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Effect of Cassia fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats
Chemico-Biological Interactions 167 (2007) 12–18
Effect of Cassia fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats Kannampalli Pradeep, Chandrasekaran Victor Raj Mohan, Kuppannan Gobianand, Sivanesan Karthikeyan ∗ Department of Pharmacology and Environmental Toxicology, Dr. A.L.M. P.G. Institute of Basic Medical Science, University of Madras, Taramani, Chennai 600113, India Received 22 September 2006; received in revised form 17 December 2006; accepted 20 December 2006 Available online 30 December 2006
Abstract The hepatoprotective and antioxidant effect of Cassia fistula Linn. leaf extract on liver injury induced by diethylnitrosamine (DEN) was investigated. Wistar rats weighing 200 ± 10 g were administered a single dose of DEN (200 mg/kg b.w., i.p.) and left for 30 days. For hepatoprotective studies, ethanolic leaf extract (ELE) of C. fistula Linn. (500 mg/kg b.w., p.o.) was administered daily for 30 days. AST, ALT, ALP, LDH, ␥-GT and bilirubin were estimated in serum and liver tissue. Lipid peroxidation (LPO), SOD and CAT were also estimated in liver tissue as markers of oxidative stress. DEN induced hepatotoxicity in all the treated animals were evident by elevated serum ALT, AST, ALP and bilirubin levels and a simultaneous fall in their levels in the liver tissue after 30 days. Induction of oxidative stress in the liver was evidenced by increased LPO and fall in the activities of SOD and CAT. ELE administration for 30 days prevented the DEN induced hepatic injury and oxidative stress. In conclusion, it was observed that ELE of C. fistula Linn. protects the liver against DEN induced hepatic injury in rats. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Diethylnitrosamine; Cassia fistula Linn.; Hepatotoxicity
1. Introduction Diethylnitrosamine (DEN), a representative chemical of a family of N-nitroso compounds has been found distributed in processed meats, tobacco smoke, and whiskey [1]. DEN has been detected in wide variety of foods like cheese, soybean, smoked, salted and dried fish, cured meat, alcoholic beverages and ground water having high level of nitrates [2]. Metabolism of certain therapeutic
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drugs is also reported to produce DEN [3]. The International Agency for Research on Cancer (IARC) has classified DEN as a probable human carcinogen, despite the lack of epidemiologic data [4]. Administration of DEN to experimental animals has been shown to cause cancer in liver and at lower incidences, in other organs as well [5,6]. It is also reported to be a hepatotoxic agent causing hepatocellular necrosis in experimental animals [7]. Considering the above factors, it is likely that human exposure to DEN is inevitable. Hence, the development of an effective hepatoprotective agent against DEN induced hepatotoxicity has become the need of the day. Medicinal plants are being used for the treatment of various diseases in different parts of the world from time immemorial. Natural products from plants, fungi,
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K. Pradeep et al. / Chemico-Biological Interactions 167 (2007) 12–18
bacteria and other organisms continue to be used in pharmaceutical preparations either as pure compounds or as extracts [8]. The use of medicinal plants is becoming popular both in developed and developing countries as the demand for developing better, safe and specific drugs is high [9]. Developing drugs from natural products may reduce the risk of toxicity and maintain its therapeutic effectiveness, when the drug is used clinically. In recent years, there has been considerable emphasis on the identification of plant products with antioxidant property, as free radicals are considered to play a major role in most of the diseases. Cassia fistula Linn. (Family: Caesalpinaceae) is commonly called Indian Labernum and is native to India, the Amazon, Sri Lanka and is extensively diffused in various countries including Mauritius, South Africa, Mexico, China, West Indies, East Africa and Brazil [10]. It is widely used for its medicinal properties, the main property being that of a mild laxative suitable for children and pregnant women. It is also a purgative due to the wax aloin and a tonic [11] and has been reported to treat many intestinal disorders [12,13]. The plant has a high therapeutic value and it exerts an antipyretic and analgesic effect [14]. C. fistula Linn. extract is used as an anti-periodic agent in the treatment of rheumatism [12,13] and the leaf extract is also used for its anti-tussive and wound healing properties [15,16]. It has been concluded that plant parts could be used as a therapeutic agent in the treatment of hypercholesterolaemia partially due to their fiber and mucilage content [17]. Antitumor [18], hepatoprotective [19], antifertility [20] and antioxidant [21–23] properties of C. fistula Linn. as well as its actions on the central nervous systems [24] and inhibitory effect on leukotriene biosynthesis [25] have also been suggested. In spite of the vast pharmacological activities of this plant, its protective effect on the liver has not been well documented. Hence, the present investigation was designed to evaluate the hepatoprotective and antioxidant properties of the ethanolic leaf extract of C. fistula Linn. against hepatotoxicity induced by DEN in rats. 2. Materials and methods 2.1. Animals Wistar albino male rats weighing 200 ± 10 g purchased from Tamil Nadu Veterinary and Animal Sciences University, Chennai, were used in this study. They were housed in polypropylene cages with 12 h light and dark cycle. Animals were fed standard pellet feed and water ad libitum. All animal experiments were
