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Effect Of Aqueous Extract Of Basil (Ocimum Basilicum) Leaves On

Nephrotoxicity Induced By Adriamycin In Male Rats: Histological And

Immunohistochemical Assessment

Hany A. Abdel Samie1 and Rania A. Ahmed*2

1 Dept. of Zoology, Faculty of Sciences, Menoufia University, Shebin El-Kom, Egypt 2Dept. of Zoology, Faculty of Science, Suez University, Suez, Egypt

* Corresponding author:Rania A. Ahmed 2

Abstract

Adriamycin, isa widely utilized anticancer drug applicable innumber ofhuman malignancies.

Adriamycin exerted considerable effectsin many organs that possibly hamper its utilization. The

beneficial action of the natural plant basil was proven against many aliments. The present work

studied the effect of aqueous extract of basil (ocimumbasilicum) on adriamycin -induced

nephrotoxicity in rats.Administration of Adriamycin intra peritoneally resulted innephrotoxicity

manifested as enlargement and congestion of blood vessels in the kidney, occurrence of

vacuolated cells, shrinkage of glomeruli, edema and leucocytic infiltration and appearance of

proteinous casts in kidney tubules. Immuno histochemically, adriamycin caused elevated

expression of bax and α-SMA in renal cells. Adriamycin treatment induced serum urea and

creatinine increase. Treatment with basil extract with adriamycin lowered the abnormalities

decreased the pro-apoptotic protein, bax and fibrosis marker, α-SMA. Again, basil moved the

levels of serum urea and creatinine towards normal. In conclusion, basiloffered protectively

against nephrotoxicity of adriamycin in male albino rats.

Keywords. Adriamycin, Basil, Kidney, Histology, Immuno histochemistry, rats

I. INTRODUCTION

Cancer, a worldwide health problem, was intensively studied because its cosmopolitan

incidence. Misra et al.(2010) recorded over ten million people are diagnosed each year. Indeed,

Gmeiner and Ghogh (2015) estimated 7.6 million annual deaths from cancer accounting for 13% of

all world deaths with mortality expected to rise to 13.1 million patients by 2030.

Despite of availability of various routes for tumor treatment, mainly surgical, chemotherapy

or radiotherapy, theses till having some drawbacks (Misra et al., 2010). Teixeira et al. (2010)

recorded the cells-non selectivity of chemotherapeutic drugs and the inadequate quantities and the

nonspecific systemic drugs distribution delivered to the cancer site makes difficulty in monitoring

the response of the body to chemotherapy (Wang et al., 2008). The intolerable cytotoxicity added to

the poor delivery of the drug to the cancer site leads to multidrug resistance (Das et al., 2009). The

treatments for cancer exerted toxicity on vital organs including liver (Thatishettyet al., 2013), testis

(Marconet al.,2008), cardiovascular (Meinardiet al., 2000)as well askidney failure (De Jonge and

Verweij, 2006).

Adriamycin (Doxorubicin), ananthra cycline anticancer antibiotics derived by chemical semi

synthesis from Streptomyces peucetius bacteria. Adriamycin works via DNA intercalation and

inhibiting the biosynthesis of macromolecules (Fornari et al., 1994) that in turnprohibits the enzyme

topoisomerase II progression entrusted for relaxing supercoils in DNA for the transcription

AW(Pommieret al., 2010). Adriamycin stabilizes the topoisomerase II complex after it broken DNA

chain for replication, therefore preventing double helix of the DNA from resealing and prevents the

replication process (Tacaretal., 2013). Adriamycin cytotoxicity may be attributed to increased

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production of quinone type free radicals(Rossi, 2013).According to Mennaet al., (2010),adriamycin

is a powerful anticancer drug but its use imposes serious dose-dependent toxicityrisk to non-target

tissues (Yeh et al., 2009). Injacet al. (2008) recorded that adriamycin generates free radicals as well

as induces oxidative stress that might be the reason forits genotoxicity and cardiotoxicity (Quileset

al., 2002).High adriamycin level may injure membranes, proteins (e.g. enzymes and receptors) and

DNA which induce cardiac dysfunction and apoptosis (Kumar et al., 2002).It was recorded by Rasha

and Abdella (2010) that a single intraperitoneal doxorubicindosein mice caused inflammatory

response and degeneration added tointense structural lesions inkidneys,liver and heart associated

with high impact in the apoptotic index (Bax/ Bcl-2) because of oxidative stress.

Huge and increasing patient numbers worldwide depend on botanical preparations to relief

and cure their illness (Jaliliet al., 2014). Girish and Pradhan (2008) highlighted alert towards

effectiveness of herbal medicines. Such natural components exhibited many pharmacologic biologic

characteristics (Hosseinimehr, 2014). Globally, plants were applied traditionally for several purposes

as anti-inflammatory and heap to protection (Ishitaet al., 2004). Nowadays, plants draw great

attention due to efficacy against various ailments including cancer prevention and therapy (Subbaraj

et al., 2013). Gupta et al. (2011) confirmed safely utilization and multi targeting plant poly phenol

derivatives for antitumor actions.

