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Hepatoprotective activity of carrot (Daucus carota L.) against carbon tetrachloride intoxication in mouse liver
~ Journal of ~, ETHNOPHARMACOIDL~ E L S E V 1E R
Journal of Ethnopharmacology47 (1995) 69-74
Hepatoprotective activity of carrot (Daucus carota L.) against carbon tetrachloride intoxication in mouse liver Anupam
Bishayee, Alok Sarkar, Malay Chatterjee*
Division of Biochemistry, Department of Pharmaceutical Technology, Jadavpur University, Calcutta 700032, India
Revision received20 March 1995; accepted 21 March 1995
Abstract
The effect of carrot extract on carbon tetrachloride (CCl4)-induced acute liver damage was evaluated. The increased serum enzyme levels (viz., glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, lactate dehydrogenase, alkaline phosphatase, sorbitol and glutamate dehydrogenase) by CCl4-induction were significantly lowered due to pretreatment with the extract. The extract also decreased the elevated serum bilirubin and urea content due to CC14 administration. Increased activities of hepatic 5'-nucleotidase, acid phosphatase, acid ribonuclease and decreased levels of succinic dehydrogenase, glucose-6-phosphatase and cytochrome P-450 produced by CC14 were reversed by the extract in a dose-responsive way. Results of this study revealed that carrot could afford a significant protective action in the alleviation of CCl4-induced hepatocellular injury. Keywords: Carbon tetrachloride; Hepatic damage; Serum and liver enzymes; Carrot extract; Hepatoprotection
1. Introduction
Carrot (Daucus carom L.) of the Apiaceae family is an annual or biannual herb mostly confined to the temperate regions of Europe, Asia and Africa. A decoction of carrot is considered as a popular remedy for jaundice in Europe (Nadkarni, 1976). Different parts of the carrot are used in Indian traditional medicine for the treatment of a broad spectrum of ailments including kidney dysfunction, asthma, dropsy, inflammation, leprosy, worm troubles, etc. (Kirtikar and Basu, 1933; * Corresponding author.
Chopra et al., 1958; Nadkarni, 1976). Carrot is widely consumed as an aphrodisiac and nervine tonic and its scraped root is used as a local stimulant for indolent ulcers (Shastri, 1952). In view of numerous physiological and pharmacological actions of carrot extract (Shet and Madaiah, 1988; Dhar, 1990; Mahran et al., 1991), attempts have been made to evaluate its effect as a protective or repairing agent against hepatocellular injury, as it is long since considered to be 'good for the liver' in traditional oriental medicine (Kirtikar and Basu, 1933). In this regard, we have previously evaluated a significant hepatoprotective potential of carrot extract in the light of inhibition of lipid
0378-8741/95/$09.50 © 1995 Elsevier ScienceIreland Ltd. All rights reserved SSDI 0378-8741(95)01254-B
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A. Bishayee et aL / Journal of Ethnopharmacology 47 (1995) 69-74
peroxidation and subsequent normalization of glutathione (GSH)-related enzymes linked with the antioxidant defence system against carbon tetrachloride (CCl4)-evoked liver damage in mice (Bishayee and Chatterjee, 1993). In the present communication, we have assessed further the hepatoprotective activity of carrot extract in vivo by monitoring the changes in several serum and liver biochemical parameters using CCl4-intoxicated mouse liver as the experimental model. The parameters studied include the serum enzyme level of glutamate oxaloacetate transaminase (SGOT, EC 2.6.1.1), glutamate pyruvate transaminase (SGPT, EC 2.6.1.2), lactate dehydrogenase (LDH, EC 1.1.1.27), alkaline phosphatase (EC 3.1.3.1), sorbitol dehydrogenase (EC 1.1.1.14) and glutamate dehydrogenase (EC 1.4.1.3); serum bilirubin and urea content; hepatic enzymatic activities of acid phosphatase (EC 3.1.3.2), acid ribonuclease (EC 3.1.27.5), succinic dehydrogenase (EC 1.3.99.1), glucose-6-phosphatase (EC 3.1.3.9) and 5 '-nucleotidase (EC 3.1.3.5); and liver cytochrome P-450 level which are known to be the most frequent and satisfactory markers for evaluating hepatocellular damage (Zylva and Oannal, 1984), especially with the known hepatotoxin CC14 (Dwivedi et al., 1990; Braide, 1991). 2. Materials and methods
2.1. Preparation of carrot extract Fresh tuber roots of carrot (500 g) were peeled, washed, cut into small pieces and homogenized in a waring blender with 2.5 volumes of distilled water. The extraction was carried out in a cold room (20 ° ± I°C) with constant stirring overnight. The homogenate was then squeezed through cheese cloth and centrifuged at 1200 g for 10 min at 0-4°C. The supernatant being the carrot extract (yield 210% w/w) was decanted and kept at 4°C until used. 2.2. Treatment of animals Male Swiss albino mice, weighing between 20-25 g and 8 weeks old, were acclimatized to conditions in the laboratory (26-28°C, 60-80% relative humidity, 12 h light/dark cycle) for 10 days prior to the commencement of the treatment, dur-