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performed in accordance with the strict guidelines prescribed by the Institutional Animal Ethical Committee (IAEC) and after getting necessary approval. 2.2. Chemicals Diethylnitrosamine (DEN) was purchased from Sigma Chemical Company, USA. All other chemicals used were of analytical grade and were purchased locally. 2.3. Collection of plant material Fresh leaves of C. fistula Linn. were collected from the Tamil Nadu Medicinal Plant Farms and Herbal Medicine Corporation Ltd. (TAMPCOL), Chennai, during the month of July–August and were authenticated by the Chief Botanist of TAMPCOL. A herbarium specimen of the leaf is deposited in Botanical Survey of India, Coimbatore and Presidency College, Chennai, India. 2.4. Preparation of ethanolic leaf extract (ELE) Freshly collected leaves were washed in tap water and then with distilled water and were shade dried for about 72 h. The dried leaves were crushed into a coarse powder. One hundred gram of the powder was soaked in 1 l of ethanol (95%) for 30 days with occasional shaking. After 30 days this ethanolic leaf extract (ELE) was filtered using Whatmann filter paper no. 1 and evaporated to dryness over a water bath at 60 ◦ C. The yield of ELE was around 19–20%. 2.5. Experimental design Rats were divided into four groups with six animals in each group. The experimental design was as follows: Group-I rats served as controls and were treated with saline orally for 30 days; Group-II rats were administered a single dose of DEN (200 mg/kg b.w., i.p.) [26] on day 0 and left for 30 days; Group-III rats were administered DEN (200 mg/kg b.w., i.p.) on day 0 followed by ELE (500 mg/kg b.w., p.o.) from day 1 till day 30; Group-IV rats were treated with ELE (500 mg/kg b.w., p.o.) alone from day 1 till day 30. The dose of ELE was selected by performing an effective dose fixation study. At the end of experimental period, animals were subjected to ether anaesthesia, blood was collected from retro orbital plexus and serum was separated by centrifugation. Animals were sacrificed by cervical decapitation and the liver was excised, washed in ice-cold saline and blotted to dryness. A 1% homogenate of the liver tis-
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sue was prepared in Tris–HCl buffer (0.1 M; pH 7.4), centrifuged and the clear supernatant used for further biochemical assays. Aspartate and alanine transaminases (AST and ALT) were assayed according to the method of Wooten [27]. The method is based on the ability of the enzymes to form pyruvate, which reacts with 2,4-dinitrophenylhydrazine in hydrochloric acid. The hydrazone thus formed turns into an orange complex in alkaline medium, which was measured at 540 nm. Alkaline phosphatase (ALP) was estimated according to King [28] where the phenol liberated by enzymatic hydrolysis (at pH 10) from disodium phenyl phosphate was estimated spectrophotometrically at 640 nm. Lactate dehydrogenase (LDH) was estimated according to the method of King [29]. The method is based on the ability of LDH to form pyruvate in the presence of NAD+ . The liberated pyruvate was estimated as described above. ␥-Glutamyl transferase (␥-GT) was estimated as described by Rosalki and Rau [30] in which the pnitroaniline liberated by the enzyme in the presence of l-␥-glutamyl-p-nitroanilide, produces a yellow colour, which was estimated spectrophotometrically at 410 nm. Total serum bilirubin (BIL) was estimated by the method of Malloy and Evelyn [31], which is based on the reaction of bilirubin with diazo reagent to form a purple compound, which was measured at 540 nm. Lipid peroxidation (LPO) was determined in the liver tissue as described by Ohkawa et al. [32]. Briefly, malondialdehyde (MDA), formed as an end product of the peroxidation of lipids was made to react with thiobarbituric acid to generate a coloured complex, which has absorption maxima at 532 nm. Superoxide dismutase (SOD) in the liver tissue was estimated according to Marklund and Marklund [33]. The degree of inhibition of the autooxidation of pyrogallol at an alkaline pH by SOD was used as a measure of the enzyme activity. Catalase (CAT) in the liver tissue was estimated according to the method of Sinha [34]. In this method, dichromate in acetic acid was reduced to chromic acetate when heated in the presence of hydrogen peroxide (H2 O2 ), with the formation of perchloric
