Brazilian Journal of Biological Sciences, 2017, v. 4, No. 7, p. 53-66. ISSN 2358-2731 http://dx.doi.org/10.21472/bjbs.040707

Anticarcinogenic potential of ethanol extract of cordifolia Roth. (: ) on diethylnitrosamine induced hepatocarcinogenesis in rats Raju Senthil Kumar¹,*, Gopal Murugananthan¹ and Balasubramanian Rajkapoor²

¹Natural Products Laboratory. Swamy Vivekanandha College of Pharmacy. Tiruchengode. Tamilnadu. India. *Email: [email protected]. ²Department of Pharmacology. Faculty of Medicine. Sebha University. Sebha. Libya.

Abstract. Roth. (Fabales: Fabaceae) has been widely used in Indian system of medicine to treat various disorders. Received Earlier studies showed that I. cordifolia (EIC) possess antioxidant, May 17, 2017 free radical scavenging and antitumour activities. The present investigation was designed to evaluate the anticarcinogenic potential Accepted of EIC against diethylnitrosamine (DEN) induced hepatocellular June 24, 2017 carcinoma in male Wistar rats. Hepatocarcinogenesis was induced by a single intraperitoneal administration of DEN (200 mg/kg) and Released the carcinogenic effect was promoted by phenobarbital given through drinking water for 16 successive weeks. EIC at the dose of June 30, 2017 200 and 400 mg/kg/day were administered orally for the entire study Open Acess period. After the end of experimental period, changes in body Full Text Article weight, the weight of liver, relative liver weight, lipid peroxidation, antioxidant, serum hepatic parameters, tumour markers, DNA, RNA and protein content were analysed. Treatment with EIC significantly increased the body weight (P < 0.01-0.001), reduced the liver weight and relative liver weight (P < 0.01-0.001), restored the altered serum hepatic parameters (P < 0.001), down-regulated the serum tumour markers such as alpha-fetoprotein and carcinoembryonic antigen (P < 0.001) when compared to DEN group. EIC treatment restored the antioxidant enzymes and significantly reduced the lipid peroxidation in DEN-treated animals (P < 0.001). EIC treatment also significantly reduced the elevated levels of nucleic acid levels and restored the protein content in liver tissues (P < 0.001). We ORCID investigated the anticarcinogenic potential of EIC against DEN- 0000-0003-1309-3886 induced HCC in rats. Chemoprotective effect of the extract might be Raju Senthil Kumar related with antioxidant, free radical scavenging and reduction of 0000-0001-6472-9173 lipid peroxidation. The results suggested that EIC would be a potent Gopal Murugananthan anticarcinogenic agent inhibiting DEN-induced hepatic carcinoma. 0000-0002-1125-7800 Keywords: Indigofera cordifolia; Diethylnitrosamine; Serum Balasubramanian hepatic parameters; Carcinoembryonic antigen; Alpha-fetoprotein; Rajkapoor Chemoprevention.

Introduction around the globe. Increase in life expectation and adopting western diet and Cancer is a worldwide challenge lifestyles, tobacco use and exposure to and remains the largest reason of death carcinogens are the principal factors for

