Ameliorative effect of methanol extract of Rubia cordifolia in N-nitrosodiethylamine-induced hepatocellular carcinoma

Abstract

Context: Rubia cordifolia Linn. (Rubiaceae) is a medicinal plant used in the ayurvedic system of medicine. It is also known as Indian Madder or Manjistha and is traditionally used as an antiinflammatory, antiseptic, and galactopurifier, but its anticancer propertis are yet not known.

Objective: The ameliorative effect of the Rubia cordifolia methanol extract on N-nitrosodiethylamine-induced experimental hepatocellular carcinogenesis in rats.

Materials and methods: Changes in liver weight, serum markers of liver damage, hydroxyl radicals, lipid peroxidation, levels of enzymic and nonenzymic antioxidants; mitochondrial and respiratory chain enzymes were also investigated using various biochemical parameters and histopathological studies. Male albino rats of Wistar strain were divided into four groups for a study period of 3 months. Animals of group I and group IV served as control and drug control, respectively. Hepatocellular carcinoma was induced in animals of groups II and III with 0.02% N-nitrosodiethylamine.

Results: Upon Rubia cordifolia methanol extract co-treatment (250, 500, and 750 mg/kg bodyweight) in group III alone levels of serum marker enzymes and antioxidants increased significantly in a dose-dependent manner. The levels of hydroxyl radicals and lipid peroxidation decreased. Mitochondrial enzymes and respiratory chain enzymes, which were decreased in N-nitrosodiethylamine-induced rats, increased significantly in RC treated rats. Further histological analysis of liver confirmed the prevention of pathological changes caused by N-nitrosodiethylamine on Rubia cordifolia supplementation.

Discussion and conclusion: These findings demonstrate that Rubia cordifolia can be a source of potent antioxidants for treatment of diseases such as cancer.

Keywords::

• Serum markers
• lipid peroxidation
• hydroxyl radicals
• reactive oxygen species
• mitochondrial and respiratory chain enzymes

Introduction

Hepatocellular carcinoma (HCC) is the most common and lethal of all cancers. A diversity of dietary, endogenous and environmental stimuli mediates hepatocellular carcinogenesis (Guyton & Kensler, 1993). Nitrosamines are a large class of compounds that act as inducers of primary liver cancer due to their environmental presence and high carcinogenic potency. They are found in foods such as meat and dairy products and in alcoholic beverages. The leather tanning, metal working, rubber and tire manufacturing processes produce high concentrations of volatile nitrosamines. It is widely accepted that metabolic activation of nitrosamines by cytochrome P450 enzymes to reactive electrophiles is required for their cytotoxic, mutagenic and carcinogenic activities (Archer, 1989). Because of its relatively simple metabolic pathway and potent carcinogenic activity, N-nitrosodiethylamine (DEN) has found wide spread use as an experimental model in the field of liver carcinogenesis (Lee & Lee, 1999). DEN has been suggested to produce oxidative stress and cellular injury due to involvement of reactive oxygen species (ROS) (Bansal et al., 2000).

ROS are potentially dangerous by products of cellular metabolism that have direct effect on cell development, growth and survival. Oxidative stress mediated by ROS due to its associated interaction with the membrane causing lipid peroxidation and DNA damage further promotes carcinogenesis. The severity and magnitude of the ROS mediated cancer problem makes it imperative to develop chemopreventive strategies, utilizing synthetic or natural agents to block initiation or to arrest progression in malignant cells (Sivaramakrishnan et al., 2008). One such natural herbal agent is Rubia cordifolia Linn. (Rubiaceae) (RC), locally known as manjistha, a perennial herbaceous climber. It grows commonly on higher ghats, Mahabaleswar, Amboli, and Maharastra states of India. It is an important medicinal plant which is used for treatment in an ayurvedic system of medicine (Pandey & Sharma, 1994). It possesses antineoplastic (Adwankar et al., 1980), immunomodulatory (Joharapurkar et al., 2003) and antiinflammatory (Antarkar et al., 1983) properties. The major active components of Rubia cordifolia are anthraquinones glycosides and include 1-hydroxy 2-methoxy anthraquinone, 1,4-dihydroxy-2-methyl-5-methoxy anthraquinone, 1,3-dimethoxy 2-carboxy anthraquinone and rubiadin (1,3-dihydroxy-2-methyl anthraquinone) (Mohana Rao et al., 2006). Considering these properties of RC, the present work was planned to give a scientific validation to the ameliorative effect of the methanol extract of RC on N-nitrosodiethylamine (DEN) induced experimental hepatocellular carcinoma in rat.