acid as an unstable intermediate. Chromic acetate thus produced was measured at 570 nm. 2.6. Statistical analysis The data was subjected to one-way ANOVA and Tukey’s multiple comparison test was performed using SPSS Statistical Package (version 7.5). Values are expressed as mean ± S.E. P-value < 0.05 was considered significant. 3. Results The activity of AST, ALT and ALP in the serum of control and experimental animals is presented in Table 1. It is observed that administration of a single dose of DEN (Group-II) to rats produced a significant increase in the activities of all the three marker enzymes, the increase being two-fold for AST and ALP, whereas it was three-fold for ALT when compared to control rats. Treatment with ELE for 30 days after DEN administration (Group-III) significantly decreased the activities of these enzymes to normalcy. The activity of ␥-GT, LDH and BIL in the serum of control and experimental animals is presented in Table 2. A similar increase in the activities of these enzymes was observed in serum of rats after administration of DEN (Group-II) when compared to control rats. ELE treatment for 30 days (Group-III) markedly decreased the activity of these enzymes in rats towards normalcy when compared to DEN treated rats. In contrast to this, the liver tissue produced a significant fall in the levels of AST, ALT and ALP (Fig. 1) and a significant increase in the levels of LDH and ␥-GT (Fig. 2) upon administration of DEN. Treatment with ELE for 30 days after DEN administration significantly reduced the altered activities of these enzymes back to normalcy. The lipid peroxidation (LPO) level in the liver tissue of control and experimental animals is presented in Fig. 3. DEN administration produced a profound elevation (three-fold) in the levels of MDA, as compared to control rats. Treatment with ELE for 30 days after
Table 1 Effect of DEN and ELE on the status of AST, ALT and ALP in serum of experimental animals Particulars
Group-I (control)
Group-II (DEN)
Group-III (DEN + ELE)
Group-IV (ELE)
AST ALT ALP
42.88 ± 1.82 42.29 ± 1.29 107.01 ± 1.23
83.52 ± 1.86 124.44 ± 5.96 a*** 211.03 ± 10.52 a***
53.90 ± 5.88 53.90 ± 5.88 b*** 151.37 ± 6.34 a** b***
55.63 ± 3.06 55.91 ± 5.47 123.28 ± 9.16
a***
b***
Results are given as mean ± S.E. for six rats. Units: AST, ALT and ALP in IU/L. Comparisons are made between: a, control rats (Group-I); b, DEN treated rats (Group-II). The symbols (*** ) and (** ) represent statistical significance at P < 0.001 and P < 0.01, respectively.
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Table 2 Effect of DEN and ELE on the status of status of ␥-GT, LDH and BIL in serum of experimental animals Particulars
Group-I (control)
Group-II (DEN)
Group-III (DEN + ELE)
Group-IV (ELE)
␥-GT LDH BIL
89.32 ± 3.64 146.69 ± 2.04 0.666 ± 0.08
201.73 ± 6.54 a*** 336.07 ± 8.52 a*** 1.92 ± 0.04 a***
96.63 ± 4.03 b*** 190.53 ± 6.98 ab*** 1.30 ± 0.04 ab***
89.46 ± 1.65 163.68 ± 2.16 0.70 ± 0.04
Results are given as mean ± S.E. for six rats. Units: ␥-GT and LDH in IU/L and BIL in mg/dL. Comparisons are made between: a, control rats (Group-I); b, DEN treated rats (Group-II). The symbol (*** ) represent statistical significance at P < 0.001.
Fig. 1. Effect of DEN and ELE on the status of AST, ALT and ALP in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (Group-I); b, compared with DEN treated rats (Group-II). The symbols (*** ) and (** ) represent statistical significance at P < 0.001 and P < 0.01, respectively. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with DEN + ELE; ( )—Group-IV: rats administered with ELE alone.
Fig. 3. Effect of DEN and ELE on lipid peroxidation in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (GroupI); b, compared with DEN treated rats (Group-II). The symbol (*** ) represent statistical significance at P < 0.001. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with DEN + ELE; ( )—Group-IV: rats administered with ELE alone.
DEN administration significantly decreased the levels of MDA, when compared with Group-II rats. The activity of SOD and CAT in the liver tissue of control and experimental rats is presented in Figs. 4 and 5, respec-
tively. There was a significant decrease in the activity of both SOD (60%) and CAT (40%) upon administration of DEN in the liver tissue, which was restored back to normalcy upon treatment with ELE for 30 days. Administration of ELE alone (Group-IV) for a period of 30 days
Fig. 2. Effect of DEN and ELE on the status of LDH and ␥-GT in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (Group-I); b, compared with DEN treated rats (Group-II). The symbols (*** ) and (* ) represent statistical significance at P < 0.001 and P < 0.051, respectively. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with a single dose of DEN + ELE; ( )—Group-IV: rats administered with a single dose of ELE alone.
Fig. 4. Effect of DEN and ELE on the activity of SOD in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (GroupI); b, compared with DEN treated rats (Group-II). The symbol (*** ) represent statistical significance at P < 0.001. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with a single dose of DEN + ELE; ( )—Group-IV: rats administered with a single dose of ELE alone.
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Fig. 5. Effect of DEN and ELE on the activity of CAT in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (GroupI); b, compared with DEN treated rats (Group-II). The symbol (*** ) represent statistical significance at P < 0.001. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with DEN + ELE; ( )—Group-IV: rats administered with ELE alone.
