HEPATOPROTECTIVE EFFECT OF d-LIMONENE AGAINST ADRIAMYCIN INDUCED HEPATOTOXICITY IN EXPERIMENTAL RATS

ABSTRACT Objective: Monoterpenes plays an essential role to fight against various diseases. Among the various monoterpenes an efficientd-Limonene plays a fundamental role to fight against hepatotoxicity caused by cancer chemotheraphy treatment. Hence the presence study is to evaluate hepatoprotective function of d-limonene against adraimycin induced hepatotoxicity. Methods: Male albino Wistar rats were administrated with ADR (15mg/Kg body weight) in six equal injection, and the protection efficacy of d-limonene (100mg/Kg body weight) was examined with reference to tissue AST level and the pathological studies was examined by microscopic study. Results: Rats treated with ADR results in elevated level of liver AST marker enzymes, whereasthe level of AST was controlled when administrated with d-Limonene. However Histopathological proof added more protective role of rats treated with d-limonene against hepatotoxicity. Conclusion:ADR administration of 15mg/Kg body weight of rats increase the level of hepatotoxicity by increasing the marker enzyme activity and show severe morphological changes. The final outcome from our result suggests that d-limonene (100mg/Kg body weight) a vibrant monoterpene act as latent hepatoprotective negotiator by attenuating ADR induced hepatotoxicity.


INTRODUCTION
Since 1960's doxorubicin or Adriamycin (ADR) an anthracycline anti-neoplastic drug widely used in treatment of major route of cancer, including Osteosarcoma [1], Soft tissue Sarcomas [2], Breast cancer [3], gastric, liver [4], esophageal carcinomas [5], Hodgkin's lymphomas and various other cancers. However, rate limiting effect of ADR on treating cancer was hampered by various side effects on chemotherapy strategies like toxicity in central nervous system [6], nephrotoxicity [7], hepatotoxicity and most awful condition leads to cardiomyopathy and congestive heart failure [8]. Various reports suggest cardiac damage and hepatotoxicity caused by ADR prion to be higher compare to other side effects [9]. ADR mediated hepatic damage is probably by the formation of free radicals causes oxidative stress and damage. However the exact mechanism for ADR causing hepatotoxicity remains to be unclear but it is believed free radical formation [10]. One of the best monocyclic monoterpened-limonene is highly present in all essential citrus fruits and citrus oil with lemon aroma. Recent studies reveals administration of d-limonenne shows low toxicity after repeated dose upto one year [11]. Administration of dlimonenen gradually inhibits lipid peroxidation and prevents free radical damage [12,13]. It also reported d-limonenen have various importat biological properties like anti oxidant potential [14,15], anti-inflammatory [16] and chemo preventive properties against numerous types of cancer [17,18]. The purpose of the study was intended to test the hypothesis whether a nutritional strategy like chronic administration of d-limonene could prevent ADR induced hepatotoxicity and hamper the free radicals production in albino rats. and all other chemicals used for biochemical estimation were used analytical grade.

Animals
Adult male albino Wistar rats of (140-160g) were used for the study. Rats were kept in polypropylene cages and maintain under standard temperature of (25±2 ) in large air conditioned room, fed with standard chow. The animals had free access to water. All the experiments were designed and conducted according to the ethical norms approved by animal

Experimental design
The experimental animals were divided into four groups, each group comprising of six animals.
Group I : Normal control rats fed with standard diet and drinking water for 6 weeks Group II : Adriamycin (15mg/kg body weight) was dissolve in normal saline and given six equal injections via, intra peritoneal.
Group III : Rats treated with d-limonene alone (100mg/kg body weight) was dissolved in corn oil and administrated intra gastric gavage for period of four weeks.

Morphometric studies
After sacrifice, the portion of liver tissue were removed surgically and rinsed completely by using normal physiological cold saline. The portion of liver tissue were fixed in 10% of buffered formalin and washed completely in 70% alcohols and embedded in paraffin wax. Cut the paraffin sections at 5µm thickness and stained with H & E for morphological assessment under light microscope.

Statistical Analysis
Results were analyzed by mean ±S.D values significance between the groups was statistically performed by one way ANOVA followed by Tukey's multiple comparisons.
Statistical comparison was followed by using graph pad prism software. P value is significantly less than 0.05. tumors. However hepatotoxicity is highly limited by dose dependent toxicity in cancer treatment condition [19]. The exact mechanism for ADR meditate toxicity in various organs wasmediated by formation of free radical leads to oxidative stress [20]. Oxygen derived free radicals causes'

Effect of d-limonene on liver marker
severe lethal damage to plasma membrane and disturbs assembly of cytoskeleton [21].
Oxidative stress redox mediating free radical production [22], mitochondrial DNA damage [23]. To regulate the over whelm of free radicals, anti-oxidant is necessary to regulate the physiological functions. Hence external source of anti-oxidant can pallitate the therapeutic strategies by controlling oxidative damage [26].
Liver is the major target site for overall metabolism and Aspartate transaminase is one of the best marker enzymes for liver damage. ADR administrated gradually decrease the activity of Aspartate transaminase level and increase the level of lipid peroxidation is highly noticeable that demonstrate the changes in cellular damages. Elevated serum alanine transaminase influence ADR toxicity, which moderately increase the outflow of these enzymes from injured liver cell.
However treatment with d-limonene (100mg/Kg body weight) gradually decreases the negative potential of ADR treated animals thus provide extensive defence against hepatic injury.
More over the histopathological finding revealed that animals treated with (100mg/kg) of dlimonene has protected hepatic tissues from necrosis induced by ADR. So it has finally revealed that the animals pre-treated with d-limonene cosseted hepatic tissues against ADR induced hepatic toxicity in experimental group of animals.

Conclusion:
Finally, the present study reveal that d-limonene plays an important role in defending against ADR induced hepatic damage or hepatotoxicity by ameliorating free radicals andiron chelating properties by scavenging lipid alkoxyl and peroxyl radical. On the basic of these finding it suggest the subsequent level of d-limonene prior to ADR will slowly reduce the impairment to live in cancer chemotherapy. Further clinical studies are abundantly requisite to find the beneficial and therapeutic functions of d-limonene in cancer treatment patients.