Materials and methods

Preparation of plant extract

Dried roots of RC were purchased from market. The plant material was identified and duly authenticated by Dr. Mathivanan, Center for Advanced Studies in Botany, Guindy Campus, University of Madras, Chennai, India. A voucher specimen of the collected sample was deposited in the departmental herbarium. Roots of RC (1 kg) were treated with 2.5 L of petroleum ether at room temperature in closed container and repeated till 7 days without allowing it to dry. The plant material was removed by vacuum filtration. The plant extract obtained from the above step was subjected to cold percolation using methanol (95%, v/v) as solvent and suitably concentrated by rotary pump vacuum evaporator and dried in a vacuum desiccator and stored at room temperature. It was dissolved in water and administered orally to rats.

Phytochemical screening

The phytochemical examination of the methanol extract was performed by standard methods and showed the presence of various phytochemical constituents (Trease & Evans, 1993).

Chemicals and reagents

N-Nitrosodiethylamine (DEN) was purchased from Sigma Aldrich, USA, and all the other chemicals used here for the study were of analytical reagent grade.

Experimental animals

Male albino rats of Wistar strain (120–150 g) were purchased from Tamil Nadu University of Veterinary Animal Sciences (TANUVAS) and acclimatized to laboratory conditions for 1 week. They were maintained in clean, sterile, polypropylene cages and fed with commercial pellet rat chow (M/s. Hindustan Lever Ltd., Bangalore, India), water ad libitum and kept in a well ventilated room with 12 h light/dark cycles throughout the experimental period. This study has been conducted as per the guidelines of the animal ethical committee of our institution (IAEC No. 01/004/02). In preliminary studies, RC was found to be non-toxic up to a dosage of 3 g/kg/body weight (data not shown) and among the various doses studied, the optimal dosage for its ameliorative efficacy was found to be 500 mg/kg body weight. Animals were divided into four groups, for a total experimental period of 90 days and studied as follows.

Experimental design

Group I: Control rats received normal diet and drinking water.

Group II: Rats were treated with DEN (0.02%) orally in drinking water.

Group III: Rats were given DEN (0.02%) in drinking water along with the methanol extract of RC (250, 500 and 750 mg/kg body weight, respectively) orally.

Group IV: Rats treated with RC alone (500 mg/kg/body weight) orally.

After 90 days, rats were anaesthetized with sodium-pentothal after overnight fasting and euthanized. Serum from control and experimental rats were separated from blood. The liver was excised immediately and kept in ice-cold saline.

Processing of liver tissue

A portion of the liver was homogenized in 0.1 M Tris buffer, pH 7.4 and used for the serum marker enzymes, enzymic and non-enzymic antioxidants, lipid peroxidation, and hydroxyl radical estimation.

Isolation of mitochondria

The mitochondrial isolation procedure used was of Johnson and Lardy (1967). A lobe of the liver was excised into ice cold solution medium (sucrose: 0.25 M; Tris-HCl: 0.005 M, pH 7.4; EDTA: 0.001 M) and were homogenized (10% w/v) using teflon homogenizer. The 10% homogenate was centrifuged at 700 × g for 5 min and the resultant supernatant was centrifuged at 30,000 × g for 10 min. The mitochondrial pellet obtained was resuspended in solution medium (1 mL/g tissue) and used for analyzing mitochondrial and respiratory chain enzymes.

