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Divya K, Tripathi J. S, Tiwari S. K, Pandey B. L. An Evaluation of Acute Toxicity and Subchronic Toxicity of Hydroethanolic Extract of “Shirishadi”- A Polyherbal Ayurvedic Compound in Rodents. Biosci Biotech Res Asia 2017;14(2).
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An Evaluation of Acute Toxicity and Subchronic Toxicity of Hydroethanolic Extract of “Shirishadi”- A Polyherbal Ayurvedic Compound in Rodents

Kajaria Divya1, Tripathi J. S1, Tiwari. S. K.1 and Pandey. B. L2

1Faculty of Ayurveda, Department of Kayachikitsa, IMS, BHU, Varanasi, India.

2Department of Pharmacology, IMS, BHU, Varanasi, India.

Corresponding Author E-mail: divyakajaria@gmail.com

DOI : http://dx.doi.org/10.13005/bbra/2480

ABSTRACT: This study was aimed at evaluating the safety profile, acute and subchronic toxicity of Shirishadi polyherbal ayurvedic compound in rodents, commonly used in the treatment of Bronchial Asthma. Acute toxicity was evaluated in wister albino rats by administering orally graded doses of extract of Albezzia lebbeck, Cyperus rotandus and Solanum xanthocarpum (Shirishadi Compound) in the dose of 2000, 5000 mg/kg, 10, and 20 g/kg body weight of animal and observed continuously for first 4 h and hourly for next 12 h, then 8 hourly for next 56 h (72 h, acute toxicity study). The study had been strictly followed the OECD guidance. Adult Wister albino rats were divided into 3 groups with 5 animals in each group and a control group, fed with doses of 5, 100 mg and 150 mg/ml once in a day for 30 days. Effect of drug was evaluated on hematological parameter after every 7 days and histological study was done after sacrificing the animals on 31st day. The median acute toxicity value (LD50) of the extract was found to be infinite and drug was found to be totally non toxic as after ingestion of 20 g no animal found to be dead. Hematological profile shows no abnormal elevation in level of Serum-Bilirubin, GOT, GPT, urea and creatinine. There was no any evidence of toxicity in histopathological studies.

KEYWORDS: Asthma; Histopathological study; LD 50 value;Polyherbal compound

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Divya K, Tripathi J. S, Tiwari S. K, Pandey B. L. An Evaluation of Acute Toxicity and Subchronic Toxicity of Hydroethanolic Extract of “Shirishadi”- A Polyherbal Ayurvedic Compound in Rodents. Biosci Biotech Res Asia 2017;14(2).

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Divya K, Tripathi J. S, Tiwari S. K, Pandey B. L. An Evaluation of Acute Toxicity and Subchronic Toxicity of Hydroethanolic Extract of “Shirishadi”- A Polyherbal Ayurvedic Compound in Rodents. Biosci Biotech Res Asia 2017;14(2). Available from: https://www.biotech-asia.org/?p=24498

Introduction

Bronchial asthma is a chronic inflammatory disease of airways. It may be allergic or nonallergic in origin. Sustain inflammation leads to remodeling of airways and cause diminution of their proper functioning.

Asthma is said to be only prevented and not cure and this fact underlies its pathophysiological distinctiveness. Asthma proves to be lethal in its acute manifestation as acute onset of severe bronchoconstriction may cause grave respiratory distress due to lack of proper oxygen saturation. Asthma whether acute or chronic always associated with permanent damage of normal architecture of airways and thus need a careful and proper management. Although contemporary medicines proves beneficial and life saving in bronchial asthma but ultimate dependence on corticosteroids and wide range of toxic side effects forces researcher to think in different dimension and search for some alternative remedy that prove beneficial in preventing as well as cure asthma.

