- Email: [email protected]
Hepatoprotective activity of Beta vulgaris against CCl4-induced hepatic injury in rats
Fitoterapia 77 (2006) 91 – 93 www.elsevier.com/locate/fitote
Hepatoprotective activity of Beta vulgaris against CCl4-induced hepatic injury in rats M. Agarwal a,*, V.K. Srivastava b, K.K. Saxena b, A. Kumar b a
Department of Pharmacology, VMMC and Safdarjung Hospital, New Delhi, India b Department of Pharmacology, LLRM Medical College, Meerut UP, India Received 11 November 2004; accepted in revised form17 November 2005 Available online 20 December 2005
Abstract Ethanolic extract of Beta vulgaris roots given orally at doses of 1000, 2000 and 4000 mg/kg exhibited significant dose-dependent hepatoprotective activity against carbontetrachloride (CCl4)-induced hepatotoxicity in rats. Hepatotoxicity and its prevention were assessed by serum markers viz. cholesterol, triglyceride, alanine amino transferase and alkaline phosphatase. D 2005 Elsevier B.V. All rights reserved. Keywords: Hepatoprotective; Beta vulgaris; CCl4
1. Introduction Despite extensive research in medical field, no drug in the modern system of medicine can be claimed to cure liver disorders, which, in many times, become fatal. Some plant extracts viz. Picrorrhiza kurroa, Andrographics paniculata, and Eclipta alba, have been reported to possess clinically useful hepatoprotective activity [1]. However, many plants remain unexplored in this regard. Beta vulgaris, a native of the coasts of Mediterranean, is extensively cultivated in Europe, America and many parts of India [2,3]. Aqueous and ethanolic extracts of B. vulgaris have been reported to posses free radical-scavenging activity, reducing the radical cations and phase II enzyme-inducing activities in murine hepatoma cell in vitro study [3]. Further, the phenolic amides isolated from the seeds of B. vulgaris produce the inhibitory effect on lipopolysaccharide-induced nitric oxide production in experimental isolated tissues in a dose-dependent manner [4]. There are some reports indicating the potential hepatoprotective, antioxidant, and antiinflammatory activity of B. vulgaris, without any scientific proof [3,5]. At present, one of the plant-derived medicines approved for use in liver cirrhosis and alcoholic liver diseases is silymarin. There are number of studies which conclude the efficacy of silymarin in these conditions [6]. Silymarin is a mixture of flavonolignans from the fruits of Silybum marianum that has been known since ancient time and recommended in traditional European and Asian medicine mainly for the treatment of liver disorder [7]. * Corresponding author. Tel.: +91 981 049 2796. E-mail address: [email protected] (M. Agarwal). 0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2005.11.004
92
M. Agarwal et al. / Fitoterapia 77 (2006) 91–93
Table 1 Effect of B. vulgaris roots’ ethanolic extract on CCl4-induced liver damage in rats Groups Group Group Group Group Group Group
Ia II III IV V VI
Treatment (mg/kg) p.o. for 7 days
Serum cholesterol (mg/dl)
Serum triglycerides (mg/dl)
ALT (mg/dl)
SAP (U/l)
Vehicle CCl4/0.3 ml (s.c.) Silymarin/25 B. vulgaris/1000 B. vulgaris/2000 B. vulgaris/4000
40.10 F 1.31 100.77 F 4.27b 37.37 F 1.04*** 90.56 F 1.13 75.81 F1.09** 68.95 F 1.09***
91.69 F 1.00 72.40 F 1.03b 86.70 F 1.16*** 78.91 F1.11** 84.36 F 1.32*** 87.99 F 0.87***
57.34 F 1.75 461.00 F 8.89b 106.7 F 2.61*** 230.52 F 7.95*** 152.12 F 3.87*** 133.03 F 1.80***
66.14 F 1.42 138.23 F 5.72b 72.74 F 1.50*** 116.84 F 2.33* 94.41 F1.13*** 86.20 F 1.04***
N = 6. Values are expressed as mean F S.D. ***P b 0.001, **P b 0.01, *P b 0.05 in comparison to CCI4-treated group. a On day 7, in place of CCl4 challenge, the animals were administered olive oil 0.3 ml s.c. b P b 0.001 in comparison to propylene glycol-treated group.
