Satish yadav project report

8/7/2019 Satish yadav project report

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IN-VITRO ANTI-CATARACT ACTIVITY OFAZADIRACHITA INDICA LEAVES EXTRACT IN

GLUCOSE-INDUCED CATARACTA

PROJECT REPORTSUBMITTED

BY

SATISH KUMAR

B.PHARMA. IV TH YEAR

UNDER THE SUPERVISION OF

Mr. ZAFAR AKBAR

M.PHARMA

UNDER CO-SUPERVISION OF

Mr. GAURAV GARG


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M.PHARMA

INSTITUTE OF BIOMEDICAL EDUCATION ANDRESEARCH

MANGALAYATAN UNIVERSITY

BESWAN, ALIGARH

2009-10

INDEX

1. Acknowledgment

2. Introduction

3. Content

4. Review of literature

5. Material & Method

6. Results

7. Dissection

8. Conduction

9. Reference


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INTRODUCTION

DefinitionA cataract is a painless clouding of the lens of the eye. The cataract develops in

along period of time leading to a gradual worsening of eyesight. Cataract can lead to blindness if it is leave untreated.

Cataracts are common in older peoples, about one third of peoples aged over 65 have cataract in one or both eyes. A cataract is a cloudy or opaque area in thenormally clear lens. Depending upon its size and location it can interfere with normalvision. (1)

Most cataracts are highly treatable. Cataract surgery is one of the most commonsurgeries performed in the United States with 95% of patients experiencing improvedvision if there are no other eye conditions present. During surgery, the doctor removesthe clouded lens and in most cases, replaces it with an artificial lens, called anintraocular lens (IOL). An IOL is a clear, plastic lens that requires no care and

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becomes a permanent part of your eye. Thick eyeglass lenses after surgery might not be needed because of the implanted lens.

Cataract is a multifactorial disease associated with several risk factors such as

aging, diabetes, malnutrition, diarrhoea, sunlight, smoking, hypertension and renalfailure1. Free radical-induced oxidative stress is postulated to be perhaps the major factor leading to senile cataract formation2. This hypothesis is supported by theanticataractogenic effect of various nutritional and physiological3,4 antioxidant inexperimental animals. Selenite cataract is a rapidly-induced, convenient model for thestudy of senile nuclear cataractogenesis. The morphological and biochemicalcharacteristics of this model have been extensively investigated; moreover, this modelshows a number of general similarities to human cataract. Thereliability and extensivecharacterization of selenite cataract makes it a useful rodent model for rapid screeningof potential anticataract agents5. Physiologic antioxidant such as pyruvate andnutritional antioxidant vitamin E, ascorbic acid and carotenoids were found to delay

the experimental cataract. ACE inhibitors have been found to afford protection fromfree radical damage in many experimental conditions6,7,8,9. Selenite cataract, firstdescribed by Ostadalova et al in 1978, is an excellent model of oxidative stress-induced cataractogenesis in vitro and in vivo 10, hence it was used in the present studyto evaluate the efficacy of lisinopril as a anticataractogenic agent.

Review of literatureHistory

The earliest record is from Bible as well as early Hindu records. (2) Earlycataract surgery was developed by Indian surgeon Sushruta(6 th century BCE). (3) TheIndian tradition of cataract surgery was perform with a special tool called the

jabomukhi salaka ,a curved needle used to loosen the lens and push the cataract out of the field of vision. (4) The eye would later be soaked with warm butter and then

bandaged. (5) Through this method was successful shushruta said that should only beused when necessary. (6)Greek physicians and philosophers traveled to India wherethese surgeries were perform by physician. (7) The removal of cataract by surgery wasalso introduced into china from India. (8) The first reference to cataract and its treatment in ancient Rome was found in 29 ADin De madicinae , The work of Latin encyclopedist Aulus cornelius celsus. (9) TheRomans pioneers in health arena particularly in eye care. (10) The Iraqi


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ophthalmologist Am mar ibn Ali of Mosul perform the first extraction of cataractusing suction. He invented a hollow metallic syringe hypodermic needle, which heapplied through the sclerotic and extracted the cataracts using suction. (11) In this choice

of eye disease written in circa 1000, he wrote of his invention of hypodermic needlehow he discovered the technique of cataract extraction while experimenting on a

patient.