ing which they received food (Hindustan Lever) and tap water ad libitum. The mice were then divided into several groups of six mice each. The normal group was given distilled water orally at 10 mg/kg body weight once daily for 7 days in succession followed by olive oil subcutaneously (s.c.) at 1 ml/kg on the last day, 1 h after distilled water feeding. The control group was administered distilled water similarly followed by 20% v/v CC14 in olive oil s.c. at 1 ml/kg on the 7th day, 1 h after distilled water administration. Experimental groups were treated orally with carrot extract (10-50 ml/kg) once daily for 7 days in succession followed by a single administration of 20% v/v CC14 in olive oil s.c. at 1 ml/kg on the last day, 1 h after carrot extract treatment. 2.3. Assessment of liver function Twenty-four hours after the s.c. administration, the mice of each group were anaesthetized with ether, and blood was collected directly from the heart. It was centrifuged at 2000 g for 10 min at 4°C to separate the serum and kept at 4°C to assay the activities of serum enzymes. SGOT and SGPT were determined by the method of Reitman and Frankel as described by Bergrneyer and Bernt (1974). LDH was measured by the method of Wroblewski and LaDue as indicated by Varley (1967a). Alkaline phosphatase was estimated according to Kind and King (1954). Activities of sorbitol and glutamate dehydrogenase were determined by the methods of Gerlach and Hiby (1974) and King (1974), respectively. Serum bilirubin and urea levels were estimated according to Malloy and Evelyn (1937) and Varley (1967b), respectively. After the collection of the blood, the liver was immediately excised, washed with cold saline, blotted, weighed, minced and homogenized in ice-cold 1.15% w/v potassium chloride in a PotterElvehjem Teflon glass homogenizer for 1 min to make a 10% w/v liver homogenate. Succinic dehydrogenase (Green et al., 1955), glucose-6phosphatase (Baginski et al., 1974) and 5'nucleotidase (Aronson and Touster, 1974) were measured in the liver homogenate. The activities of lysosomal enzymes acid phosphatase (Kind and King, 1954) and acid ribonuclease (De Duve et al.,
A. Bishayee et al. / Journal of Ethnopharmacology 47 (1995) 69- 74
1955) were determined. A liver microsomal fraction was prepared (Schneider and Hogeboom, 1950) and the cytochrome P-450 content in this fraction was measured from a reduced carbon monoxide difference spectrum (Omura and Sato, 1964). Protein was estimated by the method of Lowry et al. (1951).
2.4. Statistical analysis The significance of differences between two mean values was determined by Student's t-test. The overall response of mice to a specific type of treatment was representated by the proportion of abnormal value (PAV) (Perrissound and Weibel, 1980). This proportion, expressed in a percentage, was calculated as the ratio of the total number of abnormal individual values for the biochemical parameters to the total number of estimations carried out (number of mice in the group multiplied by number of biochemical parameters estimated).
71
3. Results
Mice treated with a single dose of CCI4 developed significant hepatic damage as observed from elevated serum levels of hepatospecific enzymes as well as severe alterations in different liver parameters (Tables 1 and 2). Activities of SGOT, SGPT, LDH, alkaline phosphatase, sorbitol and glutamate dehydrogenase in serum were increased in CC14-intoxicated control animals (Table 1). Serum bilirubin and urea levels were also enhanced upon CC14 administration alone (Table 1). Treatment with the carrot extract afforded a significant protection against CCl4-induced increase in the serum enzyme levels, bilirubin and urea contents in a dose-responsive manner (Table 1). The degree of protection was observed maximally with the highest dose of the extract. Table 2 depicts that treatment with CC14 elevated hepatic enzymatic activities of 5'-nucleotidase, acid phosphatase and
Table 1 Effect of pretreatment with carrot extract on serum biochemical responses of mice to CCI 4 Parameter
Normal (vehicle)
Control (vehicle + CC14)
Experimental (CCI4 + carrot extract) 10 ml/kg
SGOT
25 ml/kg
50 ml/kg
42.1 ± 5.2
120.4 ± 11.3
103.6 ± 6.1
72.2 ± 6.5*
59.1 4" 7.4**
23.7 ± 3.1
80.3 ± 6.2
71.4 ± 4.3
44.5 ± 5.2*
32.7 + 5.8**
105.2 ± 7.3
190.1 ± 12.2
169.4 ± 8.1
50.8 ± 5.3
139.4 ± 8.4
129.7 ± 8.7
97.3 ± 9.5*
60.7 ± 7.5**
39.8 ± 5.8
112.1 ± 10.1
95.7 ± 6.2
50.3 ± 6.9**
42.2 ± 7.2**
6.2 ± 2.1
13.7 ± 3.2
11.8 ± 3.2
9.0 ± 2.6*
7.3 + 2.8*
1.2 ± 0.5
10.7 ± 3.4
8.3 ± 3.0
5.7 ± 2.5*
3.1 ± 2.1"*
0.31 ± 0.02
0.52 ± 0.06
0.40 ± 0.04
0.37 ± 0.05
0.33 ± 0.06*
0on) SGPT
(iu/l) LDH (IU/l) Alkaline phosphatase (Units/l) Sorbitol dehydrogenase (Units/l) Glutamate dehydrogenase (Units/l) Bilirubin
140.7 ± 9.1
119.5 4- 9.7*
(me/l) Urea
(ga) Results are expressed as mean ± S.E.M. of 6 animals. Control group was compared with normal and all values were significantly different (P < 0.01). Experimental groups were compared with control: *P < 0.05 and **P < 0.01.