Basil or sweet basil (Ocimum basilicum) possesses high power against antioxidation

(Marinava and Ynishlieva, 1997).It was provenas anthelmintic, antipyretic, cardio protective and

cure for blood diseases (Bunrathepet al., 2007). Batra and Gupta (2006) indicated that

supplementation of extract of O. sanctum leaf decreased the intensity of hepatitis, focal interstitial

nephritis, hydro pericardium, lung oedema, myocarditis and lymphocytic depletion in lymphoid

organs. Studies disclosed the effectiveness of basil's oils as anti-viral (Chiang et al., 2005), antifungal

(Oxenham et al., 2005), antioxidant (Julisni and Simon, 2002; Politeoet al., 2007), anti-cancer (Bozin

et al., 2006) and anti-inflammatory agent (Rakhaet al., 2010, Rodrigues et al., 2016).Basil exerted

heap to protection against diazinon toxicity in rats' liver (Mahboub and Arisha, 2015), by heavy

metals (Sharma et al., 2002) and toxicity and apoptosis in liver caused by CCl4 in rats (Sakret al.,

2011). The current study was planned to examine basil (Ocimum basilicum) leaves extract effect on

nephro toxicity of adriamycin in male albino rats.

II. MATERIALS AND METHODS

Adriamycin (Doxorubicin)

Doxorubicin (Adriablastina produced by Carlo Erba, Milano, Italy) was purchased from a

local pharmacy, Cairo, Egypt in the form of 10 mg/ampoule.

Basil extract

A fresh leaves of Basil (Ocimum basilicum) was collected from the garden in Faculty of

Science, Menoufia University, Shebin El- Kom, Egypt. The leaves were rinsed with clean water to

remove any foreign matter. Leaves were blended with distilled water. The mixture was strained, the

merc pressed and the mixture was filtrated using filter paper. The aqueous extract was used at a dose

level of 20 ml/kg Ocimum basilicum (Offiah and Chikwendu, 1999).

Animals and treatments

Healthy adult male albino rats (Rattusnorvegicus) weighing 130 ± 5 gaged two months

obtained from the breeding center of experimental animals, Helwan, Egypt were kept under constant

temperature (24±2°C) and light/ dark cycle 12/12 hours for at least one week before and during the

experimental conditions. Rats were fed standard diet composed of 20% casein,55% corn starch, 5%

vitaminzed starch, 5% salt mixture and15% corn oil(manufactured by Egyptian Company of Oils and

Soap Kafr-El Zayat, Egypt). Water was suppliedad libitum. Experiment coincides the guide for the

care and use of laboratory animals approved by Faculty of Science, Menoufia University Egypt

(Approval No. MNSH167). Animals were divided into 4 groups of 10 rats each. Group 1is the

control; group 2was orallygiven20 mg/kg aqueous extract of O. basilicum5 days/wk for 8

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weeks;group 3 animals were injected intra peritonealy with 2 mg/kg body weight ADR in sterile

saline, once per week for 8 weeks while group 4 rats were treated with the same dose of ADR as

those of group 3 followed by oral administration of aqueous O. basilicum extract at a dose level as

group 2.

Histological preparations

Following decapitation, animals were quickly dissected, kidneys from all groups were

collected, fixed in 10% formalin, dehydrated in an ascending series of ethanol, cleared in xylene and

finally embedded in molten paraffin (melting point: 50-58°C). 5 μm – thickened sections were cut

obtainedby rotary microtome then mounted on clean glass slides. For histopathological examination,

sections were stained with Ehrlich hematoxylin and counterstained with eosin (Lillie Fulmer, 1976).

Immunohistochemicalinvestigation

For bax and α-SMA localization, wax sections were stained utilizing avidin-biotin peroxidase

method. Sections were depar affinized, endogenous peroxidase activity was blocked by H2O2in

methanol, then heated for 20 minutes in 0.01 mol/l citrate buffer using microwave pressure cooker.

Slides were leftto reach room temperature and nonspecific binding was blocked with normal horse

serum for 20 minutes. Anti-Bax (Dako) monoclonal antibodies were utilized for exposing bax.α-

SMA immune-staining was achieved by polyclonal rabbit anti-human (A3533 I g fraction; DAKO,

Glostrup, Denmark).Counterstaining was done using Mayer's hematoxylin (Cat. No. 94585,

BioGenex, Menarini Diagnostics, Antony, France).

Image analysis Analysis of the digital images was obtained via semi-quantitative scoring system (Image J

software, Java based application for analyzing images). The brown-stained immune histo chemical

expressions of both bax and α-SMA were analyzed through determining the percentage of colored

stained area / field area in five randomly high power fields at magnification of 400X.

Biochemical assays

For biochemical assays, blood samples were centrifuged at 3000 rpm for 10 minutes and

stored at -20 °C. Creatinine and urea were assessed in serum following method of Henry (1974) and

Patton and Crouch (1977), respectively.Data were expressed as mean ± standard error, analyzed using

Student’s t-test and homogeneity of variances (Levene test) applying statistical program of social science

(SPSS) software for windows. P < 0.05, value was used.

