0.0233** 0.2853f0.0185** 0.2130&0.0200 0.2193+0.0273 0.2048k0.0232 Cytochrome bS5 0.4053k0.0543 0.4281 k0.0693

* Units: I . pmoles of pi released/min./mg protein; 2. pmoles of p-nitroaniline released/min./mg protein; 3. A O.D./min./mg protein; 4. pmoles of p-nitrophenol released/min./mg protein; 5. n moles/mg protein. Values are mean+_S.D. from 6 animals. Group I1 was compared with Group I and Group 111, W, V & VI were compared with Group 11. Differences were significant only where marked (P *<0.05, ** < O . O I ) .

HEPATOPROTECTION BY PICROLIV, KUTKOSIDE AND PICROSIDE I 385

Table 2. Effect of Picroliv, Kutkoside, Picroside-I and mixture of Picroside I & Kutkoside (1:1.5) on D-galactosamine-induced changes in chemical constituents of liver in rats.

D-Galactosamine + Kutkoside+

Control D-Galactosamine Picroliv Kutkoside Picroside I Picroside I Constituent (Group I) (Group 11) (Group 111) (Group IV) (Group V) (Group VI)

(mg/g liver) DNA RNA Proteins Glycogen Total lipids Phospholipids Cholesterol Lipid peroxides"

2.31 k0.20 3.29 f 0.2 1

170.75 &- 12.68 54.30 k 5.39 34.54 1 2.21 23.36+ 2.91 9.07 k 0.68

16.521 1.56

2.41 k0.36 2.01 kO.IO** 2.93 +0.21** 2.85k0.16** 2.15 k0.14 2.59+0.09**

11 1.58*5.50** 148.91 +4.20** 136.18+4.30** 113.66k6.57 134.12+4.64** 4.12 10.74** 4.10 k0.26 3.44k 0.63 3.14k0.30 3.68+1.16

69.45*4.20** 45.99+3.34** 56.79+3.39** 67.49k6.77 61.2912.85** 44.4815.09** 26.81 k2.00** 39.36k 1.46** 37.9256.09* 38.58*0.89* 13.27+0.96** 10.57f0.31** 11.13rf;O.43** 12.11 k2.58 11.54+0.57** 23.96k 1.89** 19.78k 1.17** 20.405 1.19** 20.95f2.13** 21.02+0.97**

n moles of malonyldialdehyde formed/ 100 mg liver. Values are meanfS.D. from 6 animals each. Group I1 was compared with Group I and Group 111, IV, V and VI were compared with Group 11. P *<0.05; **<0.01. Other differences are insignificant.

Another group of animals was treated with a mixture of Picroside I and Kutkoside in the proportion in which they occur in Picroliv (1:l.S). The mixture, in a dose of 12 mg/ kg, significantly prevented changes in the activities of 5'- nucleotidase (25%), acid phosphatase (SO%), acid ribonu- clease (26%) and glucose-6-phosphatase (44%) in liver (table 1) and GPT (26%) and alkaline phosphatase (29%) in serum (fig. 1). The toxicant-induced inhibition in levels of RNA (45%) and proteins (38Y0) and increase in levels of total lipids (23u/0), phospholipids (28%), cholesterol (41%) and lipid peroxides (40%) in liver and bilirubin (32%) in serum were also prevented significantly. The effects were less marked than with 12 mg/kg Picroliv or Kutkoside alone except on acid phosphatase and phospholipids where the degree of protection was almost similar to that observed with Kutkoside.

200

160

h d .12 120 Y

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80

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T I I

Discussion

The present results show that Picroliv and its constituent, Kutkoside (12 mg/kg) prevented galactosamine-induced changes in activities of hepatic 5'-nucleotidase, glucose-6- phosphatase, acid phosphatase, acid ribonuclease and levels of RNA, proteins, lipid peroxides, total lipids, phospho- lipids and cholesterol. Picroliv was also effective in prevent- ing the toxicant-induced increase in activity of succinate dehydrogenase and decrease in level of cytochrome P450. Both these preparations also showed protective activity against elevated levels of transaminases, alkaline phospha- tase and bilirubin in serum. Picroside I, another major in- gredient of Picroliv, however, was less effective than Picroliv. Picroside I was able to prevent only the increase in activity of acid phosphatase and contents of phospholipids and lipid