A. Bishayee et al./ Journal of Ethnopharmacology 47 (1995) 69-74
72
Table 2 Effect of pretreatment with carrot extract on activities of hepatic enzymes of mice to CC14 Parameter
Suceinic dehydrogenase (t~mol indophenol reduced/rain per mg protein) Glucose-6-phosphatase (/Lmol inorganic phosphate released/rain per mg protein) 5'-Nucleotidase (/~mol inorganic phosphate released/rain per mg protein) Acid phosphatase (t~mol p-nitrophenol formed/min per mg protein) Acid ribonuclease (Units/rag protein) Cytochrome P-450 (nmol/mg protein)
Normal (vehicle)
Control (vehicle + CC14)
Experimental (CCI 4 + carrot extract) 10 ml/kg
25 ml/kg
50 ml/kg
0.62 4- 0.08
0.36 4- 0.10
0.48 + 0.12"
0.58 ± 0.13"*
0.64 4- 0.11"**
7.21 4- 0.72
4.23 4- 1.11
4.91 4- 1.67
6.74 4- 2.03**
6.85 4- 2.53**
2.30 ~- 0.21
3.51 4- 0.27
3.02 4- 0.25
2.84 4- 0.28
2.41 4- 0.30*
5.12 4- 0.32
9.37 4- 0.56
8.10 4- 0.52
7.52 4- 0.53
6.48 4- 0.59*
12.3 4- 2.1
20.7 4- 4.2
17.5 4- 3.8
16.3 4- 3.2
14.1 4- 3.6*
3.6 4- 0.6
1.6 ± 0.8
1.9 4- 0.5
2.5 4- 0.5**
2.9 4- 0.6***
Results are expressed as m e a n ± S.E.M. o f 6 animals.
Control group was compared with normal and all values were significantly different (P < 0.01). Experimental groups were compared with control: *P < 0.05, **P 0.01 and ***P < 0.001.
ribonuclease, and attenuated succinic dehydrogenase and glucose-6-phosphatase with a concurrent decline in hepatic cytochrome P-450 level. These altered biochemical features were significantly brought towards normalization by pretreatment with the carrot extract (Table 2). The maximum protection against CCl4-induced hepatic aberrations was achieved with the optimum dose of the extract. 4. Discussion and conclusions
Since the changes associated with CCl4-induced liver damage are similar to that of acute viral hepatitis (Rubinstein, 1962), CC14-mediated hepatotoxicity was taken here as the experimental model for liver injury. It has been established that CC14 is accumulated in hepatic parenchymal cells and metabolically activated by cytochrome P-450 dependent monooxygenases to form a trichloromethyl free radical (CC13) which alkylates cellular proteins (including cytochrome P-450) and other macromolecules (Recknagel, 1983) with a simulta-
neous attack on polyunsaturated fatty acids in the presence of oxygen to produce lipid peroxides (Recknagel and Ghosal, 1966) leading to liver damage (Recknagel et al., 1976). For many years now, hepatotoxic compounds such as CC14 are known to cause marked elevation in serum transaminases. In agreement with results obtained in previous investigations (Dwivedi et al., 1990; Braide, 1991), our present study elicited a significant increase in the activities of SGOT, SGPT, LDH, alkaline phosphatase, sorbitol and glutamate dehydrogenase within 24 h of exposure of the mice to a single dose of CC14 indicating considerable hepatocellular injury. Pretreatment with the carrot extract attenuated these increased enzyme activities produced by CC14 and a subsequent recovery towards normalization of these enzymes strongly suggests the possibility of carrot extract being able to condition the hepatocytes so as to cause accelerated regeneration of parenchymal cells, thus protecting against membrane fragility decreasing the leakage of marker enzymes into the circulation. Stabilization of serum biliru-
A. Bishayee et al./ Journal of Ethnopharmacology 47 (1995) 69-74
bin and urea levels through the administration of the extract is further a clear indication of the improvement of the functional status of the liver cells. The protective role of the carrot extract has also been reflected in the normal levels of hepatic succinic dehydrogenase, glucose-6-phosphatase and 5'-nucleotidase activities that were severely altered in the CC14-intoxicated mouse liver. Again, acid phosphatase and acid ribonuclease are frequently employed as the marker enzymes to assess the lysosomal changes in vivo because it is localised almost exclusively in the particles and its release parallels that of lysosomal hydrolases (Tanaka and Iizuka, 1968). A significant lower level of these enzymes, as a consequence of carrot extract pretreatment against CCl4-induced marked elevation, indicates a protection against the rupture of lysosomes and leakage of these enzymes in situ resulting from damage to the integrity of liver cell lysosomes by CC14. The inhibitory effect of CCI4 on cytochrome P450 level was also compensated by the extract through maintenance of its normal level. The role of the carrot extract in the protection of CC1amediated loss in cytochrome P-450 content may be considered as an indication of improved protein synthesis in hepatic tissue (Dwivedi et al., 1991; Mandal et al., 1993). Thus the results of the present investigation clearly demonstrate that various biochemical changes, produced in the serum and liver in mice by CCI4 treatment, were reversed or prevented by pretreatment with the carrot extract, and the resuits were statistically significant mostly in a dose of 25 ml or 50 ml/kg This finding was further confirmed by the PAV value. This value in the CCI4 control group was 83.5% in contrast to 70.3%, 40.7% and 32.4% when treated with 10, 25 and 50 ml/kg of the extract, respectively, for 7 days and subsequent exposure to CC14. It seems that the extract provided a significant hepatoprotective response and the degree of hepatoprotection improves with increasing dosage. The probable mechanism by which the carrot extract exerts its protective action against CC1ainduced hepatocellular metabolic alterations could be by the stimulation of hepatic regeneration