In an attempt to search some cheap, effective, and fast acting remedy of bronchial asthma, some ayurvedic compound are prepare and planned to be given in aerosol form through nebulization. Preclinical study of aforementioned said research trial consists of toxicity study of ayurvedic compound in animal model. The route of administration of drug selected consists of both oral and Inhalation route. Whenever an investigator administers a chemical substance to a biological system, different types of interactions can occur and a series of dose-related responses result. In most cases these responses are desired and useful, but there are a number of other effects which are not advantageous. These may or may not be harmful to the patients. The types of toxicity tests which are routinely performed bypharmaceutical manufacturers in the investigation of a new drug involve acute, sub-acute and chronic toxicity. Acute toxicity is involved in estimation of LD50 (the dose which has proved to be lethal (causing death) to 50% of the tested group of animals). Determination of acute oral toxicity is usually an initial screening step in the assessment and evaluation of the toxic characteristics of all compounds. This article reviews the methods so far utilized for the determination of median lethal dose (LD50) and the new changes which could be made. This has to go through the entire process of validation with different categories of substances before its final acceptance by regulatory bodies. The following toxicity studies were performed in the present research trial:

Acute toxicity study

Sub chronic toxicity study

Acute Lethality (Barlow et al., 2002; Bürger et al., 2005; Bruce, 1985)

Objectives

To determine Maximum Tolerated Dose (MTD) and No Observable Effect Level (NOEL).

To determine the Median Lethal Dose (LD50) after a single dose administered through oral route which is the intended route of administration in humans.

To identify potential target organs for toxicity, determine reversibility of toxicity, and identify parameters for clinical monitoring.

To help select doses for repeated-dose toxicity tests.

Duration

Mortality within 72 hours was cut-off time for acute toxicity however survival of animals were observed up to 2 weeks following single dose administration.

Parameters

(Combes, 2004)

Mortality.

General clinical observation of behavior.

Weight change.

Gross autopsic examination of dying animals.

Experimental Animal

(Crook, 2006 Daswani et al., 2006)

Adult Charles Foster Albino rats (70 +  30g) of either sex  were obtained from the Animal Research Branch of the Institute of Medical Sciences, Banaras Hindu University, Varanasi . The animals were housed in polyvinyl cages and were fed on commercial pellet diet (Amrut, Pranav Agro Industries Ltd, India). They were grouped & housed under standard conditions of temperature (22 ± 2C0), relative humidity (60 ± 5%) and 12:12 light/dark cycle, where lights on at 0700 and off at 1900 h). The saline fed group served as control and one group was treated with a standard drug in each protocol. Before experimentation, the animals were kept on fast for 24 h but water was given ad libitum except during experimental test period. During experiments, animals were also observed for any alteration in their general behavior.

All the experiments and the care of the laboratory animals were according to current ethical guidelines by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India, New Delhi.

The research protocol was approved by Institutional Research committee of Institute of Medical Sciences, BHU- India.

Plant Material and Extraction

TheplantsAlbezzia lebbeck,Cyprus rotandus & Solanum xanthocarpum,  were collected from local market of Varanasi. The identification of the drugs was done by  Prof.A.K. Singh, Department of Dravyaguna, S.S.U., Varanasi ( Identification number DG/ AKS / 604). Hydroalcoholic Extraction ( Distilled water: Ethanol =  2:1) of  drugs  was carried out by hot percolation method through soxhlet appratus.  Thereafter  extracts  were dried  using  rotatory  evaporator  and  dried  extracts  were  put  to the process of standradization.

Drug  Treatment

Standradized  extract  of   Ayurvedic  compounds  dissolved in distilled water  was orally  administered  as graded doses of 50mg, 100mg, & 150mg/ 100g of bwt of animal once daily  for 30 days for  Subchronic toxicity study. Control rats were treated with equal volume of  normal saline. The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation canula.  For Acute toxicity study, standradization extract ofShirishadi Polyherbal Ayurvedic compounds was administer as escalated doses of  200mg, 500mg, 1g & 2g/ 100g bwt to know  the LD 50 value. At each step (i.e. for each dose, starting from lowerone) three rodents were used as per OECD guideline (Guideline No.425).