Fig. 1. Effect of Picroliv, Kutkoside, Picroside I and mixture of Picroside I & Kutkoside (1:1.5) on changes in serum induced by D- galactosamine: Control O; Galactosamine treated B; Galactosamine + Picroliv (12 mg/kg) R ; Galactosamine + Kutkoside (12 mg/kg) @; Galactosamine + Picroside I (12 mg/ kg) 3; Galactosamine+mixture of Picroside I and Kutkoside (12 mg/ kg) H. Significance of changes as compared to control in galactosamine alone treated and with galactosamine treated in all other cases. P value *<0.01. 1. pmoles of pyruvate formed/min.; 2. Fmoles of p-nitrophenol released/min.

386 YOGESH DWIVEDI ET AL

peroxides in liver and alkaline phosphatase in serum. A mixture of Picroside I and Kutkoside (l:l.5), given to galact- osamine-toxicated rats in a dose of 12 mg/kg (correspond- ing to about 4.8 mg Picroside I and 7.2 mg Kutkoside) produced significant degree of protection, but this was of a lower degree than that provided by 12 mg/kg of Picroliv. Thus no synergistic action was observed when Picroside I and Kutkoside have been mixed in the proportion in which they occur in Picroliv. It has been reported by Ansari et al. ( 1 988) that the extract of Picrorhiza kurroa devoid of these glycosides is inactive. Hence, the greater efficacy of Picroliv cannot be attributed to hepatoprotective effect of its minor constituents (amounting to about 40% of the weight of Picroliv). Possibly these constituents improve absorption or bioavailability of the Picroside I and Kutkoside. Nityan- and & Kapoor (I97 1) have also reported in case of Commi- phora mukul, that the hypolipidaemic activity of a standard- ised extract was much more than could be accounted by its contents of active constituent, gugglusterones.

Galactosamine causes several changes in liver such as depletion of uridine nucleotides (Keppler et al. 1968; Decker et al. 1971; Shinozuka et al. 1973); changes in lipid, protein and amino sugar composition of plasma membrane (Far- ber & El-Mofty 1975; Petkova et al. 1987); inhibition of RNA and protein synthesis (Konishi et al. 1974; Ozturk et al. 1984) and inhibition of drug metabolizing enzymes (Oku- no et al. 1987). The changes in the activity of hepatic y- glutamyl transpeptidase and 5’-nucleotidase (the enzymes associated with the plasma membrane) and the decrease in the levels of cytochrome P450 and RNA in liver in the present study are in agreement with the reported actions of galactosamine. Some of these changes, such as altered activities of 5’-nucleotidase, y-glutamyl transpeptidase, acid phosphatase, acid ribonuclease, succinate dehydrogenase, glucose-6-phosphatase and cytochrome P450 as well as levels of RNA, proteins, lipid peroxides, total lipids, phos- pholipids and cholesterol in liver, are significantly prevented by Picroliv. Decrease in accumulation of lipid peroxides in liver of galactosamine-toxicated rats by Picroliv suggests that it may be acting as a scavanger of free radicals and as an antioxidant. Our earlier work has shown that Picroliv increases the activity of superoxide dismutase in liver dam- age induced by carbon tetrachloride (Dwivedi et al. 1990) or Plasmodium berghei infection (Chander et al. 1990). Therefore, it appears that one of the mechanisms of hepato- protective action of Picroliv is by its antioxidant action.

The dose of Picroliv selected for these studies is based on results obtained in other test models and also on extrapol- ation of therapeutic doses of Picrorhiza kurrooa used in Ayurvedic system of Indian medicine for treatment of liver disorders to rats. These results, therefore, have a clinical significance. Picroliv has been found to be safe in subacute toxicity studies and is also devoid of mutagenic or terratog- enic potential in two species of laboratory animals. Phase I clinical studies have demonstrated excellent clinical toler- ance and further studies are in progress to evaluate its efficacy in management of hepatic disorders.

Acknowledgements The authors are grateful to Dr. D. K. Kulshreshtha for

preparing Picroliv, Kutkoside and Picroside I. Y.D. is Senior Research Fellow of Council of Scientific and Industrial Research, New Delhi. Technical assistance provided by Messers S. K. Bose, N . K. Verma and Suresh Yadav is acknowledged.

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