73
through an improved synthesis of protein, or interference with the microsomal activation of C C l 4 and/or its accelerated detoxification and excretion. Which of these possibilities are the major factor(s) for the hepatoprotective role of carrot are now being studied in our laboratory. Carrot juice is reported to be rich in carotenes (Shastri, 1952). Carotenes in the carrot extract include B-carotene, c~-carotene, 3,-carotene, lycopene, cryptoxanthin, leutein, many partly degraded carotenoids such as abscisic acid, trisporic acid, B-apo-carotenals, crocetin and many common polar carotenoids, e.g., violaxanthin (Straub, 1987; Olson, 1989). Some of the above compounds have the potential to minimise the deleterious effects of free radicals including the peroxy radicals (Burton, 1989) and thereby can be ranked as hepatoprotective agents. However, which of these components is (are) actually responsible for the antihepatotoxic potential of carrot remains to be seen in the future course of our experimentations. References Aronson, N.N. Jr. and Touster, O. (1974) Isolation of rat liver plasma membrane fragment in isotonic sucrose. In: S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. 31. Academic Press, New York, pp. 90-92. Baginski, E.S., Foa, P.P. and Zak, B. (1974) Glucose-6phosphatase. In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 2. Verlag Chemie Weinheim, Academic Press, New York, pp. 876-880. Bergmeyer, H.U. and Bernt, E. (1974) Colorimetric assay of Reitman and Frankel. In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 2. Verlag Chemie Weinheim, Academic Press, New York, pp. 735-764. Bishayee, A. and Chatterjee, M. (1993) Carrot aqueous extract protection against hepatic oxidative stress and lipid peroxidation induced by acute carbon tetrachloride intoxication in mice. Fitoterapia 64, 261-265. Braide, V.B. (1991) Antihepatotoxic biochemical effects of Kolaviron, a biflavonoid of Garcinia kola seeds. Phytotherapy Research 5, 35-37. Burton, G.W. (1989) Antioxidant action of carotenoids. Journal of Nutrition 119, 109- I 11. Chopra, R.N., Chopra, I.C., Handa, K.L. and Kapur, L.D. (1958) Chopra's Indigeneous Drugs of India, 2nd Edn., U.N. Dhar and Sons Pvt. Ltd., Calcutta, p. 504. De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Tissue fractionation studies. 6. Intercellular distribution patterns of enzymes in rat liver tissue. Biochemical Journal 60, 604-617.
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A. Bishayee et al. / Journal of Ethnopharmacology 47 (1995) 69- 74
Dhar, V.J. (1990) Studies on Daucus carota seeds. Fitoterpia 61, 255-258. Dwivedi, Y., Rastogi, R., Chander, R., Sharma, S.K., Kapoor, N.K., Garg, N.K. and Dhawan, B.N. (1990) Hepatoprotectire activity of picroliv against carbon tetrachloride induced liver damage in rats. Indian Journal of Medical Research 92B, 195-200. Dwivedi, Y., Rastogi, R., Garg, N.K. and Dhawan, B.N. (1991) Prevention of paracetamol-induced hepatic damage in rats by picroliv, the standardized active fraction from Picrorhiza kurroa. Phytotherapy Research 5, 115-119. Gerlach, U. and Hiby, W. (1974) Sorbitol dehydrogenase. In: H.U. lkrgmeyer (Ed.), Methods of Enzymatic Analysis, Vol.2. Verlag Chemic Weinheim, Academic Press, New York, pp. 569-573. Green, D.E., Mii, S. and Kohout, P.M. (1955) Studies on the terminal electron transport system. I. Succinic dehydrogenase. Journal of Biological Chemistry 217, 551-567. Kind, P.R.N. and King, E.J. (1954) Estimation of plasma phosphatase by determination of hydrolysed phenol with antipyrine. Journal of Clinical Pathology 7, 322-330. King, J. (1974) Glutamate dehydrogenase. In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 2. Verlag Chemic Weinheim, Academic Press, New York, pp. 656-659." Kirtikar, K.R. and Basu, B.D. (1933) Indian Medicinal Plants, Vol. II. L.M. Basu, Allahabad, pp. 1229-1231. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. Mahran, G.H., Kadry, H.A., Issac, Z.G., Thabet, C.K., A1Azizi, M.M. and EI-Olemy, M.M. (1991) Investigation of diuretic drug plants. I. Phytochemical screening and pharmacological evaluation of Anethum graveolens L., Apium graveolens L., Daucus carota L. and Eruca sativa Mill. Phytotherapy Research 5, 169-172. MaUoy, H.T. and Evelyn, K.A. (1937) The determination of bilirubin with the photometric colorimeter. Journal of Biological Chemistry 119, 481-490. Mandal, P.K., Bishayee, A. and Chatterjee, M. (1993) Stimulation of tissue repair by Mikania cordata root extract in carbon tetrachloride-induced liver injury in mice. Phytotherapy Research 7, 103-105. Nadkarni, K.M. (1976) Indian Materia Medica, Vol. I. Popular Prokashan, Bombay, p. 442.