did not produce any significant alterations in most of the parameters investigated. 4. Discussion DEN is reported to be a well-known hepatotoxin and hepatocarcinogen. Studies have shown that hepatic metabolism of DEN generates reactive oxygen species (ROS) resulting in oxidative stress and cellular injury [35]. In the present investigation, DEN induced hepatocellular damage is clearly evidenced by the marked elevation in the activity of serum AST, ALT, ALP and ␥-GT and a simultaneous fall in the liver tissue. Serum AST, ALT, ALP and BIL are the most sensitive markers employed in the diagnosis of hepatic damage because these are cytoplasmic in location and are released into the circulation after cellular damage [36]. High serum concentrations of transaminases are taken as an index of hepatic injury where elevation of ALT is regarded as a more sensitive indicator [37] and this tendency is also known to be distinct in rodents [38]. Elevated activity of transaminases in serum observed in this study might be due to the release of these enzymes from the cytoplasm, into the blood circulation rapidly after rupture of the plasma membrane and cellular damage. This is substantiated by a simultaneous fall in the activity of these markers in the liver tissue. Free radicals released by the metabolism of DEN might have caused damage to the hepatocellular membranes, resulting in the leakage of cytosolic contents into the systemic circulation. Increase in the levels of LDH has been reported in conditions like hemolytic anemia, hepatocellular necrosis and hepatocellular carcinoma [39]. ␥-GT is considered to be a more sensitive indicator of hepatobiliary disease
and measurement of serum ␥-GT is a frequently used parameter in liver diseases [40]. The elevated activity of LDH and ␥-GT observed in this study might be due to hepatic necrosis or premalignant hepatocellular lesions induced by DEN. Treatment with ELE for 30 days significantly reduced the status of these marker enzymes in DEN treated rats. This indicates that ELE tends to prevent liver damage by suppressing the leakage of enzymes through cellular membranes by preserving the integrity of the plasma membranes thereby restoring the status of these enzymes. Membrane lipids are easily susceptible to deleterious actions of reactive oxygen species [41]. Measurement of lipid peroxidation is considered to be a convenient method to monitor oxidative membrane damage [42]. In the present study, a three-fold increase in the levels of LPO observed in liver tissue of DEN administered rats is the consequence of oxidative stress caused by DEN resulting from peroxidative membrane damage. Free radicals mediated peroxidation of membrane lipids might have resulted in loss of membrane integrity and membrane damage, leading to its rupture and subsequent release of the cytosolic contents. Previous studies have show ELE to be an effective antioxidant under in vitro conditions [22]. The fall in the levels of LPO observed after treatment with ELE in the present study is suggestive of the fact that ELE is successful in quenching the free radicals thereby inhibiting lipid peroxidation and protecting the membrane lipids from oxidative damage in the liver of rats. Hence ELE acts as an effective antioxidant in in vivo conditions also. SOD and CAT constitute a team of mutually supportive antioxidative enzymes, which provide protective defense against reactive oxygen species [43]. DEN administration decreased the activities of these antioxidant enzymes in the liver tissue by about 50%. The decline in the activities of these enzymes in the present study could be attributed to the excessive utilization of these enzymes in inactivating the free radicals generated during the metabolism of DEN. The restoration in the activities of these enzymes after ELE administration for 30 days indicates that ELE acts as an effective antioxidant. ELE possess phytochemicals like flavanoids, alkaloids and tannins, which have been shown to be effective in scavenging free radicals [23]. Therefore by acting as an alternate radical scavenger, ELE can replenish these antioxidant enzymes, thereby preventing oxidative stress. This fact is further substantiated by the decrease in the levels of LPO upon ELE administration. These results of our investigations are in accordance with that of Bhakta et al. [19,44] who have reported the hepatoprotective activity of n-hexane leaf
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extract of C. fistula Linn. against CCl4 and paracetamol induced hepatotoxicity. We have already reported the hepatoprotective effect of ELE of C. fistula Linn. against CCl4 induced hepatotoxicity in rats [45], but its efficacy against DEN induced hepatotoxicity in rats is reported here for the first time. CCl4 is metabolized by cytochrome P450 systems to yield trichloromethyl radical (CCl3 • ) and peroxy radical (OOCCl3 • ) [46], which are highly reactive oxygen species (ROS) and are capable of combining with membrane lipids and proteins in the presence of O2 to induce LPO by H• abstraction [47,48]. ELE was reported to be highly effective in scavenging ROS and thereby offering hepatoprotection against CCl4 induced hepatotoxicity in rats [45]. Studies have shown that metabolism of DEN also results in the production of ROS like H2 O2 , superoxide anions (O2 − ) and hydroxyl radical (OH• ) which has been suggested to invoke pathological conditions such as induction of hepatocellular necrosis, carcinogenicity, neoplastic changes and tumor formation in the liver during DEN administration [49,50]. Since ELE is effective in scavenging the ROS as described earlier, it can be hypothesized that ELE might have scavenged and detoxified the superoxide anions and hydroxyl radicals released during the metabolic activation of DEN, thereby inhibiting lipid peroxidation and improving the activities of hepatic markers and antioxidant enzymes. In conclusion, our present study shows that the ELE of C. fistula Linn. exhibits excellent hepatoprotective and antioxidant properties against DEN induced hepatotoxicity in rats, which is mainly attributed to the phytochemicals present in them. Acknowledgement
[5]
[6]
[7]
[8] [9]
[10] [11] [12] [13] [14]
[15]
[16]
[17]
[18]
The authors wish to thank the UGC-UWPFE Project (HS-43) for the financial assistance provided for this study.