Types of Cataracts

Age-related cataract: Most cataracts are related to aging.

Congenital cataract: Some babies are born withcataracts or develop them in childhood, often inboth eyes. These cataracts may not affect vision.If they do, they may need to be removed.

Secondary cataract: Cataracts are more likelyto develop in people who have certain medicalproblems, such as diabetes. They can also belinked to use of medications; such as steroids. Long-term unprotectedexposure to sunlight is also believed to contribute to the development of

cataracts.Traumatic cataract: Cataracts can develop soon after an eye injury, or years later.

Cataract Types: An Overview

Although most cataracts are related to aging (age-related cataract), there are other cataract types. Some of these include:

Secondary cataract

Traumatic cataract

Congenital cataract

Radiation cataract.


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Secondary Cataracts

Secondary cataracts can form after surgery for other eye problems, such as glaucoma .Cataracts can also develop in people who have other health problems, such asdiabetes . Cataracts have even been linked to steroid use.

Traumatic Cataracts

A traumatic cataract can develop after an eye injury, sometimes years later.

Congenital Cataracts

Some babies are born with cataracts or develop them in childhood, often in both eyes.These congenital cataracts may be so small that they do not affect vision. If they do,

the lenses may need to be removed.

Radiation Cataracts

Radiation cataracts -- as you might expect -- can develop after exposure to certaintypes of radiation.

Other Types of Cataracts

Learn From the Specialists

Cataracts Surgery

Cataract Surgeons

Cataracts Clinics

Cataract Lenses

Cataract Intraocular Lens

Cataract Symptoms

Cataracts Treatment

Depending on the cause of the cataracts development there are several types, other than age-related cataracts, which have been defined. They are secondary cataracts,traumatic cataracts, congenital cataracts and radiation cataracts.
http://glaucoma.emedtv.com/glaucoma/glaucoma.htmlhttp://diabetes.emedtv.com/diabetes/diabetes.htmlhttp://www.cataract.com/links/?term=cataracts+surgeryhttp://www.cataract.com/links/?term=cataract+surgeonshttp://www.cataract.com/links/?term=cataracts+clinicshttp://www.cataract.com/links/?term=cataract+lenseshttp://www.cataract.com/links/?term=cataract+intraocular+lenshttp://www.cataract.com/links/?term=cataract+symptomshttp://www.cataract.com/links/?term=cataracts+treatmenthttp://www.cataract.com/links/?term=cataracts+treatmenthttp://glaucoma.emedtv.com/glaucoma/glaucoma.htmlhttp://diabetes.emedtv.com/diabetes/diabetes.htmlhttp://www.cataract.com/links/?term=cataracts+surgeryhttp://www.cataract.com/links/?term=cataract+surgeonshttp://www.cataract.com/links/?term=cataracts+clinicshttp://www.cataract.com/links/?term=cataract+lenseshttp://www.cataract.com/links/?term=cataract+intraocular+lenshttp://www.cataract.com/links/?term=cataract+symptomshttp://www.cataract.com/links/?term=cataracts+treatment


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cataract is small, you may not notice any changes in your vision. Cataracts tend togrow slowly, so vision gets worse gradually.

Cataract is an eye disorder which is caused due to myriad of factors. Thusseveral factors lead to the development of several types. There aredifferent types of cataract, which are caused due to rare diseases or as aresult of local eye injuries or inflammation. However, the cataracts can beclassified into two major types that include age-related cataracts andcataracts present at birth. Cataracts during birth are a relatively raredisease, yet it can affect an infant.