Biochemical assays

The activities of aspartate transaminase (AST), alanine transaminase (ALT) (Mohun & Cook, 1975), alkaline phosphatase (ALP) (King, 1965a) and lactate dehydrogenase (LDH) (King, 1965b) were assayed in the serum. The activities of the enzymic and non-enzymic antioxidants superoxide dismutase (SOD) (Misra & Fridovich, 1972), catalase (CAT) (Sinha, 1972), glutathione peroxidase (GPx) (Rotruck et al., 1973), glutathione S-transferase (GST) (Habig et al., 1974), reduced glutathione (GSH) (Moron et al., 1979) were estimated in liver tissue homogenate. The levels of lipid peroxidation (Ohkawa et al., 1979) products were estimated in liver tissue homogenate. The hydroxyl radical (Cederbaum & Cohen, 1984) levels were measured in liver tissue homogenate. The levels of mitochondrial enzymes like isocitrate dehydrogenase (ICDH) (Bell & Baron, 1960), succinate dehydrogenase (SDH) (Slater & Bonner, 1952), α-ketoglutarate dehydrogenase (α-KGDH) (Reed & Mukherjee, 1969) and respiratory chain enzymes like NADH dehydrogenase (Minakami et al., 1962) and cytochrome c oxidase (Pearl et al., 1963) were also estimated in liver.

Histological examination

Histological evaluation was performed on a lobe of the liver and a portion of the specimen was fixed in 10% formalin and embedded in paraffin wax. Sections were cut at 4 µm thickness, stained with hematoxylin and eosin and viewed under light microscope for histological changes.

Results

The result of the preliminary phytochemical screening of the methanol extract of RC methanol extract revealed that presence of anthroquinone, glycosides, saponins and absence of tannins and alkaloid (Table 1).

Table 1.  Phytochemical screening of methanolic extract of Rubia cordifolia.

Test for constituents	Methanolic extract of Rubia cordifolia
Anthroquinone	+
Glycosides	+
Saponin	+
Tannins	−
Alkaloids	−

+, presence; −, absence.

Rats treated with DEN developed hepatic neoplastic nodules. Liver weight was increased significantly in DEN induced rats when compared to normal rats due to the development of nodules. RC given along with DEN for 90 days significantly prevented the formation of liver tumors and thereby the increase of the liver weight (Table 2).

Table 2.  Liver weight of control and experimental animals.

Groups	RC mg/kg b. wt.	Liver weight (g)
Control	—	2.34 ± 0.20
DEN	—	4.41 ± 0.39*
DEN + RC	250	3.75 ± 0.34^b
	500	2.99 ± 0.26^c
	750	2.68 ± 0.19^c
RC alone	500	2.31 ± 0.24^NS

Control vs. DEN *P < 0.001.

DEN vs. DEN + RC ^cP < 0.001, ^bP < 0.01.

RC alone vs. Control.

NS, Non-significant.

The activity of serum marker enzymes of clinical interest-AST, ALT and ALP increased significantly in DEN treated groups when compared to control group. The levels of serum marker enzymes were maintained at near normal level on co-treatment with RC. No significant change in AST, ALT and ALP activities were observed in the group treated with RC alone when compared with control (Table 3). There was a significant increase in LDH in DEN induced group, whereas the RC alone group showed normal levels of LDH. Rats subjected to DEN and RC regimen exhibited a significant reduction in LDH activity (Table 3).

Table 3.  Levels of serum markers of control and experimental groups of rats.

Groups	RC mg/kg b. wt.	AST IU/L	ALT IU/L	ALP IU/L	LDH IU/L
Control	—	114.6 ± 10.9	38.9 ± 3.1	6.53 ± 0.54	81.4 ± 6.8
DEN	—	324.6 ± 19.2*	117.4 ± 9.3*	13.71 ± 1.12*	110.9 ± 10.3*
DEN + RC	250	299.1 ± 16.4^b	89.5 ± 7.4^b	10.19 ± 0.99^b	101.2 ± 8.7^b
	500	179.4 ± 13.9^c	53.4 ± 4.8^c	8.73 ± 0.84^c	91.7 ± 8.4^c
	750	161.3 ± 11.4^c	44.9 ± 3.6^c	7.22 ± 0.81^c	89.4 ± 7.3^c
RC alone	500	112.0 ± 10.5^NS	37.6 ± 3.5	6.62 ± 0.74^NS	81.3 ± 6.5^NS

Values are expressed as mean ± SD for six animals. AST, ALT, ALP and LDH were expressed as IU/L.

