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Table of Contents
ORIGINAL ARTICLE
Year : 2016  |  Volume : 4  |  Issue : 1  |  Page : 9-17

Protective effect of n-butanol extract of Nyctanthes arbor-tristis on antioxidant tissue defence system against doxurubicin induced cardiotoxicity in rats


1 PG student, Department of Pharmacology, MGV’s Pharmacology College, Panchavati, Nashik-422003, India
2 Professor, Department of Pharmacology, MGV’s Pharmacology College, Panchavati, Nashik-422003, India

Date of Web Publication29-Aug-2018

Correspondence Address:
M Mohan
Professor, Department of Pharmacology, MGV’s Pharmacology College, Panchavati, Nashik-422003
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2347-6486.240047

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  Abstract 


Objective: The present investigation aims to study the protective role of n-butanol extract of Nyctanthes arbor- tristis on antioxidant tissue defense system against doxorubicin induced cardiotoxicity in rats.
Methods: Wistar rats were divided into various treatment groups. Doxorubicin (3mg/kg, i.p.) (DOX) was used to induce cardiotoxicity. N-butanol extract of Nyctanthes arbor-tristis (100 and 300 mg/kg, p.o.) and Vitamin E (25 mg/kg, p.o.) were administered for 15 days. ECG changes, biochemical parameters (LDH, SGOT, CPK), relative organ weight, antioxidant levels (SOD, GSH, LPO) and histopathological changes were recorded at the end of treatment schedule.
Results: DOX treated animals showed elevation of QRS complex and a significant (p<0.05) elevation in levels of SGOT, CPK, LDH and LPO and a significant (p<0.05) reduction in relative organ weight, GSH and SOD. Histopathological changes like nuclear changes, vacuolated myocytes, cell necrosis, dilated vessels, partial loss of myofibrils were observed in the DOX treated group. Animals co-administered with n-butanol extract of Nyctanthes arbor-tristis (100 and 300 mg/kg, p.o) and vitamin E (25 mg/kg, p.o.) in DOX treated animals showed significant (p<0.05) protection of all the above parameters as compared to DOX treated animals alone. Conclusion: The beneficial effect of Nyctanthes arbor-tristis against cardiotoxicity could be attributed to its antioxidant potential.

Keywords: Nyctanthes arbor-tristis, Antioxidant, Cardiotoxicity, Doxorubicin


How to cite this article:
Mahajan J, Mohan M. Protective effect of n-butanol extract of Nyctanthes arbor-tristis on antioxidant tissue defence system against doxurubicin induced cardiotoxicity in rats. J Integr Health Sci 2016;4:9-17

How to cite this URL:
Mahajan J, Mohan M. Protective effect of n-butanol extract of Nyctanthes arbor-tristis on antioxidant tissue defence system against doxurubicin induced cardiotoxicity in rats. J Integr Health Sci [serial online] 2016 [cited 2021 Nov 30];4:9-17. Available from: https://www.jihs.in/text.asp?2016/4/1/9/240047




  Introduction Top


Cardiotoxicity is a known side effect of numerous drugs which may lead to severe morbidity.[1] It includes a broad range of change in cardiac parameters, from blood pressure and arrhythmias to cardiomyopathy. There are several mechanisms of cardiomyopathy induced cardiotoxicity which may include cellular damage due to the formation of free oxygen radicals and stimulation of various immunogenic reactions in the presence of antigen presenting cells in the heart.[2]

The anthracycline drug Doxorubicin (DOX) is one of the most effective widely used antineoplastic agent. Its use has been restricted due to the dose- dependent cardiotoxicity which may result in myocardial damage, resulting in dilated cardiomyopathy with fatal congestive heart failure.[3]

Several hypothesis have been postulated for the development of free radicals induced myocardial injury[4] lipid peroxidation; mitochondrial damage[5]; decreased activity of sodium ions[6], K-adenosine triphosphate; vasoactive amine release; impairment in myocardial adrenergic signaling/regulation and cellular toxicity. Doxorubicin is transformed into a semiquinone free radical which reacts with molecular oxygen to produce quinone. This quinone-semiquinone cycle generates large amounts of superoxide radicals that damage the heart.[7] Antioxidants exert their effect by scavenging reactive oxygen species (ROS), and preventing the generation of ROS.[8]

Plants and plant products have been used as a source of medicine since long time. Recently, plants have received much attention as sources of biological active substances, including antioxidants.[9] It has been shown that antioxidant therapy is useful in the management of cardiotoxicity and has a protective effect against cardiovascular problems.[10] A large number of phytoconstituents from various plants are reported to have cardioprotective activity. Some of them include aloin (Aloe vera)[11]; alkaloids (Preema seratifolia)[12]; bromelian (Ananas comosus)[13]; some curcuminoides (Curcuma longa)[14]; embelicanin (Embelica officinalis)[15]; and piperine (Piper longum)[16].