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Effect of Cassia fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats Kannampalli Pradeep, Chandrasekaran Victor Raj Mohan, Kuppannan Gobianand, Sivanesan Karthikeyan ∗ Department of Pharmacology and Environmental Toxicology, Dr. A.L.M. P.G. Institute of Basic Medical Science, University of Madras, Taramani, Chennai 600113, India Received 22 September 2006; received in revised form 17 December 2006; accepted 20 December 2006 Available online 30 December 2006
Abstract The hepatoprotective and antioxidant effect of Cassia fistula Linn. leaf extract on liver injury induced by diethylnitrosamine (DEN) was investigated. Wistar rats weighing 200 ± 10 g were administered a single dose of DEN (200 mg/kg b.w., i.p.) and left for 30 days. For hepatoprotective studies, ethanolic leaf extract (ELE) of C. fistula Linn. (500 mg/kg b.w., p.o.) was administered daily for 30 days. AST, ALT, ALP, LDH, ␥-GT and bilirubin were estimated in serum and liver tissue. Lipid peroxidation (LPO), SOD and CAT were also estimated in liver tissue as markers of oxidative stress. DEN induced hepatotoxicity in all the treated animals were evident by elevated serum ALT, AST, ALP and bilirubin levels and a simultaneous fall in their levels in the liver tissue after 30 days. Induction of oxidative stress in the liver was evidenced by increased LPO and fall in the activities of SOD and CAT. ELE administration for 30 days prevented the DEN induced hepatic injury and oxidative stress. In conclusion, it was observed that ELE of C. fistula Linn. protects the liver against DEN induced hepatic injury in rats. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Diethylnitrosamine; Cassia fistula Linn.; Hepatotoxicity
1. Introduction Diethylnitrosamine (DEN), a representative chemical of a family of N-nitroso compounds has been found distributed in processed meats, tobacco smoke, and whiskey [1]. DEN has been detected in wide variety of foods like cheese, soybean, smoked, salted and dried fish, cured meat, alcoholic beverages and ground water having high level of nitrates [2]. Metabolism of certain therapeutic
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Corresponding author. Tel.: +91 44 2454 5317; fax: +91 44 2454 0709. E-mail address: [email protected] (K. Pradeep).
drugs is also reported to produce DEN [3]. The International Agency for Research on Cancer (IARC) has classified DEN as a probable human carcinogen, despite the lack of epidemiologic data [4]. Administration of DEN to experimental animals has been shown to cause cancer in liver and at lower incidences, in other organs as well [5,6]. It is also reported to be a hepatotoxic agent causing hepatocellular necrosis in experimental animals [7]. Considering the above factors, it is likely that human exposure to DEN is inevitable. Hence, the development of an effective hepatoprotective agent against DEN induced hepatotoxicity has become the need of the day. Medicinal plants are being used for the treatment of various diseases in different parts of the world from time immemorial. Natural products from plants, fungi,
0009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2006.12.011
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bacteria and other organisms continue to be used in pharmaceutical preparations either as pure compounds or as extracts [8]. The use of medicinal plants is becoming popular both in developed and developing countries as the demand for developing better, safe and specific drugs is high [9]. Developing drugs from natural products may reduce the risk of toxicity and maintain its therapeutic effectiveness, when the drug is used clinically. In recent years, there has been considerable emphasis on the identification of plant products with antioxidant property, as free radicals are considered to play a major role in most of the diseases. Cassia fistula Linn. (Family: Caesalpinaceae) is commonly called Indian Labernum and is native to India, the Amazon, Sri Lanka and is extensively diffused in various countries including Mauritius, South Africa, Mexico, China, West Indies, East Africa and Brazil [10]. It is widely used for its medicinal properties, the main property being that of a mild laxative suitable for children and pregnant women. It is also a purgative due to the wax aloin and a tonic [11] and has been reported to treat many intestinal disorders [12,13]. The plant has a high therapeutic value and it exerts an antipyretic and analgesic effect [14]. C. fistula Linn. extract is used as an anti-periodic agent in the treatment of rheumatism [12,13] and the leaf extract is also used for its anti-tussive and wound healing properties [15,16]. It has been concluded that plant parts could be used as a therapeutic agent in the treatment of hypercholesterolaemia partially due to their fiber and mucilage content [17]. Antitumor [18], hepatoprotective [19], antifertility [20] and antioxidant [21–23] properties of C. fistula Linn. as well as its actions on the central nervous systems [24] and inhibitory effect on leukotriene biosynthesis [25] have also been suggested. In spite of the vast pharmacological activities of this plant, its protective effect on the liver has not been well documented. Hence, the present investigation was designed to evaluate the hepatoprotective and antioxidant properties of the ethanolic leaf extract of C. fistula Linn. against hepatotoxicity induced by DEN in rats. 2. Materials and methods 2.1. Animals Wistar albino male rats weighing 200 ± 10 g purchased from Tamil Nadu Veterinary and Animal Sciences University, Chennai, were used in this study. They were housed in polypropylene cages with 12 h light and dark cycle. Animals were fed standard pellet feed and water ad libitum. All animal experiments were