Olson, J.A. (1989) Provitamin A function of carotenoids: the conversion of B-carotene into vitamin A. Journal of Nutrition 119, 105-108. Omura, T. and Sato, R. (1964) The carbon monoxide binding pigment of liver microsomes. Journal of Biological Chemistry 239, 2370-2378. Perrissound, D. and Weibel, I. (1980) Protective effect of (+) cyanidanol-3 in acute liver injury induced by galactosamine or carbon tetrachloride in the rat. Naunyn Schmiedeberg's Archives of Pharmacology 312, 285-290. Recknagel, R.O. (1983) A new direction in the study of carbon tetrachloride hepatotoxicity. Life Sciences 33, 401-408. Recknagel, R.O. and Ghosal, A.K. (1966) Quantitative estimation of peroxidative degeneration of rat liver microsomal and mitochondrial lipids after carbon tetrachloride poisoning. Experimental and Molecular Pathology 5, 413-426. Recknagel, R.O., Giende, E.A. and Hruszkewycz, A.M (1976) In: E.A. Pryor (Ed.), Free Radicals in Biology, Vol. III. Academic Press, New York, pp. 97-132. Rubinstein, D. (1962) Epinephrine release and liver glycogen levels after carbon tetrachloride administration. American Journal of Physiology 203, 1033-1037. Schneider, W.C. and Hogeboom, G.H. (1950) Intracellular distribution of enzymes. V. Further studies on the distribution of cytochrome c in rat liver homogenates. Journal of Biological Chemistry 183, 123-128. Shastri, B.N. (1952) Wealth of lndia - - Raw Materials, Vol. 111. C.S.I.R. Publication, New Delhi, pp. 19-23. Shet, M.S. and Madaiah, M. (1988) Lectin activity in different plant tubers, rhizomes and bulbs. Current Science 57, 1107-1110. Straub, O. (1987) In: F. Pfander (Ed.), Key to Carotenoids, 2nd Edn., Birkhauser Verlag, Basel, p. 296. Tanaka, K. and lizuka, Y. (1968) Suppression of enzyme release from isolated rat liver lysosomes by non-steroidal antiinflammatory drugs. Biochemical Pharmacology 17, 2023-2032. Varley, H. (1967a) Practical Clinical Biochemistry, 4th Edn., William Heinemann, New York, pp. 278-279. Varley, H. (1967B) Practical Clinical Biochemistry, 4th Edn., William Heinemann, New York, pp. 161-162. Zylva, J.F. and Oannal, P.R. (1984) Clinical Chemistry in Diagnosis and Treatment, 4th Edn., Publishing Pvt. Ltd., Singapur, Hong Kong and New Delhi, p. 366.
Journal of Ethnopharmacology47 (1995) 69-74
Hepatoprotective activity of carrot (Daucus carota L.) against carbon tetrachloride intoxication in mouse liver Anupam
Bishayee, Alok Sarkar, Malay Chatterjee*
Division of Biochemistry, Department of Pharmaceutical Technology, Jadavpur University, Calcutta 700032, India
Revision received20 March 1995; accepted 21 March 1995
Abstract
The effect of carrot extract on carbon tetrachloride (CCl4)-induced acute liver damage was evaluated. The increased serum enzyme levels (viz., glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, lactate dehydrogenase, alkaline phosphatase, sorbitol and glutamate dehydrogenase) by CCl4-induction were significantly lowered due to pretreatment with the extract. The extract also decreased the elevated serum bilirubin and urea content due to CC14 administration. Increased activities of hepatic 5'-nucleotidase, acid phosphatase, acid ribonuclease and decreased levels of succinic dehydrogenase, glucose-6-phosphatase and cytochrome P-450 produced by CC14 were reversed by the extract in a dose-responsive way. Results of this study revealed that carrot could afford a significant protective action in the alleviation of CCl4-induced hepatocellular injury. Keywords: Carbon tetrachloride; Hepatic damage; Serum and liver enzymes; Carrot extract; Hepatoprotection
1. Introduction
Carrot (Daucus carom L.) of the Apiaceae family is an annual or biannual herb mostly confined to the temperate regions of Europe, Asia and Africa. A decoction of carrot is considered as a popular remedy for jaundice in Europe (Nadkarni, 1976). Different parts of the carrot are used in Indian traditional medicine for the treatment of a broad spectrum of ailments including kidney dysfunction, asthma, dropsy, inflammation, leprosy, worm troubles, etc. (Kirtikar and Basu, 1933; * Corresponding author.