Nyctanthes arbor tristis Linn. (Oleaceae) is a valuable medicinal plant. The plant generally grows in tropical and subtropical regions. The plant has numerous medicinal values. Phytochemical and total phenols present in the leaves are the main sources of antioxidants, which could decrease the potential stress caused by free radicals.[17] Therefore the present study aims to elucidate the possible cardioprotective effect of n-butanol extract of Nyctanthes arbor-tristis.


  Methodology Top


Drugs

Doxorubicin (Metodox 50mg, Neon Pharmaceutical Ltd. Nashik, India) and Vit E capsule (Evion 400, MERCK) were purchased from local pharmacy. All Chemicals for biochemical assays were of analytical grade. LDH, CPK and SGOT kits were purchased from Agappe Diagnostics Pvt. Ltd., India, and Span diagnostic Ltd., respectively.

Animals

Wistar albino rats (150-200g) of either sex were obtained from Bharat Serum & Vaccines LTD, Thane. Animals were housed five per cage under standard laboratory conditions of temperature (25±2 c) with food and water provided ad libitum, relative humidity of 45-55% and 12/12 light/dark cycle. The experiments were carried out according to the guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA), New Delhi, India, and the Institutional Animal Ethical Committee (IAEC) approved protocol of this study.

Preparation of extract

Shade dried leaves of Nyctanthes arbor tristis Linn. (Oleaceae) were grinded into fine powder.100g of powder was extracted with 500 ml of methanol: water (4:1) and macerated for 7 days [18]. The extract was concentrated to remove methanol and the remaining aqueous solution was extracted with n- butanol. The butanolic fraction of Nyctanthes arbor tristis (B-NAT) produced a yield of 2.49 % w/w of extract.

Preliminary phytochemical screening

Phytochemical investigation of B-NAT extract was tested for the presence of active principle compounds such as phenol, alkaloids, flavonoids, tannins and saponins.

Determination of in-vitro antioxidant activity & total phenols

DPPH (1, 1-diphenyl-2-picryl hydrazyl) method

Scavenging free radical potential was evaluated against ethanolic solution of DPPH a stable free radical. Antioxidants react with DPPH and convert it to 1,1-diphenyl-2-picryl hydrazine (non-radical). Degree of discoloration indicates the scavenging activity of drug. The change in absorbance produced at 517nm, has been used as a measure of antioxidant activity [19],[20]. DPPH (2.365 mg) was dissolved in 10ml of 95% ethanol and B-NAT was dissolved in 95% ethanol to make the stock solution, which was diluted to give concentrations ranging from 50-800 ppm. The extracts of varying concentrations (1.5 ml) was added only in test and finally 95% ethanol (1.5 ml) was added in both blank and test. The tubes were kept aside for 20 min. The absorbance of the solutions was recorded against the respective blanks at 517 nm.[21] Ascorbic acid was used as reference standard.

Scavenging capacity= (Absorbance of sample – Absorbance of blank ) χ100

Absorbance of blank

Estimation of total phenolic content

Total soluble phenolic compounds present in extract were determined with the Folin-Ciocalteu reagent.[22],[23] The calibration curve was prepared by preparing gallic acid solutions at concentrations 0, 50, 100, 150, 250, 500 ppm in ethanol. To 0.1 ml of different concentrations of gallic acid solution, one ml of Folin-ciocalteu reagent was added. After 3 min, 3 ml of 2% Na2CO3 was added. The same concentration of extracts was also prepared. Subsequently the mixture was shaken for 2 hr at room temperature and the absorbance was measured using Shimadzu UV-2450 spectrophotometer at 760 nm. Total phenol values were expressed in terms of gallic acid equivalent (mg/g of dry mass).

Treatment schedule

Animals were randomly divided into five groups with 5 animals in each group

Group 1:- Vehicle treated (10ml /kg distilled water, p.o) for 2 weeks

Group 2:- DOX (3mg /kg, i.p) every other day for 2 weeks24

Group 3:- N-butanol fraction of Nyctanthes arbor tristis (B-NAT) (100 mg/kg, p.o) daily + DOX (3mg/kg, i.p) every other day for 2 weeks,

Group 4:- N-butanol fraction of Nyctanthes arbor tristis (B-NAT) (300 mg /kg, p.o) daily + DOX (3mg/kg, i.p) every other day for 2 weeks,

Group 5:-Vit. E (25mg/kg, p.o) daily +DOX (3mg/kg, i.p) every other day for 2 weeks.