Method Employed

(W.J. Dixon,1965 , R.D. Bruce,1985 & R.D. Combes, 2004 )

OECD guidelines number 425was followed for Acute Oral Toxicity study with  Up-and –Down- Procedure (UDP) .The concept of the up-and-down testing approach was first described by Dixon and Mood

The method is easiest to apply to materials that produce death within one or two days.  According to guideline minimum number of animal should be used for the experiment at each step. The test is divided into two parts: Limit test & Main test.

Limit Dose at 2000mg /Kg

Step Ist

One Albino rat of group A weighting was administered 200mg ofShirishadi Hydroalcoholic extract dissolved in 1ml of distilled water orally through intubation cannula.

Step IInd

The  rats were observed for 24 hour and when no toxic side effects appears, four more albino rats  were taken  in group A and administered  2000mg / Kg body weight of Shirishadi extract. The animals were watch for 48 hours. After 48 hours all the rats were survived with no toxic side effect.

Table 1: Body weight variation of treated rats with various doses of the Hydroethanolic extract of Shirishadi during Acute Toxicity Study.

Groups

Body weight  of  rats (Before Treatment) Dose  of  Drug  administered

 

Dose of Drug/ 100g  body weight Body weight  of  Rats (After treatment)
After 24hrs After 48hrs After 72 hrs
 

A

 

Ist –     90g 0.9ml  

200mg/ ml

110g 110g 110g
Iind –  70g 0.7ml 95g 100g 105g
IIIrd –  65g 0.65ml 85g 87g 100g
 

B

 

Ist  –     70g 0.7ml  

500mg/ml

90g 90g 95g
IInd –  70g 0.7ml 75g 90g 95g
IIIrd –  50 g 0.5ml 60g 62g 65g
 

C

 

Ist  –     95g 0.95ml  

1g/ml

115g 115g 120g
Iind –   75g 0.75ml 95g 97g 100g
IIIrd –  75g 0.75ml 100g 100g 100g
 

D

Ist  –     70g 0.75ml+  0.75ml 1g/ml twice within  1/2hrs interval to make total of 2g. 85g 90g 90g
Iind –   75g 0.75ml + 0.75ml 90g 90g 90g
IIIrd –  65g 0.65ml + 0.65ml 65g 65g

65g

 

Limit Test at 5000 mg/kg

Step Ist

One albino rat of Group B 100gm waas treated with 5000mg/ Kg of Shirishadi extract dissolved in distilled water and observed for 48 hrs.The ratsurvived and found healthy after 48 hrs of treatment.

Step IInd

When no observable toxic sign and symptoms appears in the albino rats treated with 5000mg/Kg of polyherbal extracts, two more albino rats in group B were added and administered  polyherbal drugs in the dose of 5000mg/Kg. The rats were observed for 48 hours.

As no sign and symptom of toxicity appeared in limit test, 12 more albino rats were taken, divided into two groups and selected for main test.

Next escalated dose of extract (Shirishadi) was decided to be 10gm/Kg body weight administered in albino rats of Group C. The rats were observed for 48 hrs and when no sing and symptom of toxicity appears six more albino rats were taken and divided into two groups namely D and were given   next escalated dose of 20gm/ Kg body weight in two divided dose at the interval of 30 minutes.

As even after administration of trial drug in the dose of 20gm/ Kg body weight, no rat die, LD50 of present trial drug is found to be infinite.

To confirm that the present trial drug is non toxic OECD guidline, Page No. 40, was followed. According to the guideline if 2gms oral administration  of  drug dose not kill the rodent  it should be considered as non –toxic.

Table 2: Effect of Hydroethanolic extract of Shirishadi Extract on various organ weights after 3days of acute toxicity study.

Dose/  100gm of animals

Lung/ 100g bwt

N= 3

Liver/ 100g bwt

N= 3

Stomach/ 100g bwt

N=3

Kidney /100 g bwt.

N= 3

Heart/ 100g bwt

N=3

Adrenal gland/ 100g bwt

N= 3

Testis/ 100g bwt.