Control vs. DEN *P < 0.001.

DEN vs. DEN + RC ^cP < 0.001, ^bP < 0.01.

RC alone vs. Control.

NS, Non-significant.

The levels of LPO and hydroxyl radicals in liver in DEN induced group were increased significantly. It was normal in drug (RC) alone group when compared with the control group. RC co-treatment inhibited the accumulation of lipid peroxidation products (TBARS) and hydroxyl radicals significantly when compared with DEN-induced rats (Figures 1 and 2). DEN induced LPO was associated with a decrease in glutathione levels and the effect was found to be statistically significant in liver. RC treatment restored the GSH level to a normal value, thereby maintaining the redox status of the cell (Table 4).

Table 4.  Antioxidant status in liver of control and experimental groups of rats.

Groups	RC mg/kg b. wt	SOD	CAT	GP x	GST	GSH
Control	—	6.18 ± 0.53	49.14 ± 2.4	3.97 ± 0.30	0.31 ± 0.03	8.95 ± 0.74
DEN	—	3.99 ± 0.27*	25.71 ± 1.9*	1.85 ± 0.11*	0.16 ± 0.01*	4.47 ± 0.49*
DEN + RC	250	4.74 ± 0.47^b	32.64 ± 2.1^b	2.11 ± 0.15^b	0.18 ± 0.01^b	5.63 ± 0.68^b
	500	6.09 ± 0.59^c	40.71 ± 2.7^c	2.98 ± 0.18^c	0.21 ± 0.02^c	7.71 ± 0.73^c
	750	6.11 ± 0.61^c	44.26 ± 3.6^c	3.16 ± 0.23^c	0.23 ± 0.03^c	7.94 ± 0.81^c
RC alone	500	6.22 ± 0.64^NS	50.29 ± 4.5^NS	3.99 ± 0.29^NS	0.29 ± 0.04^NS	8.99 ± 0.83^NS

Values are expressed as mean ± SD for six animals. SOD was expressed as units/min/mg protein. CAT was expressed as mmoles of H₂O₂ consumed/min/mg protein. GST was expressed as mg of thioester formed/min/mg protein. GSH was expressed as mg /mg protein.

Control vs. DEN *P < 0.001.

DEN vs. DEN + RC ^cP < 0.001, ^bP < 0.01.

RC alone vs. Control.

NS, Non-significant.

Figure 1.  Levels of lipid peroxidation in liver tissue homogenate. Values are expressed as mean ± SD for six animals. LPO was expressed as μmoles of MDA formed/min/mg protein in the liver tissue homogenate of control and experimental groups of rats. Control vs. DEN *P < 0.001, ^yP < 0.01, ^xP < 0.05. DEN vs. DEN + RC ^cP < 0.001, ^bP < 0.01, ^aP < 0.05. RC alone vs. Control. NS, Non-significant.

Figure 2.  Effect of RC on levels of hydroxyl radicals. Values are expressed as mean ± SD for six animals. Hydroxyl radicals are expressed as ng/mg protein in the liver tissue homogenate of control and experimental groups of rats. Control vs. DEN *P < 0.001, ^yP < 0.01, ^xP < 0.05. DEN vs. DEN + RC ^cP < 0.001, ^bP < 0.01, ^aP < 0.05. RC alone vs. Control. NS, Non-significant.

The SOD activity was decreased significantly on treatment with DEN. Co-treatment with RC resulted in higher levels of SOD when compared to DEN induced rats. The activity of CAT, GPx and GST decreased significantly in rats of the group induced with DEN. Co-administration of RC caused significant increase in the activities of CAT, GPx and GST when compared with the group treated with DEN alone (Table 4).

Decreased activities of key enzymes of the TCA cycle such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), α-ketoglutarate dehydrogenase (α-KGDH) and respiratory chain enzymes (NADH dehydrogenase and cytochrome c oxidase) in liver tissue homogenate were observed in DEN induced rats. DEN induced tumor-bearing animals treated with RC extract showed a significant increase in mitochondrial and respiratory chain enzyme activities. Rats treated with RC extract alone showed no significant variations when compared with control rats (Table 5).