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increasing the burden of cancer in the and prognosis of HCC, chemoprevention developing countries. Hepatocellular has considered being the best approach in carcinoma (HCC) is the second leading lowering the morbidity and death associated cause of cancer mortality throughout the with this disease. Chemoprevention is the world (Jemal et al., 2011). It has been modern strategy to manage primary liver joined with totally different etiologies such cancer. It is the mode of cancer as chronic hepatitis B and C infection and management during which the prevalence alcohol use (Shih et al., 2009; Kumar et al., of the disease are often prevented, delayed 2011). Because of high tolerance of liver, or reversed by the administration of one or HCC is never detected at the early phase more natural and/or synthetic agent. and treatment includes poor prognosis in Epidemiologic evidence have exposed the most of the cases, creating it a very chemopreventive effect of the many important health problem (El-Serag and medicinal and their phytochemicals Rudolph, 2007). The relapse rates of HCC in the prevention of the disease. are high and also the survival rate of the Indigofera cordifolia Roth. patient with HCC has not increased in the (Fabales: Fabaceae) it is an Indian past few decades. Surgery, including medicinal and has long been used by transplantation, is presently the foremost ethnic people and native medical successful treatment for this frightful practitioners to treat diseases like disease. rheumatism, arthritis, inflammation, The liver is the major organ that neoplasm and liver diseases. Literature involves in the metabolism of many survey revealed that crude methanol and xenobiotics; hence, it is more liable to aqueous extracts of seeds of this plant has carcinogenic insult. Diethylnitrosamine potent antioxidant, radical scavenging and (DEN) is a potent environment carcinogen wound healing activities (Tambe et al., that is entered through the food chain. DEN 2010; Sarin and Upman, 2011; Khatri et al., is synthesised endogenously and located in 2013). Earlier studies on this plant in our occupational settings, tobacco smoke, laboratory revealed that the ethanol extract processed meat, alcoholic beverages, of entire plant possesses potent antioxidant, agricultural chemicals, cosmetics and radical scavenging and antineoplastic pharmaceutical substances. It is made from properties in transplantable tumour models metabolism of certain medication. It causes (Thangavel et al., 2014; Thangavel et al., proliferative, chronic and cancerous lesions 2015). Based on the results obtained from in the liver. It will alkylate DNA with itself our previous works, an attempt was made to being converted to the extremely reactive check the anticarcinogenic impact of molecule by CYP450 dependent ethanol extract of I. cordifolia (EIC) against oxygenases and generates reactive oxygen DEN-induced phenobarbitone promoted species (ROS) inflicting oxidative stress. It hepatocarcinogenesis in rat model. also induces chromosomal aberrations, micronuclei and chromatid exchanges in the Materials and methods liver and these mutations evoked by DEN are responsible for the event of carcinoma Chemicals (Sivaramakrishnan et al., 2008; Mandal et Diethylnitrosamine (DEN) and al., 2008; Jagadeesh et al., 2009). The rat phenobarbitone were purchased from model of DEN-induced carcinoma is the Sigma Chemicals Co. (St. Louis, MO, foremost accepted and widely used USA). Carboxy methyl cellulose (CMC) experimental model to study the was procured from Loba Chemie (Mumbai, chemopreventive and chemotherapeutic India). All other chemicals used for the potential of many drugs. Human liver experiments were of analytical grade. metabolises DEN similar to that of rat liver with relevance such as genetic alteration, Animals morphology and gene expression. Male Wistar rats (150-160 g) were Considering the narrow treatment options purchased from Venkateshwara Enterprises,

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Anticarcinogenic potential of Indigofera cordifolia 55