Chopra et al., 1958; Nadkarni, 1976). Carrot is widely consumed as an aphrodisiac and nervine tonic and its scraped root is used as a local stimulant for indolent ulcers (Shastri, 1952). In view of numerous physiological and pharmacological actions of carrot extract (Shet and Madaiah, 1988; Dhar, 1990; Mahran et al., 1991), attempts have been made to evaluate its effect as a protective or repairing agent against hepatocellular injury, as it is long since considered to be 'good for the liver' in traditional oriental medicine (Kirtikar and Basu, 1933). In this regard, we have previously evaluated a significant hepatoprotective potential of carrot extract in the light of inhibition of lipid
0378-8741/95/$09.50 © 1995 Elsevier ScienceIreland Ltd. All rights reserved SSDI 0378-8741(95)01254-B
70
A. Bishayee et aL / Journal of Ethnopharmacology 47 (1995) 69-74
peroxidation and subsequent normalization of glutathione (GSH)-related enzymes linked with the antioxidant defence system against carbon tetrachloride (CCl4)-evoked liver damage in mice (Bishayee and Chatterjee, 1993). In the present communication, we have assessed further the hepatoprotective activity of carrot extract in vivo by monitoring the changes in several serum and liver biochemical parameters using CCl4-intoxicated mouse liver as the experimental model. The parameters studied include the serum enzyme level of glutamate oxaloacetate transaminase (SGOT, EC 2.6.1.1), glutamate pyruvate transaminase (SGPT, EC 2.6.1.2), lactate dehydrogenase (LDH, EC 1.1.1.27), alkaline phosphatase (EC 3.1.3.1), sorbitol dehydrogenase (EC 1.1.1.14) and glutamate dehydrogenase (EC 1.4.1.3); serum bilirubin and urea content; hepatic enzymatic activities of acid phosphatase (EC 3.1.3.2), acid ribonuclease (EC 3.1.27.5), succinic dehydrogenase (EC 1.3.99.1), glucose-6-phosphatase (EC 3.1.3.9) and 5 '-nucleotidase (EC 3.1.3.5); and liver cytochrome P-450 level which are known to be the most frequent and satisfactory markers for evaluating hepatocellular damage (Zylva and Oannal, 1984), especially with the known hepatotoxin CC14 (Dwivedi et al., 1990; Braide, 1991). 2. Materials and methods
2.1. Preparation of carrot extract Fresh tuber roots of carrot (500 g) were peeled, washed, cut into small pieces and homogenized in a waring blender with 2.5 volumes of distilled water. The extraction was carried out in a cold room (20 ° ± I°C) with constant stirring overnight. The homogenate was then squeezed through cheese cloth and centrifuged at 1200 g for 10 min at 0-4°C. The supernatant being the carrot extract (yield 210% w/w) was decanted and kept at 4°C until used. 2.2. Treatment of animals Male Swiss albino mice, weighing between 20-25 g and 8 weeks old, were acclimatized to conditions in the laboratory (26-28°C, 60-80% relative humidity, 12 h light/dark cycle) for 10 days prior to the commencement of the treatment, dur-
ing which they received food (Hindustan Lever) and tap water ad libitum. The mice were then divided into several groups of six mice each. The normal group was given distilled water orally at 10 mg/kg body weight once daily for 7 days in succession followed by olive oil subcutaneously (s.c.) at 1 ml/kg on the last day, 1 h after distilled water feeding. The control group was administered distilled water similarly followed by 20% v/v CC14 in olive oil s.c. at 1 ml/kg on the 7th day, 1 h after distilled water administration. Experimental groups were treated orally with carrot extract (10-50 ml/kg) once daily for 7 days in succession followed by a single administration of 20% v/v CC14 in olive oil s.c. at 1 ml/kg on the last day, 1 h after carrot extract treatment. 2.3. Assessment of liver function Twenty-four hours after the s.c. administration, the mice of each group were anaesthetized with ether, and blood was collected directly from the heart. It was centrifuged at 2000 g for 10 min at 4°C to separate the serum and kept at 4°C to assay the activities of serum enzymes. SGOT and SGPT were determined by the method of Reitman and Frankel as described by Bergrneyer and Bernt (1974). LDH was measured by the method of Wroblewski and LaDue as indicated by Varley (1967a). Alkaline phosphatase was estimated according to Kind and King (1954). Activities of sorbitol and glutamate dehydrogenase were determined by the methods of Gerlach and Hiby (1974) and King (1974), respectively. Serum bilirubin and urea levels were estimated according to Malloy and Evelyn (1937) and Varley (1967b), respectively. After the collection of the blood, the liver was immediately excised, washed with cold saline, blotted, weighed, minced and homogenized in ice-cold 1.15% w/v potassium chloride in a PotterElvehjem Teflon glass homogenizer for 1 min to make a 10% w/v liver homogenate. Succinic dehydrogenase (Green et al., 1955), glucose-6phosphatase (Baginski et al., 1974) and 5'nucleotidase (Aronson and Touster, 1974) were measured in the liver homogenate. The activities of lysosomal enzymes acid phosphatase (Kind and King, 1954) and acid ribonuclease (De Duve et al.,
A. Bishayee et al. / Journal of Ethnopharmacology 47 (1995) 69- 74
1955) were determined. A liver microsomal fraction was prepared (Schneider and Hogeboom, 1950) and the cytochrome P-450 content in this fraction was measured from a reduced carbon monoxide difference spectrum (Omura and Sato, 1964). Protein was estimated by the method of Lowry et al. (1951).
2.4. Statistical analysis The significance of differences between two mean values was determined by Student's t-test. The overall response of mice to a specific type of treatment was representated by the proportion of abnormal value (PAV) (Perrissound and Weibel, 1980). This proportion, expressed in a percentage, was calculated as the ratio of the total number of abnormal individual values for the biochemical parameters to the total number of estimations carried out (number of mice in the group multiplied by number of biochemical parameters estimated).