Table 5.  TCA cycle and respiratory chain enzyme activities in liver of control and experimental groups of rats.

Groups	RC mg/kg b. wt.	ICDH	SDH	α-KGDH	NADH dehydrogense	Cytochrome c- oxidase
Control	—	3.92 ± 0.25	6.34 ± 0.40	0.73 ± 0.07	29.42 ± 3.6	5.06 ± 0.63
DEN	—	1.16 ± 0.13*	3.99 ± 0.23*	0.41 ± 0.02*	11.57 ± 2.2*	2.51 ± 0.21*
DEN + RC	250	1.90 ± 0.16^b	4.36 ± 0.36^b	0.48 ± 0.04^b	17.41 ± 1.9^b	3.73 ± 0.34^b
	500	3.01 ± 0.19^c	5.18 ± 0.41^c	0.59 ± 0.06^c	21.78 ± 2.4^c	4.18 ± 0.49^c
	750	3.21 ± 0.22^c	5.63 ± 0.47^c	0.63 ± 0.06^c	23.56 ± 2.9^c	4.85 ± 0.52^c
RC alone	500	3.95 ± 0.26^NS	6.07 ± 0.55	0.69 ± 0.07	30.06 ± 3.2^NS	5.09 ± 0.71^NS

Values are expressed as mean ± SD for six animals. ICDH was expressed as m moles of αKG formed/min/mg protein. SDH was expressed as mmoles of succinate oxidized/min/mg protein. α-KGDH was expressed as mmoles of ferrocyanide/liberated/min/mg protein. NADH dehydrogenases were expressed as mmole NADH oxidized/min/mg protein. Cytochrome c oxidase was expressed as optical density × 10².

Control vs. DEN *P < 0.001.

DEN vs. DEN + RC ^cP <0 .001, ^bP < 0.01.

RC alone vs. Control.

NS-Non-significant.

Significant damage was evident to the liver cells in the DEN induced group II compared to control group I animals. On RC supplementation along with DEN caused the amelioration of damage to liver cells as evident from the histology of liver tissue (Figure 3).

Figure 3.  Histology of liver in control and experimental animals. (A) Control rats showing normal architecture. (B) DEN induced rats showing loss of architecture (both nuclear size and shape vary). (C) DEN + RC (250 mg) treated rats showing anaplastic tumor cells with scanty Cytoplasm. (D) DEN + RC (500 mg) treated rats showing scanty strands of tumor cells surrounded by large amount of neutrophils. (E) DEN + RC (750 mg) treated rats showing scanty strands of tumor cells surrounded by large amount of neutrophils. (F) RC alone (500 mg) treated rats showing normal morphology.

Discussion

The preliminary chemical constituents of Rubia cordifolia Linn. was analyzed and the results showed the bioactive properties of medicinal plants are perhaps due to the presence of various secondary metabolites such as anthroquinone, glycosides and saponins (Khandelwal, 2005).

Elevation of serum ALT and AST levels correlate with a high incidence of HCC development due to various risk factors and the prevention of liver injury decreased the incidence of HCC (Tarao et al., 1999). ALT and AST are cytosolic enzymes of the hepatocyte, and an increase in the activities of these enzymes in the serum reflects an increase in plasma membrane permeability, which in turn, is associated with liver injury (Plaa & Hewitt, 1982). In this context, we have observed a rise in the level of ALT and AST in DEN-induced rats which were found to be decreased significantly on RC treatment. The present study on the methanol extract of root of RC was found to protect and maintain the structural integrity of hepatic cells by significantly decreasing the levels of serum AST and ALT levels on treatment similar to Pradeep et al. (2007) suggesting that its hepatoprotective action might be due to a membrane-stabilizing property. ALP is mainly involved in metabolite transport across cell membranes found in a decreasing order of abundance in placenta, ileal mucosa, kidney, bone, and liver. The elevated level of serum ALP is associated mainly with liver cell damage (Moss, 1997). DEN induced rats showed elevated serum ALP, which confirms the impairment of liver function. Administration of RC along with DEN was found to significantly prevent the elevation of serum ALP levels thereby confirming the hepatoprotective action of RC. LDH is a hydrogen transfer enzyme that catalyzes the oxidation of l-lactate to pyruvate with the mediation of NAD⁺ as hydrogen acceptor. Increased LDH levels indicate that the energy demands are met by anaerobic respiration through increase in LDH activity. The increased LDH levels, as observed in the present study in the DEN-induced rats, is a direct diagnostic function of liver damage and tumor formation (Al-Attar, 2004). There was a significant decrease in enzyme activity after treatment with RC. The histological observations confirm the amount of liver damage caused by DEN and the corresponding prevention of damage and maintenance of hepatocyte architecture on RC supplementation. This corroborates with results of biochemical assays confirming the protective action of the RC extract against liver damage. The results of the present study indicate that RC acts as a potent hepatoprotective agent against DEN-induced HCC.