Therefore in the present study silymarin was used as positive control to compare the efficacy of B. vulgaris against CCl4-induced hepatotoxicity. 2. Experimental 2.1. Plant Beta vulgaris L. (Chenopodiaceae) roots were procured from the local market and authenticated by the Department of Botany, CCS University, Merrut. A voucher specimen is deposited in the Department of Pharmacology, LLRM Medical College, Merrut. 2.2. Extraction The powdered roots were macerated [8], soaked in 95% EtOH for 10 days and filtered. The solution evaporated in vacuo gave a semi-gelatinous extract (yield: 10.5%). 2.3. Animals Wistar rats of either sex, weighing 100–150 g, were used. They were housed in well-ventilated room at 23.0 F 2 8C, humidity of 65–70% and photoperiod of 12-h light/dark cycle and fed with standard rodent pellet diet with tap water ad libitum. 2.4. Induction of hepatic injury Hepatic injury was induced in rats by subcutaneous administration of a single dose of 0.3 ml/kg CC14 mixed with equal volume of olive oil on the 7th day, 2 h after the last treatment [9]. Animals were grouped as follows: GroupI: Control group, treated with vehicle (2.0 ml, p.o.) daily for 7 days, followed by olive oil treatment (0.3 ml, s.c.) on day 7. Group II: Treated with vehicle (2.0 ml, p.o.) daily for 7 days followed by CCl4 on day 7. Group III: Treated with silymarin (25 mg p.o.) daily for 7 days followed by CC14 on day 7. Groups IV, V, VI: Treated with ethanolic extract of B. vulgaris suspended in propylene glycol at doses of 1000, 2000 or 4000 mg/kg daily for 7 days followed by CC14 on day 7, respectively. On day 9, 48 h after CC14 administration, blood sample of each animal was taken from abdominal aorta under pentobarbitone anesthesia (35 mg/kg i.p.) and serum cholesterol [10], triglycerides [11], alanine amino (ALT) [12] and alkaline phosphatase (SAP) [13] were evaluated.
M. Agarwal et al. / Fitoterapia 77 (2006) 91–93
93
2.5. Statistical analysis All values are expressed as means F S.D. The results were calculated and subjected to analysis of variance (ANOVA) followed by Student’s t-test. P values of N0.05 were considered significant [14]. 3. Results and discussion It is well known that carbontetrachloride is converted by cytochrome P-450-mixed function oxygenases in smooth endoplasmic reticulum of liver into toxic metabolite, mainly trichloromethyl radical (CCI3!). This free radical in the presence of oxygen may cause peroxidation of lipids on target cell resulting in extensive damage [15]. Administration of CC14 (0.3 ml s.c.) to rats produced hepatotoxicity showed by significant increase in the serum levels of ALT and SAP as well as altered lipid profile in comparison to control group as shown in the Table 1. Ethanolic extract of B. vulgaris given at doses of 1000, 2000 and 4000 mg/kg not only prevented the rise in serum level of ALT and alkaline phosphatase but also improved serum lipid profile in a dose dependent manner. Silymarin, a well-known hepatoprotective drug, showed results comparable to that reported in the literature [16]. The constituents of B. vulgaris roots are not reported in the literature but it may be the possibility that it also contains some of the compounds found in seeds [4] and these may be responsible for hepatoprotection. Identification of chemical constituents along with their effects will be of great help in understanding the hepatoprotective activity of the ethanolic extract of B. vulgaris roots. Acknowledgements The authors thank Dr. C.D. Tripathi, Professor and Head, Department of Pharmacology VMMC Safdarjung Hospital, New Delhi, for his valuable suggestions. The study was conducted at the LLRM Medical College, Meerut. The Institutional Ethical Committee for Animal Experimentation approved the project. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