N=3

Spleen/ 100g bwt

N=3

Control 948+ 1.56 3200+ 0.08 1078+ 1.78 350+ 2.05 330+ 1.98 7.1+ 3.67 884+ 0.56 281+ 1.05
 200mg 875+ 2.34 3600+ 1.23 1136+2.3 340+1.56 320+ 2.08 7.8+ 0.68 865+ 1.78 286+ 0.56
 500mg 890+ 0.54 3450+ 1.54 1010+ 0.01 300+ 0.04 305+ 3.56 6.9+ 2.36 840+ 0.47 261+ 1.23
  1gm 930+ 0.90 3545+ 3.21 1080+ 2.78 365+ 0.64 315+ 0.01 7.5+ 1.67 884+ 0.02 290+ 0.56
 2gm 950+ 1.04 3711+ 0.21 1178+ 0.08 370+ 1.02 327+ 1.36 8.0+ 2.98 870+ 0.05 280+ 0.01

All the values are Mean +SDE , where n=3.

Table 3: Acute Toxicity study of Hydroethanolic Etract of Shirishadi in rodents;Death after 7days of treatment.

Group No. of Albino Rats Dose of  Extract No. of Dead rats % Cumulative

dead of  rats

A 3 200mg/ 100g bwt 0.00 0%
B 3 500mg/100g bwt 0.00 0%
C 3 1g/100g bwt 0.00 0%
D 3 2g/ 100g bwt 0.00 0%

Sub- Chronic Toxicity Study

(R.D.Combes,2004 & S.M.Barlow,2002)

Objectives

To establish a “no observable effect level” (NOEL).

To characterize dose-response relationships following repeated doses.

To identify and characterize specific organs affected after repeated administration.

To predict a reasonable and appropriate dose for chronic exposure studies (maximum tolerated dose or MTD).

Duration

30 days.

Test System/Animal System

Rodent

Dose Administration

Male and female Wistar albino  rats weighing 100 ± 20 g  were maintained on standard animal feeds and provided with water ad libitum. The animals were weighed and divided into seven groups of five animals each. After overnight fasting of the rats, the control group received a dose of 0.5 ml of normal saline solution orally once a day for 30 days. The treated groups respectively received the following doses: 50, 100, 150 mg/kg body weight of the hydroalcoholic extracts (Shirishadi &Bharangyadi) orally once daily for 30 days (Pieme et al., 2006; Joshi et al., 2007; Mythilypriya el al., 2007).The animals were then weighed every five days, from the start of the treatment, to note any weight variation.

Parameters

Mortality

Weight change

Signs of toxicity

Clinical pathology

Pathology and histopathology

Table 4: Variation in body weight of normal and treated rats with various doses of the Shirishadi hydroethanolic extract during 30 days of subchronic toxicity study.

Dose/Kg bwt Day  1

Bwt(gms)

Day 5

Bwt(gms)

Day 10

Bwt(mgs)

Day 15

Bwt(mgs)

Day 20

Bwt(mgs)

Day 25

Bwt(mgs)

Day 30

Bwt(mgs)

 

Control

100 101 105 110 112 113 115
105 106 108 110 113 115 118
110 112 114 116 118 120 122
102 103 105 108 110 111 112
100 100 105 106 110 110 112
 

50mg

 

115 120 122 125 128 130 134
100 105 108 115 120 122 126
110 115 120 125 130 132 135
105 108 112 115 118 122 125
110 115 118 120 125 128 132
 

100mg

110 112 115 120 125 130 132
105 110 118 120 122 125 128
100 105 108 110 112 115 118
115 120 125 128 130 132 135
120 124 130 132 135 140 142
 

150mg

100 102 105 108 110 112 115
115 115 117 119 122 125 128
120 122 124 128 130 133 135
122 125 126 130 132 135 138
108 110 112 115 118 120 122

Table 5: Body weight variation of normal and treated rats with various doses of the extract during 30 days of subchronic toxicity study.