Bangalore, India. They were maintained in 400 mg/kg body weight (Thangavel et al., standard microlon boxes and were given 2015), by oral route for sixteen consecutive standard laboratory diet and water ad weeks on five days per week. At the end of libitum. The study was conducted after experiments, blood was collected from obtaining Institutional Animal Ethical overnight fasted animals by retro-orbital Committee (IAEC) clearance and the plexus method after anaesthetized slightly animals were handled as per the Committee with anaesthetic ether and serum was for the Purpose of Control and Supervision separated out. The animals were sacrificed of Experiments on Animals (CPCSEA) and the liver was immediately removed, guidelines of Good Laboratory Practice. washed with ice-cold saline and subsequently blotted with filter paper, then Plant material and extraction weighed. A small portion of liver was fixed The whole plant of I. cordifolia was in 10% formalin solution for collected from Tiruchengode, Namakkal histopathological studies. District, Tamilnadu, in the month of November 2015. The plant was then Serum biochemical and tumour authenticated by a botanist from Botanical markers estimation Survey of India, Coimbatore and a voucher Serum was analysed for aspartate specimen is preserved in our laboratory for aminotransferase (AST), alanine further reference (Voucher specimen No. P. aminotransferase (ALT), alkaline Chem/SVCP/15-16/IC-0213). The shade phosphatase (ALP), gamma glutamate dried, powdered plant material was transpeptidase (GGTP), total cholesterol extracted with 80% v/v ethanol by and total bilirubin by using commercial kits continuous hot percolation process using (Agappe diagnostics and Ecoline soxhlet apparatus for 72 h. The extract was Diagnostic Kits, India) following the filtered and concentrated to dryness under manufacturer’s instruction. Liver tumour reduced pressure and controlled markers like Alpha-fetoprotein (AFP) and temperature (50 °C-60 °C) in a rotary Carcinoembryonic antigen (CEA) in rat evaporator. A brownish yellow solid mass serum were estimated by using obtained was stored in a vacuum desiccator commercially available ELISA kits (USCN until further use. life science Inc. Wuhan, China) according to the manufacturer’s instruction. A 10% Experimental design liver homogenate of liver tissue was The rats were divided into four prepared in cold phosphate buffer (0.05M, groups, each group consisting of twelve pH 7.4) and it was centrifuged at 10,000 animals. The liver tumour was induced in rpm for 15 min at 4 °C to get a clear groups II-IV with a single intraperitoneal injection of diethylnitrosamine (DEN) at a supernatant. Then it was used for analysis dose of 200 mg/kg body weight in saline. of lipid peroxidation (LPO) (Devasagayam Two weeks after DEN administration, the and Tarachand, 1987), catalase (CAT) carcinogenic effect was promoted by 0.05% (Sinha, 1972), superoxide dismutase (SOD) phenobarbital (PB) that was supplemented (Marklund and Marklund, 1974), to the experimental animals through glutathione peroxidase (GPx) (Rotruck et drinking water for up to 16 consecutive al., 1973) and glutathione S-transferase weeks (Rajkapoor et al., 2005). Group I (GST) (Habig et al., 1974). The liver animals served as normal control that homogenate was additionally used for the received 1 mL/kg body weight of 0.3% estimation of DNA, RNA and protein carboxymethyl cellulose (CMC) through content in liver tissues (Schinder, 1957; the oral route. Group II animals served as Burton, 1956; Lowry et al., 1951). DEN+ PB control animals and treated same Histopathological examination as group I animals. Based on our previous The portion of the liver previously studies, Group III and IV animals were fixed in formalin was embedded in paraffin, treated with EIC at the dose of 200 and

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sectioned at 5 µm and stained with rats. The results revealed that the body haemotoxylin-eosin. Light microscopy was weights were significantly diminished in used to evaluate pathological changes of the DEN + PB treated group (P < 0.001). The liver. animals treated with plant extract at the dose of 200 mg/kg showed a moderately Statistical analysis significant increment in body weight in The values were expressed as mean comparison to the DEN + PB group ± SEM. Statistical analysis was performed (P < 0.01). At the same time, the group by one-way analysis of variance (ANOVA) treated with 400 mg/kg showed a followed by Tukey multiple comparison significant increase in the body weight test using InStat Prism version 4.0. P value when compared to tumour control group < 0.05 was considered as significant. (P < 0.001). However, no significant difference was ascertained between the Results tested dose levels. The gain in body weight and protection from weight loss by the Effect of EIC on body weight extract treatment indicates the nourishing Anticarcinogenic potential of EIC and chemoprotective properties of the was assessed by finding out the body extract. The body weight changes are given weight changes in liver neoplasm bearing in Figure 1.

Figure 1. Effect of EIC on body weight changes in liver tumour bearing animals. Number of animals used per group = 12. Values are expressed as Mean ± SEM. aP<0.001 vs Control; bP < 0.001, cP < 0.01 vs DEN + PB. Data were analysed by one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test.

Effect of EIC on liver weight and animals (P < 0.001) when compared to relative liver weight normal control. The extract treatment Figure 2 illustrates liver weight and considerably (P < 0.001 to P < 0.05) inhibit relative liver weight changes from the the gain in the liver weight and relative normal, DEN + PB as well as extract liver weight. No significant weight treated animals. A significant increase in difference was observed between the tested liver weight and relative liver weight was dose levels. ascertained in DEN + PB alone treated

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Anticarcinogenic potential of Indigofera cordifolia 57

Figure 2. Effect of EIC on liver weight and relative liver weight of different groups. Number of animals used per group = 12. Values are expressed as Mean ± SEM. aP < 0.001 vs Control; bP < 0.001, cP < 0.01, dP < 0.05 vs DEN + PB. Data were analysed by one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test.