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3. Results
Mice treated with a single dose of CCI4 developed significant hepatic damage as observed from elevated serum levels of hepatospecific enzymes as well as severe alterations in different liver parameters (Tables 1 and 2). Activities of SGOT, SGPT, LDH, alkaline phosphatase, sorbitol and glutamate dehydrogenase in serum were increased in CC14-intoxicated control animals (Table 1). Serum bilirubin and urea levels were also enhanced upon CC14 administration alone (Table 1). Treatment with the carrot extract afforded a significant protection against CCl4-induced increase in the serum enzyme levels, bilirubin and urea contents in a dose-responsive manner (Table 1). The degree of protection was observed maximally with the highest dose of the extract. Table 2 depicts that treatment with CC14 elevated hepatic enzymatic activities of 5'-nucleotidase, acid phosphatase and
Table 1 Effect of pretreatment with carrot extract on serum biochemical responses of mice to CCI 4 Parameter
Normal (vehicle)
Control (vehicle + CC14)
Experimental (CCI4 + carrot extract) 10 ml/kg
SGOT
25 ml/kg
50 ml/kg
42.1 ± 5.2
120.4 ± 11.3
103.6 ± 6.1
72.2 ± 6.5*
59.1 4" 7.4**
23.7 ± 3.1
80.3 ± 6.2
71.4 ± 4.3
44.5 ± 5.2*
32.7 + 5.8**
105.2 ± 7.3
190.1 ± 12.2
169.4 ± 8.1
50.8 ± 5.3
139.4 ± 8.4
129.7 ± 8.7
97.3 ± 9.5*
60.7 ± 7.5**
39.8 ± 5.8
112.1 ± 10.1
95.7 ± 6.2
50.3 ± 6.9**
42.2 ± 7.2**
6.2 ± 2.1
13.7 ± 3.2
11.8 ± 3.2
9.0 ± 2.6*
7.3 + 2.8*
1.2 ± 0.5
10.7 ± 3.4
8.3 ± 3.0
5.7 ± 2.5*
3.1 ± 2.1"*
0.31 ± 0.02
0.52 ± 0.06
0.40 ± 0.04
0.37 ± 0.05
0.33 ± 0.06*
0on) SGPT
(iu/l) LDH (IU/l) Alkaline phosphatase (Units/l) Sorbitol dehydrogenase (Units/l) Glutamate dehydrogenase (Units/l) Bilirubin
140.7 ± 9.1
119.5 4- 9.7*
(me/l) Urea
(ga) Results are expressed as mean ± S.E.M. of 6 animals. Control group was compared with normal and all values were significantly different (P < 0.01). Experimental groups were compared with control: *P < 0.05 and **P < 0.01.
A. Bishayee et al./ Journal of Ethnopharmacology 47 (1995) 69-74
72
Table 2 Effect of pretreatment with carrot extract on activities of hepatic enzymes of mice to CC14 Parameter
Suceinic dehydrogenase (t~mol indophenol reduced/rain per mg protein) Glucose-6-phosphatase (/Lmol inorganic phosphate released/rain per mg protein) 5'-Nucleotidase (/~mol inorganic phosphate released/rain per mg protein) Acid phosphatase (t~mol p-nitrophenol formed/min per mg protein) Acid ribonuclease (Units/rag protein) Cytochrome P-450 (nmol/mg protein)
Normal (vehicle)
Control (vehicle + CC14)
Experimental (CCI 4 + carrot extract) 10 ml/kg
25 ml/kg
50 ml/kg
0.62 4- 0.08
0.36 4- 0.10
0.48 + 0.12"
0.58 ± 0.13"*
0.64 4- 0.11"**
7.21 4- 0.72
4.23 4- 1.11
4.91 4- 1.67
6.74 4- 2.03**
6.85 4- 2.53**
2.30 ~- 0.21
3.51 4- 0.27
3.02 4- 0.25
2.84 4- 0.28
2.41 4- 0.30*
5.12 4- 0.32
9.37 4- 0.56
8.10 4- 0.52
7.52 4- 0.53
6.48 4- 0.59*
12.3 4- 2.1
20.7 4- 4.2
17.5 4- 3.8
16.3 4- 3.2
14.1 4- 3.6*
3.6 4- 0.6
1.6 ± 0.8
1.9 4- 0.5
2.5 4- 0.5**
2.9 4- 0.6***
Results are expressed as m e a n ± S.E.M. o f 6 animals.
Control group was compared with normal and all values were significantly different (P < 0.01). Experimental groups were compared with control: *P < 0.05, **P 0.01 and ***P < 0.001.