III. RESULTS

Histological results

Examination of control kidneys revealed normal structure (Fig. 1a). Basil extract had no

effects on tissue of the kidney. Adriamycin treatment for 4 weeks caused enlargement and

congestion of blood vessels in, appearance of some vacuolated cells and shrinkage of glomeruli

(Fig.1b). Added to that, edema, presence of widened blood vessel and slight leucocytic infiltration

were observed (Fig.1c and d). ADR treatment for 8 weeks induced degeneration of renal tubules with

their lumina filled with proteinous casts and increased intra glomerular space in addition to

degeneration of the glomeruli (Fig 2a &b). Interestingly, treatment with basil with adriamycin

decreased the abnormalities in the kidney (Fig.2c). Table 1 summarized the histo pathological

alterations in different animal groups.

Immunohistochemical results

Regarding bax expression, renal cells of control and basil-treated rats were negatively stained

(Fig.3a). 4 weeks adriamycin treatment strongly increase bax expression in renal tubular cells

reaching maximum following 8 weeks treatment (Fig.3b). However, such strong positive baximmuno

reactivity was declined in animals subjected to the dual treatment of basil and adriamycin (Fig.3c).

Moreover, control and basil exposed- rats exhibited α-SMA immune- staining in smooth muscle of

renal arterioles (Fig.4a). Adriamycin treatment for 4 weeks induces increase in expression of α-SMA

was observed in the tubules where as maximal expression was recorded after 8 weeks (Fig.4b).

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However, treatment with basil reversed the abnormality, where a decrease in expression of α-SMA

was encountered (Fig.4c).

Image analysis

Data in table 2declared that ADR group exhibited statistically higher significant difference (p

≤ 0.05)in mean % area of α-SMA in cortex of the kidney comparing with the other groups.

Treatment with ADR and basil revealed insignificant staining % area. Similarly, significant

expression in bax % area was recorded in rats given ADR compared with ADR plus basil group.

Biochemical results

It is clear in figure 5 that there isa significant increase in creatin in einsera of ADR treated-

rats compared with control or basil groups in all experimental periods. Treating animals with ADR

and basil created a significant creatinine depression when compared with ADR group. Similarly,

blood urea significantly elevated following ADR treatment while rats treated with basil and ADR

had significantly decreased urea level in comparison with ADR group in allperiods(Fig.6).

IV. DISCUSSION

The increasing need forth erapies of cancer with no harmful actions on non-cancerous cells

targeting only malignant cells has opened the horizon for new era of utilizing the natural components

to ameliorate their side effects. Adriamycin toxicity limits its application in chemical therapy (Al-

Syaad and Ibrahim, 2014). It was indicated thatadriamycin toxicity is because of production of free

radicals (Yagmurcaet al., 2004) causedinjury to cells (Saadet al., 2001) which is demonstrated by

lipid peroxidation andoxidation of protein in tissue (Karamanet al., 2006). Adriamycin induces

severe damage in organs including testis, liver, heart andkidneys (Gokcimenet al., 2007). In this

study,the toxic actionsof adriamycin was investigated on kidneys expressed in changes of

histological and immunohistochemical inspection as well as measuring kidney function serum

parameters.

Nephrotoxicity constitutes an important side effect of anthracyclines. Findings obtained in

this work indicated that adriamycin administration induced congestion of blood vessels and

vacuolization of renal cells. However, the exact mechanism of doxorubicin nephro toxicity is

unclear. It is suggested it is mediated through formation of free radicals, iron-dependent oxidative

damage of macromolecules and peroxidation of membrane lipid (Liu et al., 2007). In this work

shrinkage of glomeruli, dilation of blood vessel and cellular vacuolation were recorded in renal cells.

Similarly, Fajardoet al. (1980) demonstrated cytoplasmic vacuoles in the renal tubule cells. Added to

that, Al-Syaad and Ibrahim (2014) indicated that extended adriamycin administration caused

extensive glomerular lesions, tubular dilatation, renal glomeruli vacuolization and atrophy of the

glomeruli. In addition, Wapstraet al. (1999) recorded alterations in rats' kidneys as increased

permeability of glomerular capillaries and tubular atrophy by doxorubicin treatment. Podjarnyet al.

(1993) and Jovanovicet al. (1996) indicated that adriamycin caused rats nephrotoxicity. Pedrycz et al.

(2005) found that a single intraperitoneal adriamycin dose of 5mg/kg b w given to mothers rats at 4

weeks before pregnancy caused dilatation of the urinary space and increased thickness of basement

membranes in fetal kidneys' glomeruli, damaged or completely destroyed epithelial cells and dilated

the tubular lumen. In this work, adriamycin caused edema, widened blood vessel and leucocytic

infiltration. In agreement with these results, Deepa and Varalakshmi (2005) and Injac and Strukelji

(2008) found that nephro toxicity following doxorubicin manifested in glomerular atrophy and

inflammatory reaction in rats kidney tissues. More confirmatory, Ayla et al. (2011) indicated

glomerular vacuolization and degeneration, thickening and untidiness of glomerular andtubular

capillary basement membranesin tissue of doxorubicin- treated rats.

From the immune histochemical point of view, adriamycin treatment for 4 weeks increased

expression of bax in renal cells reaching maximum after 8 weeks. More importantly,

immunohistochemical findings confirm the structuralfindings where degeneration reflected by

increase in apoptotic index mainly bax expression. Bax protein promotes apoptosis through elevating

susceptibility to stimuli-inducingapoptosis (Tsamandas et al., 2003).Coincides with these findings

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what was announced by Pedrycz et al. (2005) who registered high level of bax/ Bcl-2 in the liver of

rat fetuses whose mothers treated with a single dose of adriamycin before pregnancy. The current

results extend and confirm previous data ofRasha and Abdella (2010)who reported that doxorubicin

administration resulted in elevatedbax expression level in liver and kidney. Experiments conducted

by Franco et al. (2001) revealed that doxorubicin application causes elevation in pre-apoptotic bax

and caspase-3 immunoreactivity accompanied by lipid peroxidation and increasing MDA levels in

kidney.

