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 I^st

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

Step II^nd

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.

Limit Test at 5000 mg/kg

Step I^st

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 II^nd

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.

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.

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.

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

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   Click here to View figure

Table 6 : Effect of Shirishadi Compound on Biochemical profile

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

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

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 Click here to View figure

Figure 2. Measuring diet for animals during for toxicity studies sub- chronic toxicity study Click here to View figure

Figure 3: Showing animals taking diet during toxicity studies Click here to View figure

Figure 4: Different concentration of drugs Click here to View figure

Figure 5: Samples of organ specimens collected after dissection for histopathological studies Click here to View figure

Figure 6: Different organ after dissection of albino rats in Acute toxicity study. Click here to View figure

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

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.   Click here to View figure

Figure 9: Showing normal Histology of testis tissues treated with Shirishadi compound in Acute toxicity study.   Click here to View figure

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

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).   Click here to View figure

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

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

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   Click here to View figure

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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