ribonuclease, and attenuated succinic dehydrogenase and glucose-6-phosphatase with a concurrent decline in hepatic cytochrome P-450 level. These altered biochemical features were significantly brought towards normalization by pretreatment with the carrot extract (Table 2). The maximum protection against CCl4-induced hepatic aberrations was achieved with the optimum dose of the extract. 4. Discussion and conclusions
Since the changes associated with CCl4-induced liver damage are similar to that of acute viral hepatitis (Rubinstein, 1962), CC14-mediated hepatotoxicity was taken here as the experimental model for liver injury. It has been established that CC14 is accumulated in hepatic parenchymal cells and metabolically activated by cytochrome P-450 dependent monooxygenases to form a trichloromethyl free radical (CC13) which alkylates cellular proteins (including cytochrome P-450) and other macromolecules (Recknagel, 1983) with a simulta-
neous attack on polyunsaturated fatty acids in the presence of oxygen to produce lipid peroxides (Recknagel and Ghosal, 1966) leading to liver damage (Recknagel et al., 1976). For many years now, hepatotoxic compounds such as CC14 are known to cause marked elevation in serum transaminases. In agreement with results obtained in previous investigations (Dwivedi et al., 1990; Braide, 1991), our present study elicited a significant increase in the activities of SGOT, SGPT, LDH, alkaline phosphatase, sorbitol and glutamate dehydrogenase within 24 h of exposure of the mice to a single dose of CC14 indicating considerable hepatocellular injury. Pretreatment with the carrot extract attenuated these increased enzyme activities produced by CC14 and a subsequent recovery towards normalization of these enzymes strongly suggests the possibility of carrot extract being able to condition the hepatocytes so as to cause accelerated regeneration of parenchymal cells, thus protecting against membrane fragility decreasing the leakage of marker enzymes into the circulation. Stabilization of serum biliru-
A. Bishayee et al./ Journal of Ethnopharmacology 47 (1995) 69-74
bin and urea levels through the administration of the extract is further a clear indication of the improvement of the functional status of the liver cells. The protective role of the carrot extract has also been reflected in the normal levels of hepatic succinic dehydrogenase, glucose-6-phosphatase and 5'-nucleotidase activities that were severely altered in the CC14-intoxicated mouse liver. Again, acid phosphatase and acid ribonuclease are frequently employed as the marker enzymes to assess the lysosomal changes in vivo because it is localised almost exclusively in the particles and its release parallels that of lysosomal hydrolases (Tanaka and Iizuka, 1968). A significant lower level of these enzymes, as a consequence of carrot extract pretreatment against CCl4-induced marked elevation, indicates a protection against the rupture of lysosomes and leakage of these enzymes in situ resulting from damage to the integrity of liver cell lysosomes by CC14. The inhibitory effect of CCI4 on cytochrome P450 level was also compensated by the extract through maintenance of its normal level. The role of the carrot extract in the protection of CC1amediated loss in cytochrome P-450 content may be considered as an indication of improved protein synthesis in hepatic tissue (Dwivedi et al., 1991; Mandal et al., 1993). Thus the results of the present investigation clearly demonstrate that various biochemical changes, produced in the serum and liver in mice by CCI4 treatment, were reversed or prevented by pretreatment with the carrot extract, and the resuits were statistically significant mostly in a dose of 25 ml or 50 ml/kg This finding was further confirmed by the PAV value. This value in the CCI4 control group was 83.5% in contrast to 70.3%, 40.7% and 32.4% when treated with 10, 25 and 50 ml/kg of the extract, respectively, for 7 days and subsequent exposure to CC14. It seems that the extract provided a significant hepatoprotective response and the degree of hepatoprotection improves with increasing dosage. The probable mechanism by which the carrot extract exerts its protective action against CC1ainduced hepatocellular metabolic alterations could be by the stimulation of hepatic regeneration
73
through an improved synthesis of protein, or interference with the microsomal activation of C C l 4 and/or its accelerated detoxification and excretion. Which of these possibilities are the major factor(s) for the hepatoprotective role of carrot are now being studied in our laboratory. Carrot juice is reported to be rich in carotenes (Shastri, 1952). Carotenes in the carrot extract include B-carotene, c~-carotene, 3,-carotene, lycopene, cryptoxanthin, leutein, many partly degraded carotenoids such as abscisic acid, trisporic acid, B-apo-carotenals, crocetin and many common polar carotenoids, e.g., violaxanthin (Straub, 1987; Olson, 1989). Some of the above compounds have the potential to minimise the deleterious effects of free radicals including the peroxy radicals (Burton, 1989) and thereby can be ranked as hepatoprotective agents. However, which of these components is (are) actually responsible for the antihepatotoxic potential of carrot remains to be seen in the future course of our experimentations. References Aronson, N.N. Jr. and Touster, O. (1974) Isolation of rat liver plasma membrane fragment in isotonic sucrose. In: S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. 31. Academic Press, New York, pp. 90-92. Baginski, E.S., Foa, P.P. and Zak, B. (1974) Glucose-6phosphatase. In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 2. Verlag Chemie Weinheim, Academic Press, New York, pp. 876-880. Bergmeyer, H.U. and Bernt, E. (1974) Colorimetric assay of Reitman and Frankel. In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Vol. 2. Verlag Chemie Weinheim, Academic Press, New York, pp. 735-764. Bishayee, A. and Chatterjee, M. (1993) Carrot aqueous extract protection against hepatic oxidative stress and lipid peroxidation induced by acute carbon tetrachloride intoxication in mice. Fitoterapia 64, 261-265. Braide, V.B. (1991) Antihepatotoxic biochemical effects of Kolaviron, a biflavonoid of Garcinia kola seeds. Phytotherapy Research 5, 35-37. Burton, G.W. (1989) Antioxidant action of carotenoids. Journal of Nutrition 119, 109- I 11. Chopra, R.N., Chopra, I.C., Handa, K.L. and Kapur, L.D. (1958) Chopra's Indigeneous Drugs of India, 2nd Edn., U.N. Dhar and Sons Pvt. Ltd., Calcutta, p. 504. De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Tissue fractionation studies. 6. Intercellular distribution patterns of enzymes in rat liver tissue. Biochemical Journal 60, 604-617.
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