The present findings revealedan elevation ofα-SMA in kidney of ADR-treated rats while

basil exertedsome protection. Similarly, RuChunet al. (2009) indicated that adriamycin induced

nephrosis and greatly elevated the immune-expression of α-SMA in rats renal tissue while this

expression was lowered by Yiqixue Qufengshi Fang and Sinomenine treatment. A recent study

conducted by Mustafa et al. (2015) indicated that doxorubicin increased the expression of α-SMA.

These effects were lessened using natural antioxidant Coenzyme Q10 (CoQ10) or artichoke.

Recently, Xu et al. (2016) indicated that dalbergioidin (DAL) extracted from Urariacrinita greatly

improved doxorubicin-caused nephro toxicity and modified α-SMA expression.

ADR has toxic action on the kidneys, thus causes acute renal failure that is a limiting factor

for its beneficial application (Ayla et al., 2011). In this work, ADR significantly increased blood urea

and creatinine after treatment for 8 weeks. In agreement, Mansour et al. (1999) recorded arise of

serum urea post- 24 and 48 h treatment with single dose of doxorubicin (15 mg/kg) causing

nephrotoxicity. Wang et al. (2000) observed creatinine clearance depression in animals one week

following injection of adriamycin's single dose via the tail vein. According to Nilesh et al. (2010)

elevated levels of creatinine and urea in serumof doxorubicin- treated rats could be due to intrinsic

acute renal failure. Jambhulkar et al. (2014) indicated that adriamycin (10mg/kg body weight)

administered twice caused increase in serum creatinine andurea.

A large body of evidence collected throughin vitro preclinical studies showed the

effectiveness of phyto chemicals in retarding tumors growth and eliciting anti-inflammatory and

antioxidant responses (Bland, 1996).Worth noting according to articles search that no available

literature dealing with synergistic effect of basil on adriamycin in kidney histology, immune histo

chemistry or the selected biochemical parameter that possibly indicates the value of the present

work.

Results presented hereemphasized the basil efficiency in ameliorating / or protecting

nephrotoxicity exerted by adriamycin which manifested in improving histological structure,

restoration of bax and α-SMA expression in kidney. Additionally, serum creatinine and urea levels

were restored directed towards normal levels. In agreement with these results, Sakr and Al-Amoudi

(2012) indicated that pyrethroid insecticide deltamethrin- inducedrats' nephrotoxicity.Deltamethrin

caused degeneration of lining epithelium of kidney tubules, atrophy, dilation and congestion of blood

vessels, inflammatory leuco cytic cells in the intertubular spaces added to marked increase in urea

and creatinine whereas basil administration reversed these alterations. Karamala et al. (2011)

indicated protection byO. basilicum against mercury and lead toxicity nephrotoxicty. In addition,

dietary treatment withO. basilicum normalized the high creatininelevel indiabetic rats serum as was

recorded by Suanarunsawat and Songsak (2005).

Enzymatic and non-enzymatic antioxidants serve as an important biological defense against

environmental pollutants (Tabrez and Ahmed, 2011).The degree of toxicity expressed by various

xenobiotics could be judged by measuring the concentration and activities of product of lipid

peroxidation (e.g. malondialdhde) and oxidative stress with antioxidant system (e.g. superoxide

dismutases).The antioxidant property of basil was tested by many researchers. Ramesh and

Satakopan (2010) reported that administration of Ocimum sanctumpre- and post- cadmium

intoxication resulted in a significant depression in LPO values and significant elevation

inCAT,GSH,SOD, GPx, and ascorbate levels. Moreover, Kahilo et al.(2015) indicated that

Ocimumbasilicum elevated the antioxidant activity (SOD, CAT, GSH, GPx, GST) and decreased

cholesterol, TAG level, Blood urea and ALT following exposing broilers chicken to gibberllic acid.

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Treating animals with diazinon and O. basilicumextract at induced reduction in testicular alterations

caused by diazinon. Moreover, SOD and CAT were increased while MDA was decreased

confirmingefficiency of extract obtained from O. basilicum leaves in loweringthe reproductive

alterations induced by diazinon in albino rats due to the strong antioxidant chracteristics of its

components (Sakr and Abdel Samie, 2015).

Conclusively, ADR caused structural alterations in kidney structure of rats which is reflected

by increase inexpression of both bax and α-SMA in addition to creatinine and urea in the serum.

Treatment with basil extract in conjuction with ADR ameliorated most of the histopathological

anomalies and restored the immunohistochemical and biochemical parameters towards normal. It is

plausible to apply natural products to manipulate the side effects of chemodrugs when their use is

unavoidable.