Lipid peroxidation is a chain reaction process that involves the participation of free radical species and has been shown to induce alterations in several cellular functions. In the present study, lipid peroxidation products (MDA) were significantly increased following DEN treatment. RC inhibited accumulation of lipid peroxidation products in liver. DEN-induced lipid peroxidation was associated with a decrease in glutathione levels and the effect was found to be statistically significant in liver as similar to Bansal et al. (2007). RC treatment restored the GSH level to a normal value, thereby maintaining the redox status of the cell (Table 3).

The level of GSH inside the cell depends primarily on the rate of synthesis and therefore on the pool of cysteine, glycine and glutamate and their assembling enzyme and secondly on the rate of its utilization via glutathione S-transferase and glutathione peroxidase/glutathionereductase system. The remarkably low concentration of one of the most potent antioxidants, GSH on DEN treatment, contributes to impairment in the defense mechanism against oxygen attack (Pradeep et al., 2007; Sehrawat & Sultana, 2006). An in vitro study has shown that binding of carcinogen metabolites to nuclear DNA is inhibited by conjugation with glutathione (Herse et al., 1980). Therefore, the enzymes may be important in the inhibition of hepatomas (Kimihiko & Ichiro, 2002).

The relative deficiency of SOD and catalase could result in decreased detoxification of O₂⁻ in tumors (Tisdale & Mahmoud, 1983). This could result in cell death from membrane damage arising from peroxidative reaction of polyunsaturated fatty acids (lipid peroxidation) and attack of ROS on protein and nucleic acids. The relatively unreactive O₂⁻ is converted by superoxide dismutase (SOD) into superoxide radical which in turn takes part in the “Fenton reaction” using transition metal ions (copper or iron) as catalysts to produce the highly reactive hydroxyl radical. The hydroxyl radical is an extremely reactive oxidizing radical that will react with most biomolecules at diffusion controlled rates. The hydroxyl free radical is several orders of magnitude more reactive towards cellular constituents than superoxide radicals and hydrogen peroxide (Halliwell & Gutteridge, 1992). In the present study, there was a multi-fold increase in the levels of hydroxyl free radicals in DEN-induced animals that was neutralized to near normalcy in RC co-treated ones, thus indicating prevention of increased ROS production by RC by its capacity to scavenge free radicals.

The lower level of GSH-Px, together with that of CAT, might favor accumulation of free radicals, which may induce the neoplastic condition. These reduced levels of SOD, CAT and GPx may permit the accumulation of superoxide or hydrogen peroxide within a tumor, which may facilitate advantageous growth of neoplastic cells. The simultaneous administration of RC with DEN induction maintained the SOD, CAT and GPx, GST activities probably by decreasing the levels of ROS like hydroxyl radicals as evidenced in the case of garlic and silymarin (Shaarawy et al., 2009).