Electrocardiography (ECG)

ECG was recorded by inserting needle lead 2 electrodes under skin of anaesthetized animals using Chart 5.0, ADI instruments. QRS complex, QT intervals and ST intervals were measured.[25]

Estimation of cardiac marker enzymes

Blood samples were collected soon after ECG recordings by cardiac puncture from each animal and the serum obtained by centrifugation was used for determination of Lactate dehydrogenase -LDH [26] , Serum Glutamic Oxaloactetic transaminase-SGOT [27] , Creatine Phosphokinase-CPK.[28]

Relative organ weight

After blood withdrawal, the heart was dissected out, washed with ice cold 0.9% saline and weighed.

Estimation of in-vivo antioxidants

After weighing, the heart tissue was homogenized with ice cold 0.1M tris HCL buffer (7.5) using Remi homogenizer to give a 10% homogenate for the determination of In vivo antioxidant parameters.

Lipid peroxidation (LPO)

0.5 ml of 0.6 % Thiobarbituric acid (TBA) and 125 μL of 20% trichloroacetic acid (TCA) were mixed with 250 μL of cardiac tissue homogenate. The mixture was heated for 30 min in boiling water bath then cooled and centrifuged at 3000 rpm for 10 min. at 4°C. The absorbance was measured at 535 nm against reagent blank.[29]

Reduced glutathione (GSH)

1 ml of homogenate was added to 1ml of 10% TCA and centrifuged. 1 ml of supernatant was treated with 0.5 ml of Ellman’s reagent (19.8 mg of 5, 5’- dithiobisnitro benzoic acid (DTNB) in 100 ml of 1% sodium citrate) and 3ml of phosphate buffer (pH-8). The color developed was measured at 412 nm.[30]

Superoxide dismutase activity (SOD)

The assay of SOD was based on the ability of SOD to inhibit spontaneous oxidation of adrenaline to adrenochrome.0.05 ml, Supernatant was added to 2.0 ml of carbonate buffer and 0.5 ml of 0.01 Mm EDTA solution. The reaction was initiated by addition of 0.5 ml of epinephrine and auto oxidation of adrenaline to adrenochrome was measured at 480 nm. The change in absorbance for every minute was measured against blank. The results were expressed as unit of SOD activity (mg/wet tissue).[31]

Histopathological studies

The heart tissue from each group were excised and immediately fixed in 10% buffered formalin. The ventricular mass was sectioned from the apex to the base of the heart, which was embedded in paraffin after being dehydrated in alcohol and subsequently cleared with xylene. Five–micrometer thick serial histological sections were obtained from the paraffin blocks and stained with haematoxylin and eosin. The sections were examined under light microscope and photomicrographs were taken under 10X.[32]

Statistical analysis

The results were expressed as mean ± SEM. Statistical analysis was done using one-way analysis of variance, followed by Dunnett’s multiple comparison tests. P < 0.05 was considered significant.


  Results Top


Preliminary phytochemical screening

Phytochemical screening of B-NAT showed the presence of flavonoids, alkaloids and phenols.

DPPH Assay

The bleaching of DPPH absorption (517 nm) by test extract is representative of its capacity to scavenge free radicals. Hence DPPH-scavenging activity of B- NAT extract was taken as the parameter to check their antioxidant potential. The extract exhibited significant antioxidant activity in DPPH assay with IC50 value of 177μg/ml.

Total Phenolic contents

Phenols are highly effective free radical scavengers and antioxidants. Consequently the antioxidant activities of extract could be compared to the total phenolic contents, with good correlation. The total phenolic content was found to be 304.25 %w/w of gallic acid equivalent.

ECG

Animals treated with DOX showed a significant (p<0.05) elevation of QRS complex as compared to vehicle treated animals. Co-administration with B- NAT (300 mg/kg) & vitamin E (25mg/kg) significantly (p<0.05) decreased the elevation of QRS complex [Figure 1]A.
Figure 1: Effect of N-butanol fraction of Nyctanthes arbor-tristis-B-NAT (100 and 300 mg/kg p.o.) on (A) ECG (B) Lactate dehydrogenase -LDH level (C) Serum glutamate oxaloacetic transaminase-SGOT level (D) Creatnine Phosphokinase-CPK level in doxorubicin induced cardiotoxicity in Wistar rats. #P<0.05 as compared to vehicle treated group, *P<0.05 as compared to DOX treated control group.