Figure1: a.Kidney cortex of a control rat showing normal-structured glomeruli (G) and intact renal tubules (R),

(X300).b, c and d: Kidney of ADR-treated rat for 4 weeks. b: showing enlarged and congested renal vein (bv), presence

of few vacuolated cells (arrowhead) and condensation of renal glomeruli (X200). c. Degeneration of renal tubules with

and edema (AD), degenerated glomerulus (DG) and presence of widened blood vessel and slight leucocytic infiltration

(LI). (X200). d. Kidney cortex showing degeneration of renal tubules (R), enlarged blood vessel (bv) and leucocytic

infiltrations (LI), (X300)

e Figure2:a & b. Kidney cortex of a rat treated with ADR for 8 weeks. a: Showing degeneration of renal tubules with

lumen filled with proteinous casts (X300). b. Showing degeneration of glomeruli (DG). (X200).c. Kidney cortex of a rat

treated with both ADR and basil showing improvement in structure of renal tubules and glomeruli (G) despite of some

glomeruliwhichwere still shrunken. (X200).

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Figure 3a.Section of kidney of control rat showing negative expression of bax (baximmunohistochemical stain, X200).

b.Section of kidney of ADR treated rat showing strong positive expression of bax in the epithelia of renal tubules (arrow

head) (baximmunohistochemical stain, X200).c. Section of kidney of a rat treated with ADR and basil showing weak

expression of bax in the epithelia of renal tubules (arrow head) (baximmunohistochemical stain, X200).

Figure4. a.Section in kidney of a control rat showing α-SMA immune- staining in smooth muscle of renal arterioles

(X200).b. Section of kidney of ADR treated rat showing an increase in expression of α-SMA in renal cells (x200).c.

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Section of kidney of ADR and basil- treated rat showing decrease in expression of α-SMA positive fibroblastic renal cells

(x200).

Table 1 Quantitative assessment of histopathological changes in kidney of different animal groups.

+ Mild (5–10% severity), + + Moderate (10–25% severity), + + +Severe (25–50% severity)

Figure 5.Change in creatinine in different animal groups* Difference from control and basil- treated animals

0

0.5

1

1.5

2

2.5

Animal group

Cre

atin

ine(

mg/

dl)

controlBasilADR ADR+Basil

0

5

10

15

20

25

30

35

40

45

Animal group

Ure

a n

itro

gen

(m

g/d

l)

controlbasilADRADR+basil

Animal Group Tubular Leucocytic Glomeruli Degeneration cast infiltrations Atrophy

Control

Basil

Adriamycin

Adreiamycin+

Basil

- - +

-

- - +

-

+++ ++ +++

++

+ + +

+

*

*

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Figure 6. Change in urea in different animal groups* Difference from control and basil- treated animals

Table 2: Mean % area occupied by bax and α-SMA in the rat kidney in all animal groups.

Data was expressed in mean ± SD.

*: Statistically significant at p ≤ 0.05 compared with other groups.

**: Statistically significant at p ≤ 0.05 compared with ADR group.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

REFERENCES

1. Al-Syaad K, Ibrahim E H. (2014). The protective effects of the aqueous extract of saliva against biochemical and

histopathological changes in kidney and liver of male rats treated with the anticancer drug doxorubicin. Res J

Pharmaceut Biol Chem Sci RJPBCS . 5: 237- 45..

2. Ayla, S.; Seckin, I.; Tanriverdi, G.; Cengiz, M.; Eser, M.; Soner, B.C. and Oktem, G. (2011). Doxorubicin

induced nephrotoxicity: Protective effect of nicotinamide. International J Cell Biol . (2011): 1- 9..

3. Batra M, Gupta RP (2006). Effects of Ocimum sanctum leaf on pathology and immune response in chickens

experimentally infected with hydropericardium syndrome. J Indian Vet Pathol. 30:4750-8..

4. Bland JS (1996) . Phytonutrition, phytotherapy, and phytopharmacology. AlternTher Health Med . 2: 73–6.

5. Bozin B, Mimica-Dukic N, Simin N, Anackov G (2006). Characterization of the volatile composition of essential

oils of some lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J Agric Food Chem.

54: 1822–8.

6. Bunrathep S, Palanuvej C,Ruangrungsi N (2007). Chemical compositions and antioxidative activities of essential

oils from four Ocimum species endemic to Thailand. J Health Res . 3: 201-6.

7. Chiang L C, Ng L T, Cheng P W, Chiang W, Lin C C (2005). Antiviral activities of extracts and selected pure

constituents of Ocimum basilicum. Clin Exp Pharmacol Physiol. 32:811-6.

8. Das M, Mohanty C, Sahoo SK (2009). Ligand-based targeted therapy for cancer tissue. Expert Opin Drug Deliv.

6:285-304..

9. De Jonge MJ, Verweij J (2006) .Renal toxicities of chemotherapy. Seminars in Oncol. 33: 68–73.[9]

10. Deepa PR, Varalakshmi P (2005) . Biochemical evaluation of the inflammatory changes in cardiac, hepatic and

renal tissues of Adriamycin-administered rats and the modulatory role of exogenous heparinderivative treatment.

Chem Biol Interact. 156: 93-100..

11. Fajardo LF, Eltringham J R, Stewart JR, Klauber M R (1980). Adriamycin nephrotoxicity. Lab Invest. 43: 242–53.

12. Fornari FA, Randolph JK, Yalowich JC, Ritke MK, Gewirtz D A (1994) . Interference by doxorubicin with DNA

unwinding in MCF-7 breast tumor cells. Mol Pharmacol. 45: 649–56..