The various types of Cataract include primary cataracts such as age-related cataracts as well as secondary cataracts, traumatic cataracts,congenital cataracts and radiation cataracts. Although most cataracts arerelated to aging, the other types of cataracts develop due to variousfactors. The cataracts are classified by etiology, such as age-relatedcataract can be categorized as Immature Senile Cataract (IMSC) where thelens gets partially opaque and the disc view is hazy. Mature SenileCataract (MSC) where the lens get completely and there is no disc view.Hypermature Senile Cataract (HMSC) is also Liquefied cortical matterwhich is known as Morgagnian Cataract.

Secondary cataracts are those which develop in an individual after certainsurgery. This type can also affect a person for various eye problems too.An individual who was treated for glaucoma or a different eye problem ordisease can be affected by cataract. Cataracts may develop in person whohas health problems, such as diabetes. In addition to that, Cataracts areformed due to excessive or prolonged use of steroids.

Traumatic cataract is among the types of Cataract that develop after asevere eye injury. An individual may experience this eye disorderimmediately or just after damage caused to the eye. Moreover, traumaticcataracts and can occur due to blunt trauma to the eye or from exposureof the eye to alkaline chemicals. Traumatic cataracts are also associatedwith a penetrating eye injury. Congenital cataract is one of the types thataffect babies during birth. This type of eye disorder can develop inchildhood too, often in both eyes. However, these cataracts are small anddo not affect vision. If the cataracts do cause vision disturbances in thechild, this type of Cataract can be rectified by removing the lenses of theaffected person and replaced with a synthetic lens.

The most common symptoms of a cataract are:

Fading or yellowing of colours.


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Needing brighter light to read.

Double or multiple vision in one eye(this symptom often goes away as thecataract grows).

Cloudy or blurry vision.

Poor night vision.

Problems with light.

These can include headlights that seem too bright at night; glare fromlamps or very bright sunlight; or a halo around lights.

Frequent prescription changes in your eyeglasses or contact lenses.

These symptoms can also be a sign of other eye problems. If you have any of thesesymptoms check with your eye doctor.

Causes of cataract

Cataracts are caused by changes in the lens protein of the eye which them cloudy.There are various factors that may cause the risk of cataract - Diabetes mellitus - persons with DM are at higher risk for cataract.Drugs - certain drugs are associates in development of cataract. These include -

(1) Corticosteroids. (2)

Chlorpromazine and other phenothiazine related medication.Ultra violet radiation - studies have shown that there is an increased chance of cataract

formation with unprotected exposure to U.V. radiation.Smoking - An association between smoking and increased number opacities has


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reported . Alcohol - Several studies haveshown increased cataract formation in patients with higher

alcohol consumption compared with people who have lower or no alcohol

consumption.Nutritional deficiency - there is direct association between cataract formation andlow level of antioxidant. Ex. vita- A, vita-C, vita-E and carotenoids. Antioxidantshave a significant effect on decreasing cataract development. (12)

Diagnosis of cataractCataract can be diagnosed through a comprehensive eye examination .this examinationmay include -(1) Visual acuity test - this eye chart test measures how well you seen at various

distances i.e. to determine to what extent a cataract may be limiting clear vision atdistance and near.

(2) Dilated eye exam - drops are placed in eye to widen or dilate the pupil. The eyecare professional uses a special magnifying optic nerve for signs of change andother eye problem.

(3) Tonometry - an instrument measures the pressure inside the eye. Numbering thedrops may be applied to the eye for this test.

(4) Refraction - to determine the need for changes in an eye glass or contact lens prescription.(5) Supplemental testing for color vision and glare sensitivity. (13)

Cataract Treatments

A cataract is an eye condition that produces cloudy vision in patients in their 50s, 60s,and older; unfortunately, all men and women will develop cataracts if they live longenough. Cataracts form on the lens of the eye as we age because the protein in the lens

begins to clump together, resulting in cloudy patches of vision. As the cataract grows,vision worsens. Fortunately, cataract treatment options can improve patients' quality of life.


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Glasses

Cataracts can make it difficult for patients to read and see well while driving at night.When the symptoms of cataracts first present themselves, visual aids such as glasses,

bifocals, and magnification devices can assist patients in seeing clearly.