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Cardiac marker enzymes

Doxorubicin treated animals showed significant (p<0.05) increase in the levels of Lactate dehydrogenase-LDH [Figure 1]B, Serum glutamate oxaloacetic transaminase-SGOT [Figure 1]C and Creatine Phosphokinase-CPK [Figure 1]D as compared to vehicle treated group. Animals treated with B-NAT (100 and 300 mg/kg) & vitamin E (25 mg/kg) in DOX treated animals showed significant (p<0.05) decrease in cardiac marker enzymes as compared to DOX treated group.

Heart weight

There was a significant decrease in heart weight in DOX treated animals. Co-administration with B- NAT (100 and 300) in DOX treated animals significantly (p<0.05) ameliorated the decrease in heart weight as compared to DOX treated control group [Figure 2]A.
Figure 2: Effect of N-butanol fraction of Nyctanthes arbor-tristis -B-NAT (100 and 300 mg/kg p.o.) on (A)relative organ weight LPO (B) levels (C) SOD levels (D) GSH levels in DOX induced cardiotoxicity in Wistar rats. All data are analyzed by one way ANOVA followed by Dunnet’s test. #P<0.05 as compared to vehicle treated group, *P<0.05 as compared to DOX treated control group.

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Antioxidant enzymes

In antioxidant enzymes study DOX treated animals significantly (P<0.05) increased the levels of lipid peroxidative indices (LPO) and significantly (P<0.05) decreased the levels of reduced glutathione (GSH), superoxide dismutase (SOD) as compared to vehicle treated group in heart tissue. Animals pre- treated with B-NAT(100 and 300 mg/kg) & vitamin E (25mg/kg) in DOX treated animals significantly reversed all the above changes as compared to DOX treated animals alone.[Figure 2] B,[Figure 2]C,[Figure 2]D

[Figure 3]A Vehicle shows normal architecture, [Figure 3]B group treated with DOX (3 mg/kg) showed vacuolated myocytes, partial loss of myofibrils, cell necrosis and edema, [Figure 3]C group treated with B-NAT (100 mg/kg) showed milder degree of vacuolated myocytes, partial loss of myofibrils and nuclear changes , [Figure 3]D group treated with B-NAT (300 mg/kg) showed mononuclear inflammation. [Figure 3]E group treated with Vitamin E (25 mg/kg) showed mononuclear inflammation and increased fibroblasts.
Figure 3: Histopathological examination (10X magnification) of heart tissue.

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  Discussion Top


Plants and plant products have been used as a source of medicine since time immemorial. Recently, plants have received much attention as sources of biological active substances, including antioxidants. Despite the wide use of DOX in the treatment of cancer patients, its mechanism of action is still not well known. Several mechanisms seem to account for the effects of this anthracycline, both in terms of anticancer action and of cardiac and other organ toxicity.[33] It is widely accepted that oxidative stress and the production of free radicals are involved in DOX action, in relation to both anticancer effects and toxicity. Thus, it has been reported that DOX leads to direct oxidative injury to DNA and generates lipid peroxidation.[34],[35] In relation to DOX cardiotoxicity, it is of valuable to note that heart tissue is very sensitive to free radical damage among other reasons, because of its high oxidative metabolism and because its antioxidant defenses are fewer than those of other organs, such as liver.[36] The present study evaluated the protective effect of n- butanol extract of Nyctanthes arbor-tristis on antioxidant tissue defence system against DOX induced cardiotoxicity in rats. DOX treated group of animals showed significant (P<0.05) elevation of QRS complex, LDH, SGOT, CPK and LPO; and significant (P<0.05) reduction in relative organ weight, GSH and SOD of heart tissue. Animals co- administered with B-NAT (100 and 300 mg/kg) and vitamin E (25 mg/kg) in DOX (3 mg/kg) treated animals showed significant (P<0.05) reversal of ECG, morphological and biochemical changes as compared to DOX treated animals alone. In histopathological examination, heart tissue showed nuclear changes, vacuolated myocytes, cell necrosis, dilated vessels and partial loss of myofibrils in the DOX treated group. B-NAT (100 and 300 mg/kg, p.o.) treated heart tissue partially protected the histological changes. Thus, phenolic rich B-NAT can ameliorate the effects of DOX induced cardiotoxicity as assessed by it morphological, biochemical, & histopathological features.


  Conclusion Top


The present investigation demonstrates the potent therapeutic value of B-NAT (100 and 300 mg/kg) in ameliorating the cardiotoxicity induced by DOX in rats. The beneficial effect of Nyctanthes arbor-tristis could be attributed to its antioxidant effect.



 
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