Effect of EIC on serum hepatic parameters Effect of EIC on antioxidant From Figure 3, it is clearly evident enzyme levels that DEN + PB treatment caused significant DEN + PB treatment in rats elevation of liver serum markers. In the significantly attenuated the antioxidant DEN + PB treated group, the level of ALT, defence enzyme levels in the liver AST, ALP, GGTP, total cholesterol and homogenate (P < 0.001). Treatment with total bilirubin were considerably elevated EIC at the tested dose levels significantly (P < 0.001). In contrast, the groups treated elevated the antioxidant enzymes like with EIC at the tested dose levels, was able catalase, superoxide dismutase, glutathione to reverse all the elevated serum hepatic peroxidase and glutathione S-transferase parameters to close normal (P < 0.001 to levels in the liver tumour induced rats. The 0.05). The results indicate that the extract levels of antioxidant enzymes are displayed potentially defends the liver cells from in Figure 5. carcinogenic insult. Effect of EIC on lipid Effect of EIC on serum tumour peroxidation markers The level of lipid peroxidation was Effect of EIC on serum liver estimated from the liver homogenate of tumour specific markers such as alpha- experimental animals. Administration of fetoprotein (AFP) and carcinoembryonic DEN + PB led to a significant increase antigen (CEA) are presented in Figure 6. (P < 0.001) in the level of lipid DEN + PB treatment significantly elevated peroxidation. Treatment of rats with EIC at the levels of AFP and CEA (P < 0.001), a dose of 200 and 400 mg/kg markedly whereas, the treatment with EIC prevented the DEN + PB elicited alterations significantly reduced the raised levels of of lipid peroxidation. Figure 4 illustrated these serum specific tumour markers. the impact of EIC on lipid peroxidation levels of different treatment groups.

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Figure 3. Effect of EIC on serum hepatic parameters of different treatment groups. Number of animals used per group = 12. Values are expressed as Mean ± SEM. aP < 0.001 vs Control; bP < 0.001, cP < 0.01, dP < 0.05 vs DEN + PB. Data were analysed by one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test.

Figure 4. Effect of EIC on lipid peroxidation levels of different treatment groups. Number of animals used per group = 12. Values are expressed as Mean ± SEM. aP < 0.001 vs Control; bP < 0.001 vs DEN + PB. Data were analysed by one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test.

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Figure 5. Effect of EIC on antioxidant enzyme levels of different treatment groups. Number of animals used per group = 12. Values are expressed as Mean ± SEM. aP<0.001 vs Control; bP<0.001, cP<0.01 vs DEN + PB. Data were analysed by one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test. SOD, units/min/mg protein; CAT, μ mole of H2O2 consumed/min/mg protein; GPx, μ moles of GSH oxidized/min/mg protein; GST, μ moles of CDNB conjugation formed/min/mg protein.

Figure 6. Effect of EIC on serum tumour markers of different treatment groups. Number of animals used per group = 12. Values are expressed as Mean ± SEM. aP < 0.001 vs Control; bP < 0.001 vs DEN + PB. Data were analysed by one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test.

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60 Anticarcinogenic potential of Indigofera cordifolia

Effect of EIC on DNA, RNA and 0.01). In contrast, the groups treated with protein content EIC at the dose of 200 and 400 mg/kg It is clearly evident from the Figure decreased the elevated levels of DNA and 7 that DEN + PB caused significant RNA and increase the protein level towards elevation of DNA and RNA and decreases normalisation (P < 0.01 and P < 0.05). in liver tissue protein levels (P < 0.001 to

Figure 7. Effect of EIC on DNA, RNA and protein content of different treatment groups. Number of animals used per group = 12. Values are expressed as Mean ± SEM. aP < 0.001, bP < 0.01 vs Control; cP < 0.01 vs DEN + PB. Data were analysed by one-way ANOVA followed by Tukey-Kramer Multiple Comparison Test.

Histopathological study (Figure 8B). The architecture of liver The histopathological examinations sections co-administered with EIC 200 support the results obtained from serum mg/kg showed normal architecture with hepatic enzymes and serum liver tumour some liver cells and minimum specific markers. Figure 8A revealed the inflammatory cell infiltration around portal normal architecture (Control) and hepatic triads with few neoplastic hepatocytes cells with granulated cytoplasm, small (Figure 8C). The rats treated with EIC 400 uniform nucleus and nucleolus. The mg/kg showed normal architecture with animals treated with DEN + PB showed very few pre-neoplastically transformed loss of architecture and cancerous cells cells and hepatocytes maintaining normal arranged in lobules with the inflammatory architecture which was almost comparable collection and small bile duct proliferation to the control group (Figure 8D).