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performed in accordance with the strict guidelines prescribed by the Institutional Animal Ethical Committee (IAEC) and after getting necessary approval. 2.2. Chemicals Diethylnitrosamine (DEN) was purchased from Sigma Chemical Company, USA. All other chemicals used were of analytical grade and were purchased locally. 2.3. Collection of plant material Fresh leaves of C. fistula Linn. were collected from the Tamil Nadu Medicinal Plant Farms and Herbal Medicine Corporation Ltd. (TAMPCOL), Chennai, during the month of July–August and were authenticated by the Chief Botanist of TAMPCOL. A herbarium specimen of the leaf is deposited in Botanical Survey of India, Coimbatore and Presidency College, Chennai, India. 2.4. Preparation of ethanolic leaf extract (ELE) Freshly collected leaves were washed in tap water and then with distilled water and were shade dried for about 72 h. The dried leaves were crushed into a coarse powder. One hundred gram of the powder was soaked in 1 l of ethanol (95%) for 30 days with occasional shaking. After 30 days this ethanolic leaf extract (ELE) was filtered using Whatmann filter paper no. 1 and evaporated to dryness over a water bath at 60 ◦ C. The yield of ELE was around 19–20%. 2.5. Experimental design Rats were divided into four groups with six animals in each group. The experimental design was as follows: Group-I rats served as controls and were treated with saline orally for 30 days; Group-II rats were administered a single dose of DEN (200 mg/kg b.w., i.p.) [26] on day 0 and left for 30 days; Group-III rats were administered DEN (200 mg/kg b.w., i.p.) on day 0 followed by ELE (500 mg/kg b.w., p.o.) from day 1 till day 30; Group-IV rats were treated with ELE (500 mg/kg b.w., p.o.) alone from day 1 till day 30. The dose of ELE was selected by performing an effective dose fixation study. At the end of experimental period, animals were subjected to ether anaesthesia, blood was collected from retro orbital plexus and serum was separated by centrifugation. Animals were sacrificed by cervical decapitation and the liver was excised, washed in ice-cold saline and blotted to dryness. A 1% homogenate of the liver tis-
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sue was prepared in Tris–HCl buffer (0.1 M; pH 7.4), centrifuged and the clear supernatant used for further biochemical assays. Aspartate and alanine transaminases (AST and ALT) were assayed according to the method of Wooten [27]. The method is based on the ability of the enzymes to form pyruvate, which reacts with 2,4-dinitrophenylhydrazine in hydrochloric acid. The hydrazone thus formed turns into an orange complex in alkaline medium, which was measured at 540 nm. Alkaline phosphatase (ALP) was estimated according to King [28] where the phenol liberated by enzymatic hydrolysis (at pH 10) from disodium phenyl phosphate was estimated spectrophotometrically at 640 nm. Lactate dehydrogenase (LDH) was estimated according to the method of King [29]. The method is based on the ability of LDH to form pyruvate in the presence of NAD+ . The liberated pyruvate was estimated as described above. ␥-Glutamyl transferase (␥-GT) was estimated as described by Rosalki and Rau [30] in which the pnitroaniline liberated by the enzyme in the presence of l-␥-glutamyl-p-nitroanilide, produces a yellow colour, which was estimated spectrophotometrically at 410 nm. Total serum bilirubin (BIL) was estimated by the method of Malloy and Evelyn [31], which is based on the reaction of bilirubin with diazo reagent to form a purple compound, which was measured at 540 nm. Lipid peroxidation (LPO) was determined in the liver tissue as described by Ohkawa et al. [32]. Briefly, malondialdehyde (MDA), formed as an end product of the peroxidation of lipids was made to react with thiobarbituric acid to generate a coloured complex, which has absorption maxima at 532 nm. Superoxide dismutase (SOD) in the liver tissue was estimated according to Marklund and Marklund [33]. The degree of inhibition of the autooxidation of pyrogallol at an alkaline pH by SOD was used as a measure of the enzyme activity. Catalase (CAT) in the liver tissue was estimated according to the method of Sinha [34]. In this method, dichromate in acetic acid was reduced to chromic acetate when heated in the presence of hydrogen peroxide (H2 O2 ), with the formation of perchloric