DocShop can help you find an eye care specialist in your area today.

DocShop can help you find an eye care specialist in your area today.

Lens Removal Surgery

When the symptoms of cataracts become so great the patients have difficulty doingroutine tasks, it is time to consider undergoing cataract surgery . During the first step of cataract surgery, the clouded lens of the eye is removed.

Lens Replacement Surgery

After the lens is removed, your cataract surgeon will replace it with a plastic-basedlens. The synthetic lens will work with your eyes to produce improved vision.Depending upon the type of lens that you have implanted, you may or may not need touse glasses to see near objects after cataract surgery.

Premium IOLsPremium intraocular lenses such as ReSTOR, ReZoom, and crystalens areadvanced lenses that provide patients with vision that is clear when viewing near anddistant objects. Although premium IOLs are more expensive than other lensreplacement options, they can completely eliminate patients' need for glasses.

Contact a Cataract SurgeonIf your cataracts are preventing you from driving, reading, and other activities, contact a cataract surgeon in your area to learn about your available treatment options. It may

be time to undergo lens removal and replacement cataract surgery.

A person affected with cataract should cover his eye with sunglasses to escapethe UV rays refraining from smoking is another good idea. It would be advised to gofor regular eye check up. The best treatment is removing the affected lens by surgeryand putting an artificial one lens in its place. (14)


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Cataract surgeries are of two types(a) Intra capsular surgery - In this; the surgery removes the lens capsule as well as

the lens. In recent surgeries it is not much performed.(b) Extra capsular surgery - In this surgery; the cloudy lens is removed but the most

part of The lens is as it is.Unfortunately there is no medication has been found to slow down the growth of

cataract so far. There is not any medication that can clear a clouded lens .(15)

Plant profile Azadirachta Indica -

Botanical : Azadiracta IndicaFamily: MeliaceaeSynonyms : Neem, Margosa

Azadirachta

Taxonomical Classification :-Kingdom - PlantaeDivision - MagnoliophytaClass - SapinadalesFamily - MeliaceaeGenus - AzadirahatraSpecs - Indica

Part Used Dried fruits & Leaves.


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Habitat India, East Africa, Shri Lanka, Thyland, Bangladesh, Pakistan, SouthAfrica, Malacia.

Constituents - Scientist Siddique Sali muzza man siddique . Extracted three bitter compounds from neem oil which he named as nimbin, nimbinin, nimidinandthe seeds contains secondary metabolite azadirachtin. Leaves contain beta-sitosterol,stigmasterol, salanin (antifeedant) & meliantriol (antifeedant). (16)

Medicinal uses-use as antimicrobial

use as insecticideuse as spermicidaluse as nematicideuse as antifeedant

Biological activity reported-Use as anthelmenticUse as antifungal & antimalerialUse as antidiabetUse as antibacterial and anti viralUse as sedativeUse as antifertility agent

Neem oil is used as preparation of cosmetics shop, shampoo, balm and cream.Use as mosquito repellant.

Material and Methods

E: Tissue Parameters


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The animals were sacrificed and the heart was dissected out andweighed. It was then processed for:

1) Lipid peroxidation or malondialdehyde (MDA) formation

2) Endogenous antioxidant

a) Reduced glutathione (GSH)b) Superoxide dismutase(SOD)

3) Total proteins content.

Removal and processing of tissues for variousestimations

ReagentsPhosphate Buffered Saline Ph (7.4)

1.38 gm of disodium ethylenediaminetetraacetic acid, 0.19 gm of potassium dihydrogen phosphate and 8 gm of sodium chloridewas dissolved in 900 ml of distilled water and adjusted pH usingdilute hydrochloric acid. The volume was adjusted to 1000 mlusing distilled water.

1. Sucrose solution (0.25 M)

85.58 gms of sucrose was dissolved in 200 ml of water anddiluted to 1000 ml with distilled water.