Subramoniam A, Pushpangadan P. Indian J Pharmacol 1999;31:166. Chopra RN, Nayar SL, Chopra C. Glossary of Indian medicinal plants. New Delhi7 CSIR; 1956. Prajapati ND, Purohit SS, Sharma AK, Kumar TA. A handbook of medicinal plants. A complete source book: Agorbios; 2003. p. 88. Wettasinghe M, Bolling B, Plhak L, Xiao H, Parkin K. J Agric Food Chem 2002;50:6704. Duke JA, Bogenschutz-Godwin MJ, Du J, Cellier P-AK. Handbook of medicinal herbs. Boca Raton, FL7 CRC Press;. 2002. p. 68. Saller R, Meier R, Brignoli R. Drugs 2001;61:2035. Skottova N, Krecman V. Physiol Res 1998;47:1. Riebling RW, Walker GC. Extraction and extractive Remington’s pharmaceuticals science, 15th ed. Pennsylvania Mack Publishing Company; 1975. p. 1509. Saraswat B, Visen PKS, Patnaik GK, Dhawan BN. Indian J Exp Biol 1993;31:316. Zlatkis A, Zak BB, Boyle GJB. J Lab Clin Med 1953;41:86. VanHandle E, Zilversmit DB. J Lab Clin Med 1957;50:152. Reitman S, Frankel S. Am J Clin Pathol 1957;28:56. Kind PRN, King EJ. J Clin Pathol 1971;7:322. Ghosh MN. Fundamentals of experimental pharmacology, 2nd ed. Calcutta7 Scientific Book Agency;. 1984. p. 154. Recknagel RO. Trends Pharmacol Sci 1983;4:129. Bhattacharyya D, Pandit S, Mukherjee R, Das N, Sur TK. Indian J Physiol Pharmacol 2003;47:435.
Hepatoprotective activity of Beta vulgaris against CCl4-induced hepatic injury in rats M. Agarwal a,*, V.K. Srivastava b, K.K. Saxena b, A. Kumar b a
Department of Pharmacology, VMMC and Safdarjung Hospital, New Delhi, India b Department of Pharmacology, LLRM Medical College, Meerut UP, India Received 11 November 2004; accepted in revised form17 November 2005 Available online 20 December 2005
Abstract Ethanolic extract of Beta vulgaris roots given orally at doses of 1000, 2000 and 4000 mg/kg exhibited significant dose-dependent hepatoprotective activity against carbontetrachloride (CCl4)-induced hepatotoxicity in rats. Hepatotoxicity and its prevention were assessed by serum markers viz. cholesterol, triglyceride, alanine amino transferase and alkaline phosphatase. D 2005 Elsevier B.V. All rights reserved. Keywords: Hepatoprotective; Beta vulgaris; CCl4
1. Introduction Despite extensive research in medical field, no drug in the modern system of medicine can be claimed to cure liver disorders, which, in many times, become fatal. Some plant extracts viz. Picrorrhiza kurroa, Andrographics paniculata, and Eclipta alba, have been reported to possess clinically useful hepatoprotective activity [1]. However, many plants remain unexplored in this regard. Beta vulgaris, a native of the coasts of Mediterranean, is extensively cultivated in Europe, America and many parts of India [2,3]. Aqueous and ethanolic extracts of B. vulgaris have been reported to posses free radical-scavenging activity, reducing the radical cations and phase II enzyme-inducing activities in murine hepatoma cell in vitro study [3]. Further, the phenolic amides isolated from the seeds of B. vulgaris produce the inhibitory effect on lipopolysaccharide-induced nitric oxide production in experimental isolated tissues in a dose-dependent manner [4]. There are some reports indicating the potential hepatoprotective, antioxidant, and antiinflammatory activity of B. vulgaris, without any