acid as an unstable intermediate. Chromic acetate thus produced was measured at 570 nm. 2.6. Statistical analysis The data was subjected to one-way ANOVA and Tukey’s multiple comparison test was performed using SPSS Statistical Package (version 7.5). Values are expressed as mean ± S.E. P-value < 0.05 was considered significant. 3. Results The activity of AST, ALT and ALP in the serum of control and experimental animals is presented in Table 1. It is observed that administration of a single dose of DEN (Group-II) to rats produced a significant increase in the activities of all the three marker enzymes, the increase being two-fold for AST and ALP, whereas it was three-fold for ALT when compared to control rats. Treatment with ELE for 30 days after DEN administration (Group-III) significantly decreased the activities of these enzymes to normalcy. The activity of ␥-GT, LDH and BIL in the serum of control and experimental animals is presented in Table 2. A similar increase in the activities of these enzymes was observed in serum of rats after administration of DEN (Group-II) when compared to control rats. ELE treatment for 30 days (Group-III) markedly decreased the activity of these enzymes in rats towards normalcy when compared to DEN treated rats. In contrast to this, the liver tissue produced a significant fall in the levels of AST, ALT and ALP (Fig. 1) and a significant increase in the levels of LDH and ␥-GT (Fig. 2) upon administration of DEN. Treatment with ELE for 30 days after DEN administration significantly reduced the altered activities of these enzymes back to normalcy. The lipid peroxidation (LPO) level in the liver tissue of control and experimental animals is presented in Fig. 3. DEN administration produced a profound elevation (three-fold) in the levels of MDA, as compared to control rats. Treatment with ELE for 30 days after
Table 1 Effect of DEN and ELE on the status of AST, ALT and ALP in serum of experimental animals Particulars
Group-I (control)
Group-II (DEN)
Group-III (DEN + ELE)
Group-IV (ELE)
AST ALT ALP
42.88 ± 1.82 42.29 ± 1.29 107.01 ± 1.23
83.52 ± 1.86 124.44 ± 5.96 a*** 211.03 ± 10.52 a***
53.90 ± 5.88 53.90 ± 5.88 b*** 151.37 ± 6.34 a** b***
55.63 ± 3.06 55.91 ± 5.47 123.28 ± 9.16
a***
b***
Results are given as mean ± S.E. for six rats. Units: AST, ALT and ALP in IU/L. Comparisons are made between: a, control rats (Group-I); b, DEN treated rats (Group-II). The symbols (*** ) and (** ) represent statistical significance at P < 0.001 and P < 0.01, respectively.
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Table 2 Effect of DEN and ELE on the status of status of ␥-GT, LDH and BIL in serum of experimental animals Particulars
Group-I (control)
Group-II (DEN)
Group-III (DEN + ELE)
Group-IV (ELE)
␥-GT LDH BIL
89.32 ± 3.64 146.69 ± 2.04 0.666 ± 0.08
201.73 ± 6.54 a*** 336.07 ± 8.52 a*** 1.92 ± 0.04 a***
96.63 ± 4.03 b*** 190.53 ± 6.98 ab*** 1.30 ± 0.04 ab***
89.46 ± 1.65 163.68 ± 2.16 0.70 ± 0.04
Results are given as mean ± S.E. for six rats. Units: ␥-GT and LDH in IU/L and BIL in mg/dL. Comparisons are made between: a, control rats (Group-I); b, DEN treated rats (Group-II). The symbol (*** ) represent statistical significance at P < 0.001.
Fig. 1. Effect of DEN and ELE on the status of AST, ALT and ALP in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (Group-I); b, compared with DEN treated rats (Group-II). The symbols (*** ) and (** ) represent statistical significance at P < 0.001 and P < 0.01, respectively. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with DEN + ELE; ( )—Group-IV: rats administered with ELE alone.
Fig. 3. Effect of DEN and ELE on lipid peroxidation in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (GroupI); b, compared with DEN treated rats (Group-II). The symbol (*** ) represent statistical significance at P < 0.001. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with DEN + ELE; ( )—Group-IV: rats administered with ELE alone.
DEN administration significantly decreased the levels of MDA, when compared with Group-II rats. The activity of SOD and CAT in the liver tissue of control and experimental rats is presented in Figs. 4 and 5, respec-
tively. There was a significant decrease in the activity of both SOD (60%) and CAT (40%) upon administration of DEN in the liver tissue, which was restored back to normalcy upon treatment with ELE for 30 days. Administration of ELE alone (Group-IV) for a period of 30 days
Fig. 2. Effect of DEN and ELE on the status of LDH and ␥-GT in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (Group-I); b, compared with DEN treated rats (Group-II). The symbols (*** ) and (* ) represent statistical significance at P < 0.001 and P < 0.051, respectively. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with a single dose of DEN + ELE; ( )—Group-IV: rats administered with a single dose of ELE alone.
Fig. 4. Effect of DEN and ELE on the activity of SOD in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (GroupI); b, compared with DEN treated rats (Group-II). The symbol (*** ) represent statistical significance at P < 0.001. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with a single dose of DEN + ELE; ( )—Group-IV: rats administered with a single dose of ELE alone.
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Fig. 5. Effect of DEN and ELE on the activity of CAT in liver tissue of experimental animals. Results are given as mean ± S.E. for six rats. Comparisons are made between: a, compared with control rats (GroupI); b, compared with DEN treated rats (Group-II). The symbol (*** ) represent statistical significance at P < 0.001. ( )—Group-I: control rats administered with saline alone; ( )—Group-II: rats administered with a single dose of DEN alone; ( )—Group-III: rats administered with DEN + ELE; ( )—Group-IV: rats administered with ELE alone.