13. Franco Z, Graziella P, Paola P (2001). Role of the sugar moiety in the pharmacological activity of anthracyclines:

development of a novel series of disaccharide analogs. Bioc Fm Pharmacol. 61:933–8.

14. Girish C,Pradhan SC (2008). Drug development for liver diseases: focus on picroliv, ellagic acid and curcumin.

Fundam Clin Pharmacol. 22:623-32..

15. Gmeiner W H,Ghosh S (2015). Nanotechnology for cancer treatment. Nanotechnol Rev . 3: 111–22.

16. Gokcimen, A.;Cim, A.;Tola, H.T.;Bayram, D.;Kocak, A.;Ozgüner, F. and Ayata, A (2007). Protective effect of N-

acetylcysteine, caffeic acid and vitamin E on doxorubicin hepatotoxicity. Hum Exp Toxicol . 26: 519-25..

17. Gupta SC, Kim JH, Kanappan R, Reuter S, Dougherty PM, Aggarwal BB (2011) . Role of nuclear factor- kB-

mediated inflammatory pathways in cancer-related symptoms and their regulation by nutritional agents. Experiment

Biol Med. 236: 658-71..

18. Henry RJ (1974) . Clin. Chem., Principles and Technics (2nd Ed), Harper and Row. 548-551.

19. Hosseinimehr SJ (2014) . Beneficial effects of natural products on cells during ionizing radiation. Rev Environ

Health. 29: 341-53..

20. Injac R, Strukelji B (2008) . Recent advances in protection against doxorubicin-induced toxicity. Technol Cancer

Res Treat . 7: 497-516..

Group I

(Control)

(n = 5)

Group II

(basil)

(n = 5)

Group III

(ADR)

(n = 5)

Group IV

(ADR +basil)

(n = 5)

α-SMA 2.2 ± 0.8 2.5 ± 1.1 16.2± 1.4* 5.5 ± 1.7**

Bax 1.1 ± 0.50 1.3 ± 0.60 18.5± 2.1* 7.4 ± 1.4**

International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 03, Issue 3, [March- 2017] e-ISSN: 2394-5532, p-ISSN: 2394-823X

@IJAPSA-2017, All rights Reserved Page 108

21. Injac, R.; Perse, M.; Obermajer, N.; Djordjevic-Milic, V.; Prijatelj, M.; Djordjevic, A.; Cerar, A. and Strukelj, B

(2008) .Potential hepatoprotective effects of fullerenol C60 (OH) 24 in doxorubicin-induced hepatotoxicity in rats

with mammary carcinomas. Biomaterials. 29(24-25):3451-60..

22. Ishita C, Kaushik B, Uday B, Banerjee R(2004). Turmeric and curcumin biological actions and medicinal

applications. Curr Sci . 87:44- 53..

23. Jalili C, Salahshoor MR, Moradi S, Pourmotabbed A, Motaghi M (2014) . The therapeutic effect of the aqueous

extract of boswellia serrata on the learning deficit in kindled rats. Int J Prev Med . 5:563-8..

24. Jambhulkar S, Deshireddy S, Jestadi DB, Periyasamy L (2014). Quercetin attenuating doxorubicin induced hepatic,

cardiac and renal toxicity in male albino Wistar Rats. Am J Phytom Clin Therapeut AJPCT.2: 985-1004..

25. Jovanovic, D.; Djukanovic, I.; Susic, D.; Funduk, G.; Jovanovic, Z.; Dragojlovic, Z.; Boydanovic, G. and Starcevic-

Bonzovic, Z (1996) . The effect of captopril on the development of adriamycin nephropathy in rats with

spontaneous arterial hypertension. Srp Arh Celok Lek . 124: 47-9.

26. Julisni HR, Simon JE (2002) . Trends in new crops and new uses, J. Janick, Ed., ASHS Press, Alexandria, VA, p.

575.6.7.

27. Kahilo K, El kany H, Sadek K, Kheir-Eldeen A (2015) . Antioxidant and immunostimulant effects of basil

(Ocimum basilicum) against gibberllic acid and auxin Supplemention in broilers ration. Global Veterinaria.15 (3):

289-95.

28. Karamala SK, Srilatha C, Anjaneyulu Y,Rao TS, Sreenivasulu D, Pidugu AP (2011) . Hematobiochemical changes

of lead poisoning and amelioration with Ocimum sanctum in wistar albino rats. Vet World. 4:260–3..

29. Karaman A, Fadillioglu E, Turkmen E, Tas E, Yilmaz Z (2006) . Protective effects of leflunomide against ischemia

reperfusion injury of the rat liver. Pediatr Surg Int. 22: 428–34.

30. Kumar D, Lou H, Singal PK (2002). Oxidative stress and apoptosis in heart dysfunction. Herz. 27: 662–8.[30]

31. Lillie RD, Fulmer HM (1976). Histopathological Technique and Practical Histochemistry. 4th ed. New York,

McGraw Hill.

32. Liu LL, Li QX, Xia L, Li J,Shao L (2007) . Differential effects of dihydropyridine calcium antagonists on

doxorubicininduced nephrotoxicity in rats. Toxicology. 231:81–90.