Dose/ Kg bwt Day 1 Day 5 Day 10 Day 15 Day 20 Day 25 Day 30
Control

50mg

100mg

150mg

103+ 1.88

108+ 2.54

110+ 3.53

113+4.04

104+2.16

112+ 2.69

114+3.44

114+4.14

107+1.74

116 +2.607

119+ 3.83

116+ 3.86

110+1.67

1202.36

122+3.79

120+4.08

112+ 1.46

124+ 2.28

126+3.89

122+4.02

113+1.77

126+2.05

128+ 3.86

125+4.23

115+ 1.98

130+2.06

132+ 3.70

127+4.20

All the values are Mean +SDE , where n=5.

Figure 1: Percentage increase in weight of the control and animals treated withdifferent doses of Shirishadi hydroethanolic extract Figure 1: Percentage increase in weight of the control and animals treated withdifferent doses of Shirishadi hydroethanolic extract

 

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Table 6 : Effect of Shirishadi Compound on Biochemical profile

Parameter

After 15 days After 30 days
Control 50mg/

Kgbwt

100mg/

Kgbwt

150mg/

Kbwt

Control 50mg/

Kgbwt

100mg/

Kgbwt

150mg/

Kbwt

B.Urea 30.2 ± 2.78 36 ± 5.86 33.6± 2.73 44.6± 4.82 46.7± 4.87 39.2 ± 4.89 30.4± 1.43 50.6± 6.54
S.Creatinine 0.5± 0.05 0.5 ± 0.45 0.6± 0.09 0.5± 0.02 0.68± 0.09 0.52 ± 0.02 0.5 ± 0.05 0.68± 0.08
S.Bilirubin (Total) 0.6± 0.78 0.5±  0.45 0.74± 0.08 0.78± 0.12 0.5± 0.02 0.68± 0.04 0.5± 0.02 0.70± 0.12
S.Bilirubin(Direct) 0.5 ±0.54 0.5 ± 0.05 0.42 ± 0.06 0.56 ± 0.20 0.52± 0.03 0.48 ± 0.09 0.5± 0.08 0.5± 0.05
SGOT (AST) 150± 4.57 100 ± 1.25 143± 7.40 163.7 ± 14.12 150.8± 3.35 123.3± 9.40 107.1± 8.67 168.8± 5.89
SGPT (ALT) 60± 4.38 73.6 ± 2.56 43.4± 5.41 68± 1.90 40.3± 5.36 65.48± 5.29 69.4± 10.63 70± 4.35

All the values are Mean +SDE , where n=5.

Table 7: Sub-Chronic Toxicity study in albino rats with Shirishadi extract

Groups No. of Rats Dose of extract No.of  Dead Rats % Cumulative dead of Rats
A 5 Control 0 0%
B 5 50mg/ Kgbwt 0 0%
C 5 100mg/Kgbwt 1 20%
D 5 150mg/ Kgbwt 2 40%

Results and Discussion

The result shows that Shirishadi polyherbal compound is innocuous and very safe for therapeutic use (Yashada, 2006). There were no abnormalities detected in histopathological study nor any

biochemical abnormalities were identified in organ sample of sacrificed rodents after acute toxicity study. In chronic treatment with higher doses, death rate showed 20 and 40% cumulative increase. However there is no evidence of organic toxicity in the animals. Studies may be performed in very exclusive conditions to evaluate the issue of long term safety. The acute toxicity study of the extract (Shirishadi compound) indicated no changes in the behaviour and in the sensory nervous system responses in the animals. Also no adverse gastrointestinal effects were observed in the albino rats. The LD50 calculated for the drug is found to be infinite (according to OECD guidelines) as no rat died even after oral administration of dose 20 g/kg bwt (Table 1). Histopathalogical study of viscera’s showed no microscopic or macroscopic abnormality without any change in colour, no congestion, no necrosis or any other sign of toxicity (Plates 6, 7, 8, 9 and 10).