did not produce any significant alterations in most of the parameters investigated. 4. Discussion DEN is reported to be a well-known hepatotoxin and hepatocarcinogen. Studies have shown that hepatic metabolism of DEN generates reactive oxygen species (ROS) resulting in oxidative stress and cellular injury [35]. In the present investigation, DEN induced hepatocellular damage is clearly evidenced by the marked elevation in the activity of serum AST, ALT, ALP and ␥-GT and a simultaneous fall in the liver tissue. Serum AST, ALT, ALP and BIL are the most sensitive markers employed in the diagnosis of hepatic damage because these are cytoplasmic in location and are released into the circulation after cellular damage [36]. High serum concentrations of transaminases are taken as an index of hepatic injury where elevation of ALT is regarded as a more sensitive indicator [37] and this tendency is also known to be distinct in rodents [38]. Elevated activity of transaminases in serum observed in this study might be due to the release of these enzymes from the cytoplasm, into the blood circulation rapidly after rupture of the plasma membrane and cellular damage. This is substantiated by a simultaneous fall in the activity of these markers in the liver tissue. Free radicals released by the metabolism of DEN might have caused damage to the hepatocellular membranes, resulting in the leakage of cytosolic contents into the systemic circulation. Increase in the levels of LDH has been reported in conditions like hemolytic anemia, hepatocellular necrosis and hepatocellular carcinoma [39]. ␥-GT is considered to be a more sensitive indicator of hepatobiliary disease
and measurement of serum ␥-GT is a frequently used parameter in liver diseases [40]. The elevated activity of LDH and ␥-GT observed in this study might be due to hepatic necrosis or premalignant hepatocellular lesions induced by DEN. Treatment with ELE for 30 days significantly reduced the status of these marker enzymes in DEN treated rats. This indicates that ELE tends to prevent liver damage by suppressing the leakage of enzymes through cellular membranes by preserving the integrity of the plasma membranes thereby restoring the status of these enzymes. Membrane lipids are easily susceptible to deleterious actions of reactive oxygen species [41]. Measurement of lipid peroxidation is considered to be a convenient method to monitor oxidative membrane damage [42]. In the present study, a three-fold increase in the levels of LPO observed in liver tissue of DEN administered rats is the consequence of oxidative stress caused by DEN resulting from peroxidative membrane damage. Free radicals mediated peroxidation of membrane lipids might have resulted in loss of membrane integrity and membrane damage, leading to its rupture and subsequent release of the cytosolic contents. Previous studies have show ELE to be an effective antioxidant under in vitro conditions [22]. The fall in the levels of LPO observed after treatment with ELE in the present study is suggestive of the fact that ELE is successful in quenching the free radicals thereby inhibiting lipid peroxidation and protecting the membrane lipids from oxidative damage in the liver of rats. Hence ELE acts as an effective antioxidant in in vivo conditions also. SOD and CAT constitute a team of mutually supportive antioxidative enzymes, which provide protective defense against reactive oxygen species [43]. DEN administration decreased the activities of these antioxidant enzymes in the liver tissue by about 50%. The decline in the activities of these enzymes in the present study could be attributed to the excessive utilization of these enzymes in inactivating the free radicals generated during the metabolism of DEN. The restoration in the activities of these enzymes after ELE administration for 30 days indicates that ELE acts as an effective antioxidant. ELE possess phytochemicals like flavanoids, alkaloids and tannins, which have been shown to be effective in scavenging free radicals [23]. Therefore by acting as an alternate radical scavenger, ELE can replenish these antioxidant enzymes, thereby preventing oxidative stress. This fact is further substantiated by the decrease in the levels of LPO upon ELE administration. These results of our investigations are in accordance with that of Bhakta et al. [19,44] who have reported the hepatoprotective activity of n-hexane leaf
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extract of C. fistula Linn. against CCl4 and paracetamol induced hepatotoxicity. We have already reported the hepatoprotective effect of ELE of C. fistula Linn. against CCl4 induced hepatotoxicity in rats [45], but its efficacy against DEN induced hepatotoxicity in rats is reported here for the first time. CCl4 is metabolized by cytochrome P450 systems to yield trichloromethyl radical (CCl3 • ) and peroxy radical (OOCCl3 • ) [46], which are highly reactive oxygen species (ROS) and are capable of combining with membrane lipids and proteins in the presence of O2 to induce LPO by H• abstraction [47,48]. ELE was reported to be highly effective in scavenging ROS and thereby offering hepatoprotection against CCl4 induced hepatotoxicity in rats [45]. Studies have shown that metabolism of DEN also results in the production of ROS like H2 O2 , superoxide anions (O2 − ) and hydroxyl radical (OH• ) which has been suggested to invoke pathological conditions such as induction of hepatocellular necrosis, carcinogenicity, neoplastic changes and tumor formation in the liver during DEN administration [49,50]. Since ELE is effective in scavenging the ROS as described earlier, it can be hypothesized that ELE might have scavenged and detoxified the superoxide anions and hydroxyl radicals released during the metabolic activation of DEN, thereby inhibiting lipid peroxidation and improving the activities of hepatic markers and antioxidant enzymes. In conclusion, our present study shows that the ELE of C. fistula Linn. exhibits excellent hepatoprotective and antioxidant properties against DEN induced hepatotoxicity in rats, which is mainly attributed to the phytochemicals present in them. Acknowledgement
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The authors wish to thank the UGC-UWPFE Project (HS-43) for the financial assistance provided for this study.
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