33. Mahboub FA, Arisha SM(2015) . Hepatoprotective effect of Ocimum basilicum extract against the toxicity of

diazinon in albino rats: Histopathological and immunohistochemical evaluation World J Pharm Sci. 3: 790-9.

34. Mansour MA, El-kashef HA, Al-shabanah OA (1991). Effect of captopril on doxorubicin-induced nephrotoxicity in

normal rats. Pharmacol Res. 39:233-7.

35. Marcon L, Hales BF, Robaire B (2008). Reversibility of the effects of subchronic exposure to the cancer

chemotherapeutics bleomycin, etoposide, and cisplatin on spermatogenesis, fertility, and progeny outcome in the

male rat. J Androl. 29:408–17..

36. Marinava EM, Ynishlieva N V (1997). Antioxidative activity of extracts from selected species of the family

lamiaceae in sunflower oil. Food Chem. 58: 245- 8..

37. Meinardi, M.T.; Gietema, J.A.; van, der. Graaf, W.T.; van, Veldhuisen, D.J.; Runne, M.A.; Sluiter, W.J.; de Vries,

E.G.; Willemse, P.B.; Mulder, N.H.; van den Berg, M.P.; Koops, H.S. and Sleijfer, D.T (2000) . Cardiovascular

morbidity in long-term survivors of metastatic testicular cancer. J Clin Oncol . 18: 1725–32..

38. Menna P, Salvatorelli E, Minotti G (2010) . Anthracycline degradation in cardiomyocytes: a journey to oxidative

survival. Chem Res Toxicol. 23:6–10..

39. Misra R, Acharya S, Sahoo SK (2010) . Cancer nanotechnology: application of nanotechnology in cancer therapy.

Drug Discov Today .15 (19/20): 842- 50..

40. Mustafa HN, El Awdan SA, Hegazy GA,Abdel Jaleel GA (2010). Prophylactic role of coenzyme Q10 and Cynara

scolymus L on doxorubicin-induced toxicity in rats: Biochemical and immunohistochemical study. Indian J

Pharmacol. 47:649-56

41. Nilesh S, Amit J, Vaishali U, Sujit K, Sachin K, Ravindra P (2010). Protective effect of Lepidium sativum against

doxorubicin-induced nephrotoxicity in rats. RJPBCS . 1: 42-9.

42. Offiah, V.N. and Chikwendu, U.A. Antidiarrhoeal effects of Ocimum gratissimum leaf extract in experimental

animals. J Ethnopharmacol., 1999, 68(1-3):327-330.

43. OxenhamS K, Svoboda KP, WaltersD R(2005). Antifungal Activity of the Essential Oil of Basil (Ocimum

basilicum). J Phytopath. 153: 174–80.

44. Patton C, Crouch S (1977). Determination of serum urea. Anal Chem. 49: 464-469.

45. Pedrycz A, Boratynski Z, Wieczorski M,Visconti J (2005) . Ultrastructural and immunohistochemical evaluation

ofapoptosis in foetal rat liver after adriamycin administration. Bull Vet Inst Pulawy . 49: 475-8.

46. Podjarny E, Bernheim JL, Pomeranz A, Rathaus M, Green J,Bernheim J (1993) . Effect of timing of

antihypertensive therapy on glomerular injury: Comparison between captopril and diltiazem. Nephrol Dial

Transplant . 8: 501-6. .

47. Politeo O, Jukic M,Milos M (2007) . Chemical composition and antioxidant capacity of free volatile aglycones from

basil (Ocimum basilicum L.) compared with its essential oil. Food Chem 2007; 101: 379–85.

48. Pommier Y, Leo E, Zhang H, Marchand C (2010). DNA topoisomerases and their poisoning by anticancer and

antibacterial drugs. Chem Biol. 17: 421–33.

International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 03, Issue 3, [March- 2017] e-ISSN: 2394-5532, p-ISSN: 2394-823X

@IJAPSA-2017, All rights Reserved Page 109

49. Quiles J L, Huertas J R, Battino M, Mataix J, Ramirez Tortosa M C (2002) . Antioxidant nutrients and adriamycin

toxicity. Toxicology2002; 180: 79-95.

50. Rakha P, Sharma S,Parle M (2010) . Anti - inflammatory potential of the seeds of Ocimum basilicum Linn. in rats.

Asian J Bio Sci. 5: 16-8. .

51. Ramesh B, Satakopan VN (2010) . Antioxidant activities of hydroalcoholic extract of Ocimum sanctum against

cadmium induced toxicity in rats. Indian J Clin Biochem . 25: 307–10.

52. Rasha AR, Abdella EM (2010) . Modulatory Effects of Rosemary Leaves Aqueous Extract on Doxorubicin-

Induced Histological Lesions, Apoptosis and Oxidative Stress in Mice. IJCP . 1: 1-22.

53. Rodrigues, L.B.;Oliveira, Brito. Pereira.Bezerra. Martins. A.;Cesário, F.R.;Ferreira, E. Castro. F.;de Albuquerque,

T.R.;Martins, Fernandes. M.N.;Fernandes, da Silva. B.A.;Quintans, Júnior. L.J.;da Costa, J.G.;Melo, Coutinho.

54. H.D.;Barbosa, R. andAlencar, de Menezes. I.R (2016) . Anti-inflammatory and antiedematogenic activity of the

Ocimum basilicum essential oil and its main compound estragole: In vivo mouse models. Chem Biol Interact.