scientific proof [3,5]. At present, one of the plant-derived medicines approved for use in liver cirrhosis and alcoholic liver diseases is silymarin. There are number of studies which conclude the efficacy of silymarin in these conditions [6]. Silymarin is a mixture of flavonolignans from the fruits of Silybum marianum that has been known since ancient time and recommended in traditional European and Asian medicine mainly for the treatment of liver disorder [7]. * Corresponding author. Tel.: +91 981 049 2796. E-mail address: [email protected] (M. Agarwal). 0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2005.11.004
92
M. Agarwal et al. / Fitoterapia 77 (2006) 91–93
Table 1 Effect of B. vulgaris roots’ ethanolic extract on CCl4-induced liver damage in rats Groups Group Group Group Group Group Group
Ia II III IV V VI
Treatment (mg/kg) p.o. for 7 days
Serum cholesterol (mg/dl)
Serum triglycerides (mg/dl)
ALT (mg/dl)
SAP (U/l)
Vehicle CCl4/0.3 ml (s.c.) Silymarin/25 B. vulgaris/1000 B. vulgaris/2000 B. vulgaris/4000
40.10 F 1.31 100.77 F 4.27b 37.37 F 1.04*** 90.56 F 1.13 75.81 F1.09** 68.95 F 1.09***
91.69 F 1.00 72.40 F 1.03b 86.70 F 1.16*** 78.91 F1.11** 84.36 F 1.32*** 87.99 F 0.87***
57.34 F 1.75 461.00 F 8.89b 106.7 F 2.61*** 230.52 F 7.95*** 152.12 F 3.87*** 133.03 F 1.80***
66.14 F 1.42 138.23 F 5.72b 72.74 F 1.50*** 116.84 F 2.33* 94.41 F1.13*** 86.20 F 1.04***
N = 6. Values are expressed as mean F S.D. ***P b 0.001, **P b 0.01, *P b 0.05 in comparison to CCI4-treated group. a On day 7, in place of CCl4 challenge, the animals were administered olive oil 0.3 ml s.c. b P b 0.001 in comparison to propylene glycol-treated group.
Therefore in the present study silymarin was used as positive control to compare the efficacy of B. vulgaris against CCl4-induced hepatotoxicity. 2. Experimental 2.1. Plant Beta vulgaris L. (Chenopodiaceae) roots were procured from the local market and authenticated by the Department of Botany, CCS University, Merrut. A voucher specimen is deposited in the Department of Pharmacology, LLRM Medical College, Merrut. 2.2. Extraction The powdered roots were macerated [8], soaked in 95% EtOH for 10 days and filtered. The solution evaporated in vacuo gave a semi-gelatinous extract (yield: 10.5%). 2.3. Animals Wistar rats of either sex, weighing 100–150 g, were used. They were housed in well-ventilated room at 23.0 F 2 8C, humidity of 65–70% and photoperiod of 12-h light/dark cycle and fed with standard rodent pellet diet with tap water ad libitum. 2.4. Induction of hepatic injury Hepatic injury was induced in rats by subcutaneous administration of a single dose of 0.3 ml/kg CC14 mixed with equal volume of olive oil on the 7th day, 2 h after the last treatment [9]. Animals were grouped as follows: GroupI: Control group, treated with vehicle (2.0 ml, p.o.) daily for 7 days, followed by olive oil treatment (0.3 ml, s.c.) on day 7. Group II: Treated with vehicle (2.0 ml, p.o.) daily for 7 days followed by CCl4 on day 7. Group III: Treated with silymarin (25 mg p.o.) daily for 7 days followed by CC14 on day 7. Groups IV, V, VI: Treated with ethanolic extract of B. vulgaris suspended in propylene glycol at doses of 1000, 2000 or 4000 mg/kg daily for 7 days followed by CC14 on day 7, respectively. On day 9, 48 h after CC14 administration, blood sample of each animal was taken from abdominal aorta under pentobarbitone anesthesia (35 mg/kg i.p.) and serum cholesterol [10], triglycerides [11], alanine amino (ALT) [12] and alkaline phosphatase (SAP) [13] were evaluated.