GSTs are a group of multifunctional proteins localized mainly in the hepatic cytosol and they participate in detoxification reactions of electrophilic compounds and contribute to the mechanism of cancer chemopreventive agents via induction. A detoxification pathway, which plays a major role in cellular defenses against genetic damage by electrophiles, is the thiol tripeptide glutathione together with glutathione S-transferase (Ketterer, 1988) that interact with DEN metabolites and make them less toxic. Individual deficiencies in the protection afforded by phase II detoxification enzymes, including the GSTs, are reported to increase the risk of cancer (Strange et al., 1998). This unique feature of induction of GST activity possibly by RC as evident from our study may also be responsible for its antioxidant, hepatoprotective and anticarcinogenic effect.

DEN-induced tumor-bearing animals treated with RC extract showed a significant increase in mitochondrial enzyme and respiratory chain enzyme activities. Rats treated with RC extract alone showed no significant variations when compared with control rats (Table 4). This study confirms that the chemopreventive effect of RC is found to be more effective in the initiation phase of carcinogenesis. Similar results have been observed with other chemopreventive agents. For example, sodium selenite, by maintaining TCA cycle enzymes (Thirunavukkarasu et al., 2001) acts as a chemopreventive agent in DEN induced hepatocellular carcinoma. The significant decrease in the activity of liver succinate dehydrogenase suggests that anaerobic metabolism was favored over aerobic oxidation of glucose through Krebs cycle in order to mitigate the energy crisis for survival (Argiles & Bioeto, 1988).

Enhanced lipid peroxidation damaged the membranous structure of mitochondria, which was indicated by the loss of lamellae, and increased oxidation of exogenously added NADH. Loss in membrane integrity altered the activities of the tricarboxylic acid cycle enzymes and levels of cytochromes (Josephine & Keshav, 2002). Similarly, Remus and Firman (1995) reported that the decrease in TCA cycle action might be related to a decline in the activity of α-ketoglutarate dehydrogenase and pyruvate dehydrogenase. Our study confirms that RC was effective in maintaining mitochondrial integrity by increasing the enzymatic activities of TCA cycle, which was initially decreased in DEN-induced animals to normal levels on co-treatment.

Mitochondria carry out a variety of processes of which oxidative phosphorylation is the most important. Detoxification of oxygen via its reduction to water by cytochrome oxidase takes place in the mitochondria. Cytochrome c oxidase and NADH dehydrogenase are enzymes involved in the electron transport chain and are located in the inner mitochondrial membrane. Their role is ultimately linked to the production of useful energy rich compounds such as ATP.

Lipid peroxidation has been reported to decrease the rate of oxygen consumption as well as the activity of cytochrome c oxidase in mitochondria (Ishankhodzhaev et al., 1987). The respiratory process involves the transport of electrons via cytochromes to molecular oxygen. Variations in cytochrome concentrations may affect the transport of electrons via the transport chain and thereby alter energy production of mitochondria. It has been reported that reduction in functioning of mitochondrial energy production would impair protein synthesis and energy production. A decrease in mitochondrial cytochrome content could result in the concomitant loss of oxidative phosphorylation capacity (Schultz & Chan, 2001). The close correlation reaction preceding the phase of mitochondrial MDA production between the decline of mitochondrial respiration and the activity of complex III suggests an impairment of respiratory chain in the bc1region, representing one of the functional events in the casual peroxidation reaction (Yau-Huei et al., 2001). Upon RC supplementation, the above biochemical changes were reverted in DEN induced animals.

Conclusion

In conclusion, all these observations clearly indicate a significant ameliorative effect of RC extract against DEN-induced carcinogenesis. From our study, it is concluded that RC suppressed the free radical processes by scavenging hydroxyl radicals. Our results underline the capacity of RC to modulate the levels of LPO and significantly increase the endogenous antioxidant defense mechanisms in DEN-induced hepatocellular carcinoma. Our results also show that the significant increase in the levels of serum markers in DEN-induced animals was prevented by RC treatment. From the results obtained, we suggest that RC may be developed as an effective chemotherapeutic agent. Further studies to characterize the active principles and to elucidate the molecular mechanism of action are in progress.

Acknowledgments

The authors wish to thank Dr. V. Mathivanan, Center for Advanced Studies in Botany, University of Madras, Guindy campus, Chennai, India, for authenticating the plant, Rubia cordifolia.

Declaration of interest

The authors declare no conflicts of interest.