257:14-25.

55. Rossi S ED (2013) . Australian Medicines Handbook. Adelaide: The Australian Medicines Handbook Unit Trust.

56. RuChun, Y,YongJun W,Ying L,YingHua Z,XiaoLing Z,ChunPing L (2009) . Effects of YiqixueQufengshiFang on

expression of α-SMA and ROCK1 in kidney tissue of rats with adriamycin nephrosis. China J Traditional Chinese

Med Pharm . 24: 104-7..

57. Saad SY, Najjar TA, Al-Rikabi AC (2001) . The preventive role of deferoxamine against acute doxorubicin-induced

cardiac, renal and hepatic toxicity in rats. Pharmacol Res. 43:211–8.

58. Sakr S A ; Al-Amoudi MW (2012). Effect of leave extract of Ocimum basilicum on deltamethrin induced

nephrotoxicity and oxidative stress in albino rats. J Appl Pharm Sci .62: 22-7..

59. Sakr S A, Abdelsamie H A (2015) . Protective effect of ocmuim basilicum leaves extract on diazinon-induced

reproductive toxicity and oxidative stress in albino rats. EJPMR. 2: 375-83..

60. Sakr SA, El-Abd SF, Osman M, Kandil AS, Helmy MS (2011) . Ameliorative effect of aqueous leave extract of

Ocimum basilicum on Ccl4 - induced hepatotoxicity and apoptosis in Albino rats. J American Sci. 7: 116-27..

61. Sharma MK, Kumar M, Kumar A (2002) . Ocimum sanctum leaves extract provides protection against mercury

induced toxicity in Swiss albino mice. Indian J Exp Biol. 40: 1072–82..

62. Suanarunsawat T, Songsak T (2005) . Anti-hyperglycemic and anti-hyperlipidemic effect of dietary supplement of

white Ocimum sanctum Linn. before and after STZ-induced diabetes mellitus. Int. J Diabetes and Metabolism2005;

13:18–23.

63. Subbaraj GK, Kulanthaivel L, Rajendran R, Veerabathiran R (2013) . Ethanolic extract of carumcarvi (eecc)

prevents nnitrosodiethylamine induced Phenobarbital promoted hepatocarcinogenesis by modulating antioxidant

enzymes. International J Pharm Pharmaceutl Sci. 5(Supp1):195-9.

64. Tabrez S,Ahmad M (2011) . Some enzymatic/ nonenzymatic antioxidants as potential stress biomarkers of

trichloroethylene, heavy metal mixture, and ethyl alcohol in rat tissues. Environ Toxicol . 26:207-16..

65. Tacar O, Sriamornsak P, Dass CR (2013) . Doxorubicin: an update on anticancer molecular action, toxicity and

novel drug delivery systems. The J Pharm Pharmacol. 65: 157–70.

66. Teixeira VM, Silva AC, Lopes CM (2010) . Role of nanotechnology in cancer treatment and technology. Revista

da Faculdade de Ciências da Saúde. 7: 224-32..

67. Thatishetty AV, Agresti N, O'Brien CB (2013) . Chemotherapy-induced hepatotoxicity. Clin Liver Dis .17: 671–86.

68. Tsamandas, A.; Thomopoulos, K.; Zolota, V.; Kourelis, T.; Karatzas, T.; Ravazoula, P.; Tepetes, K. and Petsas, T

(2003) . Potential role of Bcl-2 and Bax m-RNA and protein expression in chronic hepatitis type B and C: A

clinicopathologic study. Mod Pathol. 16: 1237-88..

69. Wang X, Yang L, Chen Z, Dong M, Shin MD (2008) . Application of nanotechnology in cancer therapy and

imaging. CA Cancer J Clin . 58: 97–110.

70. Wang Y, Wang, YP, Tay YC, Harris DCH. Progressive Adriamycin nephropathy in mice (2000) : sequence of

histologic and immunohistochemical events. Kidney Int . 58:1797-1804.

71. Wapstra FH, van Goor H,de Jong PE, Navis G, de Zeeuw D (1999) . Dose of doxorubicin determines severity of

renal damage and responsiveness to ACE-inhibition in experimental nephrosis. J Pharmacol Toxicol Methods.

41(2-3):69-73..

72. Xu D,Dong Y,Chen M,Fan J,Dong Y, Xu C (2016) . Dalbergioidin ameliorates doxorubicin-induced renal fibrosis

by suppressing the TGF-ß signal pathway. Mediators of Inflammation. (In press).

73. Yagmurca M, Erdogan H, Iraz M, Songur A, Ucar M, Fadillioglu E (2004) . Caffeic acid phenethyl ester as a

protective agent against doxorubicin nephrotoxicity in rats. Clin Chim Acta. 348 (1-2): 27-34.

74. Yeh, Y.C.; Liu, T.J.; Wang, L.C.; Lee, H.W.; Ting, C.T.; Lee, W.L.; Hung, C.J.; Wang, K.Y. and Lai, H.C (2009) .

A standardized extract of Ginkgo biloba suppresses doxorubicin-induced oxidative stress and p53-mediated

mitochondrial apoptosis in rat testes. Br J Pharmacol. 156:48–61..