M. Agarwal et al. / Fitoterapia 77 (2006) 91–93
93
2.5. Statistical analysis All values are expressed as means F S.D. The results were calculated and subjected to analysis of variance (ANOVA) followed by Student’s t-test. P values of N0.05 were considered significant [14]. 3. Results and discussion It is well known that carbontetrachloride is converted by cytochrome P-450-mixed function oxygenases in smooth endoplasmic reticulum of liver into toxic metabolite, mainly trichloromethyl radical (CCI3!). This free radical in the presence of oxygen may cause peroxidation of lipids on target cell resulting in extensive damage [15]. Administration of CC14 (0.3 ml s.c.) to rats produced hepatotoxicity showed by significant increase in the serum levels of ALT and SAP as well as altered lipid profile in comparison to control group as shown in the Table 1. Ethanolic extract of B. vulgaris given at doses of 1000, 2000 and 4000 mg/kg not only prevented the rise in serum level of ALT and alkaline phosphatase but also improved serum lipid profile in a dose dependent manner. Silymarin, a well-known hepatoprotective drug, showed results comparable to that reported in the literature [16]. The constituents of B. vulgaris roots are not reported in the literature but it may be the possibility that it also contains some of the compounds found in seeds [4] and these may be responsible for hepatoprotection. Identification of chemical constituents along with their effects will be of great help in understanding the hepatoprotective activity of the ethanolic extract of B. vulgaris roots. Acknowledgements The authors thank Dr. C.D. Tripathi, Professor and Head, Department of Pharmacology VMMC Safdarjung Hospital, New Delhi, for his valuable suggestions. The study was conducted at the LLRM Medical College, Meerut. The Institutional Ethical Committee for Animal Experimentation approved the project. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
Subramoniam A, Pushpangadan P. Indian J Pharmacol 1999;31:166. Chopra RN, Nayar SL, Chopra C. Glossary of Indian medicinal plants. New Delhi7 CSIR; 1956. Prajapati ND, Purohit SS, Sharma AK, Kumar TA. A handbook of medicinal plants. A complete source book: Agorbios; 2003. p. 88. Wettasinghe M, Bolling B, Plhak L, Xiao H, Parkin K. J Agric Food Chem 2002;50:6704. Duke JA, Bogenschutz-Godwin MJ, Du J, Cellier P-AK. Handbook of medicinal herbs. Boca Raton, FL7 CRC Press;. 2002. p. 68. Saller R, Meier R, Brignoli R. Drugs 2001;61:2035. Skottova N, Krecman V. Physiol Res 1998;47:1. Riebling RW, Walker GC. Extraction and extractive Remington’s pharmaceuticals science, 15th ed. Pennsylvania Mack Publishing Company; 1975. p. 1509. Saraswat B, Visen PKS, Patnaik GK, Dhawan BN. Indian J Exp Biol 1993;31:316. Zlatkis A, Zak BB, Boyle GJB. J Lab Clin Med 1953;41:86. VanHandle E, Zilversmit DB. J Lab Clin Med 1957;50:152. Reitman S, Frankel S. Am J Clin Pathol 1957;28:56. Kind PRN, King EJ. J Clin Pathol 1971;7:322. Ghosh MN. Fundamentals of experimental pharmacology, 2nd ed. Calcutta7 Scientific Book Agency;. 1984. p. 154. Recknagel RO. Trends Pharmacol Sci 1983;4:129. Bhattacharyya D, Pandit S, Mukherjee R, Das N, Sur TK. Indian J Physiol Pharmacol 2003;47:435.