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Figure 8. Histopathological study of liver sections of different treatment groups. (A) Control group animals showing normal architecture with granulated cytoplasm, small uniform nucleus and nucleolus. (B) DEN + PB induced hepatocellular carcinoma bearing animals showing loss of architecture and cancerous cells arranged in lobules with the inflammatory collection and small bile duct proliferation. (C) Administration of EIC at the dose of 200 mg/kg on HCC bearing animals showing normal architecture with some liver cells and minimum inflammatory cell infiltration around portal triads with few neoplastic hepatocytes. (D) Treatment with EIC 400 mg/kg on HCC bearing animals showing normal architecture with very few pre-neoplastically transformed cells and hepatocytes maintaining normal architecture.

Discussion metabolic activation of DEN by cytochrome P450 enzyme to reactive Medicinal plants and natural electrophiles is needed for their cytotoxic, products have long been used to prevent mutagenic and carcinogenic activity. Due to and treat several diseases; hence they are its comparatively easy metabolic pathway sensible candidates for the development of and potent carcinogenic activity, DEN has newer drugs. Among the anticancer drugs, found widespread use as an experimental most of the lead molecules are derived from model in the field of carcinogenesis and in natural sources. Different screening chemoprevention (Borbath et al., 2010). strategies are available to study the In the current investigation, the anticancer and chemopreventive efficacy of effect of ethanolic extract of I. cordifolia in the test drugs. The current study showed body weight changes in HCC was carried that the oral administration of EIC prevents and showed a significant result in treatment. the hepatocarcinogenesis and development Liver cancer could cause complex of cancer that is elicited by DEN and PB in metabolic disturbances in both human and experimental rats. It is widely accepted that experimental animals leading to loss of

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weight and tissue wasting. There is a hepatocellular damage that was observed considerable loss in body weight in HCC from a significant increase in the serum bearing animals as compared to control rats liver marker enzymes like AST, ALT, ALP and the reduction in body weight (Glory and GGTP. This kind of elevations was and Thiruvengadam, 2012). The body additionally observed by many research weights steadily increased after treatment groups (Glory and Thiruvengadam, 2012; with extract that indicates that the extract Roy and Gadad, 2016). The raised levels of reduces the neoplasm burden and changes those markers are considerably reduced to in energy metabolism and additionally near normal in HCC bearing animals shows anticarcinogenic efficiency. Relative treated with plant extract that show the liver weight is a crucial parameter in anticarcinogenic activity of the extract. The judging the pathological conditions of the elevated levels of lipids in tumor-bearing liver. Relative liver weight was animals may be due to decreased activities considerably raised in DEN-treated animals of lipid metabolising enzymes. This because of the increase in liver weight predicts the defective catabolism rather than while not increase in weight of the tumour an elevated synthesis of lipoprotein that bearing animals treated with the plant appears to be the cause. The total bilirubin extract. The increase in relative liver weight level is a biomarker for liver injury. The might be because of the liver damage by accumulation of bilirubin in the serum may DEN (Roy and Gadad, 2016). The be a result of decreased biliary excretion administration of EIC attenuated the liver when the conjugation of bilirubin in the weight and relative liver weight that shows liver rather than the results of an increased the rehabilitating capability of extract in bilirubin load caused by haemolysis. In respect with chemopreventive efficiency. hepatic tumours, haemolysis and the Our results are in rational agreement with deranged liver function end up in the previous report (Ghosh et al., 2012). hyperbilirubinemia. In the present Biochemical liver marker enzymes investigation, the HCC bearing animals are used to screen particularly tumour showed an elevation in levels of serum conditions for differential diagnosis, bilirubin which can be due to the leak of the prognosis, monitoring the progress and for plasma membrane and loss of functional assessing the response to therapy. These integrity of cell membranes in liver enzymes are more unique and changes in (Sivaramakrishnan et al., 2008; Chen et al., their activities reflect the effect of the 2012). The groups treated with EIC showed proliferation of cells with growth potential significant results, reducing the elevated and its metabolic turnover. The increase in level that indicates the restoration of their activities is shown to be in reasonable bilirubin level as well as total cholesterol correlation with the number of transformed level. cells in cancer conditions. These enzymes DEN + PB treatment increase the serve as an index of liver cell injury and degree of lipid peroxidation in carcinogenic might be used to determine or confirms process is also attributable to abnormal liver diseases. The comparatively similar levels of reactive oxygen species (ROS) elevations of the liver-specific enzymes are that react with membrane lipids. considered because of the most sensitive Measurement of tissue malondialdehyde markers in the diagnosis of hepatocellular (MDA), one of the end products of lipid damage and loss of functional integrity of peroxidation, is often used as an indirect the cell membrane. The carcinogenesis index for assessing the extent of lipid process in the liver additionally affecting peroxidation in tissues (Pracheta et al., the liver cells with a subsequent breakdown 2011). The treatment with EIC down in membrane architecture of the cells ends regulates the malonaldehyde (MDA) levels up in their release into serum wherever their as compared with HCC bearing animals. levels goes high (Ramakrishnan et al., Thus it is convenient to suggest that the 2007). In the present investigation, animals extract positively has a beneficial impact on treated with DEN + PB causes DEN + PB induced HCC. The presence of