Figure 1a: Showing different groups of animal

Figure 1a: Showing different groups of animal

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Figure 2. Measuring diet for animals during for toxicity studies sub- chronic toxicity study

Figure 2. Measuring diet for animals during for toxicity studies sub- chronic toxicity study

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Figure 3: Showing animals taking diet during toxicity studies

Figure 3: Showing animals taking diet during toxicity studies

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Figure 4: Different concentration of drugs

Figure 4: Different concentration of drugs

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Figure 5: Samples of organ specimens collected after dissection for histopathological studies

Figure 5: Samples of organ specimens collected after dissection for histopathological studies

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Figure 6: Different organ after dissection of albino rats in Acute toxicity study.

Figure 6: Different organ after dissection of albino rats in Acute toxicity study.

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Figure 7: Sample of lungs showing no macroscopic changes ( colour, texture etc.) in albino rat treated with 2gm of Shirishadi Compound. Figure 7: Sample of lungs showing no macroscopic changes ( colour, texture etc.) in albino rat treated with 2gm of Shirishadi Compound.

 

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Biochemical profile including –Liver function test (LFT), Renal function test (RFT) show no abnormal change substantiated that drug has no renal or hepatotoxicity (Table 2). Percentage cumulative dead after acute toxicity study was found to be 0% (Table 3).

Figure 8: Microphotograph of rat’s lung (Shirishadi compound treated group) Showing normal alveoli & lung parenchyma (40x) in Acute toxicity study. Figure 8: Microphotograph  of rat’s lung  (Shirishadi compound treated group) Showing  normal alveoli & lung parenchyma (40x) in Acute toxicity study.

 

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Figure 9: Showing normal Histology of testis tissues treated with Shirishadi compound in Acute toxicity study. Figure 9: Showing normal Histology of testis tissues treated with Shirishadi compound in Acute toxicity study.

 

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Figure 10: Showing normal histology of rat’s Stomach tissuse treated with Shirisahdi compound in acute toxicity study(40x). Figure 10: Showing normal histology of rat’s Stomach tissuse treated with  Shirisahdi compound in acute toxicity study(40x).

 

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Figure 11. Showing histology of liver tissuses of albino rat treatedwith Shirishadi compound in Acute toxicityStudy.Microphotograph reveals normal hepatocytes, central vein & portal tract (40x). Figure 11. Showing histology of liver tissuses of albino rat treatedwith  Shirishadi compound in Acute toxicityStudy.Microphotograph reveals normal hepatocytes, central vein & portal tract (40x).

 

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Figure 12: Showing normal histology of lung tissuse of albino rat treated with Shirishadi Compound in Sub-chronic toxicity study (40x). Figure 12: Showing normal histology of lung tissuse of albino rat treated with Shirishadi Compound in Sub-chronic toxicity study (40x).

 

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 Figure 13: Showing histology of Kidney tissues of albino rat treated withShirishadi compound in Sub- chronic toxicity study (40x). Figure 13: Showing histology of Kidney tissues of albino rat treated withShirishadi compound in Sub- chronic toxicity study (40x).

 

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Figure 14: Showing histology of lung tissue after acute toxicity study.Many small, bubble-like alveoli can be seen in the thistissue, as well as a small bronchiole running obliquely Figure 14: Showing histology of lung tissue after acute toxicity study.Many small, bubble-like alveoli can be seen in the thistissue, as well as a small bronchiole running obliquely

 

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Body weight of albino rat’s increases propionate with dose of drug but become stabilized at higher doses (Figure 1). As there was increase intake of feed with increasing dose of drug, the increase in body weight was indicative of anabolic effect (Tables 4 and 5). Biochemical analysis showed no toxic or abnormal changes suggesting that drug is safe for long duration internal use (Table 6). There was 40% cumulative death in group treated with 150 mg of Shirishadi extract during subchornic study whereas 20% (Table 7) in group treated with 100 mg of extract.

As there were no morbid signs found prior to death and in addition to this there was no any evidence of toxicity in histopathological studies (Plates 11, 12, 13 and 14) excludes the possibility of death due to toxic effect of drug.

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