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flavonoids could contribute this effect the CEA assay could be a higher indicator because they are established to be potent of liver cancer. In the present study, a inhibitors of conjugated dienes and are significant increase in serum CEA levels ready to inhibit lipid peroxidation. following DEN treatment was associated Phenobarbital is a tumour promoter and it is with a production rate of tumours, their a powerful inhibitory effect on cellular location, stage, size, vascularity and antioxidant defence systems like SOD, differentiation. Reduction in CEA CAT, GST and GPx (Yadav and Bhatnagar, expression in extract treated groups 2007). SOD accelerates the transformation indicates the attenuated proliferation rate of of superoxide anions to hydrogen peroxide, liver tumours. Our findings are in good whereas catalase or GPx converts hydrogen agreement with earlier reports peroxide to water. A sharp fall in these (Ramakrishnan et al., 2007; Jagan et al., antioxidant enzymes will be attributable to 2008). increased free radicals production Nucleic acids play an important throughout the metabolism of DEN and role during neoplastic transformation. The phenobarbital. Upon treatment with EIC, a determination of nucleic acid content was rise in the levels of antioxidant enzymes more important with regards to biological was observed. Additionally, our earlier and functional aspects of the tumour. research on this plant found that it acts as a Nucleic acid content is an index of potent free radical scavenging, antioxidant proliferative activity in tumour conditions. enzyme-inducing and having anticancer Additionally, their content is found to be an properties (Thangavel et al., 2014; independent factor of prognosis since the Thangavel et al., 2015). The plant extract size of the tumour often correlates with the additionally contains rich amount of nucleic acid content of tumour. flavonoids, phenolics, ascorbic acid and Deoxyribonucleic acid (DNA) and tannins which could modulate the oxidative ribonucleic acid (RNA) synthesis were status via free radical scavenging and studied in order to determine the effects of antioxidant enhancing activities in rat liver. EIC on nucleic acid synthesis in HCC. It The most commonly used tumour has been observed that the tumour growth markers for the diagnosis of hepatoma corresponds to the elevated levels of DNA (HCC) is alpha-fetoprotein (AFP) that and RNA synthesis. The level of DNA and could be a distinctive immunomodulatory RNA of liver found to be progressively glycoprotein, which is generally made by increased in HCC bearing animals. The the immature hepatocytes in the fetus. increased nucleic acid synthesis in tumour Approximately in 70 % of the patients with bearing animals was found to decrease primary HCC, the serum AFP level could when the animals were treated with EIC. In also be markedly elevated. It has been the present investigation, it has also been recognised that exposure of animals with observed that decreased protein contents in DEN increases the circulating AFP levels. plasma of HCC bearing animals appear In our study, the results showing a which could be attributed to the impaired significant rise in levels of AFP in tumour hepatic function resulting from infiltration bearing animals which were found to be with the tumour. The liver is an important reduced in extract treated animals and our site of protein synthesis and it has the results are more consistent with the highest rate of synthesis of tissue proteins. previous studies (Sivaramakrishnan et al., Recycling of amino acids has been 2008; Singh et al., 2009; Taha et al., 2010; decreased in tumour conditions resulting in El Miniawy et al., 2014). Carcinoem- the enhanced efflux of these amino acids bryonic antigen (CEA) is an oncofetal from the tissues. Thus, the host responds to glycoprotein that is expressed in normal increased tumour load by increasing tissue mucosal cells and is over-expressed in liver protein breakdown. The administration of and colon cancers. It is frequently detected EIC to the HCC bearing group resumed the in high concentrations in the serum of protein level to near normal. people with liver tumours. It is clear that

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DEN + PB treatment induced inducer in the epithelial cells of blood histologic changes in the liver like fatty vessels then on adhering, invasion and infiltration, focal necrosis and hepatocytes metastasis of tumour cells. They having hyperchromatic nuclei and these additionally exhibit the antitumour effect by changes are indicative of carcinoma. cell cycle arrest and programmed cell death Further, histopathological studies showed (Man et al., 2010). An antitumour and the normal architecture, mild congested chemopreventive effect of the extract is sinusoids and absence of hepatoma cells in also because of the presence of those the livers of animals treated with the extract phytochemical constituents. Hence, the and DEN compared to those treated with chemopreventive potential of ethanolic DEN alone. All these results indicate that extract of I. cordifolia is also due to its EIC has a chemopreventive impact against antioxidant, free radical scavenging and the DEN-induced liver tumour. amelioration of lipid peroxidation together Our previous studies on this plant with the elevation of antioxidant defence have been revealed that the plant extract system. was rich on several phytochemical compounds like sterols, tannins, phenolics, Conclusion flavonoids, alkaloids, terpenoids and saponins. Several such compounds are best- In conclusion, exposure with DEN known to possess potent antitumor clearly evoked liver tumour formation. EIC properties (Kintzios, 2006). The plant treatment ameliorated the level of hepatic extract additionally exerts good radical injury in DEN-treated animals. Our scavenging and antioxidant activities in findings suggested that EIC was effective in numerous in vitro models (Thangavel et al., the treatment of chemically induced liver 2014). The extract has potent antitumour tumours in rats. The antitumour and activity in animal models and it enhances chemopreventive mechanisms that the antioxidant enzyme system in tumour concerned in the inhibition of HCC bearing animals (Thangave et al., 2015). contained the down-regulation of free Flavonoids are known to possess radicals via antioxidant defence mechanism antimutagenic and antitumour effect. In as well as suppression of inflammatory addition to antitumour activity, flavonoids mediators concerned in carcinogenesis. exert growth inhibitory effects on many These findings were of great importance for malignant tumour cell lines in vitro. the invention and development of new and Moreover, flavonoids have a safe medicine for the treatment of liver chemopreventive role in cancer through cancer. Additional studies are warranted to signal transduction in cell proliferation and grasp the in-depth mechanism of action development of newer blood vessels which can result in the identification of (Weber et al., 1996; Fotsis et al., 1997). active molecules from I. cordifolia against Flavonoids could inhibit carcinogenesis by DEN and PB induced hepatocarcinoma. inhibiting the metabolic activation of the carcinogen to its reactive intermediate, Conflict of interest statement causing the enzymes involved in the detoxification of the carcinogen and We declare that there are no binding to reactive forms of carcinogens, conflicts of interest. thereby preventing their interaction with vital cellular targets like DNA, RNA and References proteins. In addition, plant flavonoids might conjointly inhibit tumour promotional Borbath, I.; Leclercq, I. A.; Sempoux, C.; events as mentioned above. It is probably Abarca-Quinones, J.; Desaeger, C.; Horsmans, that flavonoids could emerge as a definite Y. Efficacy of lanreotide in preventing the group of antitumour agents. Saponins are occurrence of chemically induced hepatocellular carcinoma in rats. Chem. Biol. Interact., found beneficial targeted on inhibition of v. 183, p. 238-248, 2010. tumour angiogenesis by suppressing its

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