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2. Tris hydrochloric buffer (10mM, pH 7.4)

1.21 gm tris was dissolved in 900 ml of distilled water and the pHwas adjusted to 7.4 with 1M hydrochloric acid. The resultingsolution was diluted to 1000 ml with distilled water.

Procedure

The animals were sacrificed, after blood collection: heart wasquickly transferred to ice-cold phosphate buffered saline (pH 7.4). Itwas blotted free of blood and tissue fluids, weighed on a Single PanElectronic Balance (Precisa 205 ASCS). The hearts were cross-chopped with surgical scalpel into fine slices, suspended in chilled0.25M sucrose solution and quickly blotted on a filter paper. Thetissues were then minced and homogenized in chilled trishydrochloride buffer (10mM, pH 7.4) to a concentration of 10% w/v.Prolonged homogenization under hypotonic condition was designedto disrupt, as far as possible, the structure of the cells so as torelease soluble proteins. The homogenate was centrifuged at 7000rpm at 25 minutes using Remi C-24 high speed cooling centrifuge.

The clear supernatant was used for the determination of MDA, GSHand total protein.

Tissue estimations

The tissue levels of lipid peroxidation (MDA content) and ReducedGlutathione (GSH) were estimated as biomarkers of cardiotoxicity.

1 ) Determination of Lipid Peroxidation (MDA


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content)

It was estimated using the method described by Slater and Sawyer

(1971).

Reagents

1. Thiobarbituric acid (0.67% w/v)

0.67gm of thiobarbituric acid was dissolved in 50 ml of distilledwater and the final volume was made upto 100 ml with hotdistilled water.

2. Trichloroacetic acid (10% w/v)

10gms of trichloroacetic acid was dissolved in 60 ml of distilledwater and the final volume was made upto 100 ml with distilledwater.

3. Standard Malondialdehyde stock

solution (50mM)

A standard malondialdehyde stock solution was prepared bymixing 25 l of 1,1,3,3-tetraethoxypropane upto 100 ml withdistilled water. 1.0 ml of this stock solution was diluted up to 10ml to get solution containing 23 l of malondialdehyde/ml. Oneml of this stock solution was diluted up to 100 ml to get a working

standard solution containing 23ng of malondialdehyde/ml.

Procedure


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2.0 ml of the tissue homogenate (supernatant) was added to2.0 ml of freshly prepared 10% w/v trichloroacetic acid (TCA) and themixture was allowed to stand in an ice bath for 15 minutes. After 15

minutes, the precipitate was separated by centrifugation and 2.0 mlof clear supernatant solution was mixed with 2.0 ml of freshlyprepared thiobarbituric acid (TBA). The resulting solution was heatedin a boiling water bath for 10 minutes. It was then immediatelycooled in an ice bath for 5 minutes. The colour developed wasmeasured at 532nm against reagent blank. Different concentrations(0-23nM) of standard malondialdehyde were taken and processed asabove for standard graph.The values were expressed as nM of MDA/mg protein.

2 ) Assay of endogenous antioxidant (Superoxide

dismutase i.e. SOD)

Superoxide Dismutase (SOD)

Superoxide dismutase was estimated using the method developedby Misra and Fridovich (1972).

Reagents

1. Carbonate Buffer (0.05M, pH 10.2)

16.8gms of sodium bicarbonate and 22gms of sodium carbonate wasdissolved in 500ml of distilled water and the volume was made upto


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1000ml with distilled water.

2. Ethylenediaminetetra acetic acid (EDTA) solution(0.49M)

1.82gm of EDTA was dissolved in 200ml of distilled water and thevolume was made upto 1000ml with distilled water.

3. Hydrochloric acid (0.1N)

8.5ml of conc. hydrocloric acid was mixed with 500ml of distilledwater and the volume was made upto 1000ml with distilled water.

4. Epinephrine solution (3mM)

0.99gm epinephrine bitartarate was dissolved in 100ml of 0.1Nhydrocloric acid and the volume was adjusted to 1000ml with 0.1Nhydrocloric acid.

5 . Superoxide Dismutase (SOD) standard (100 U/L)

1mg (1000 U/mg) of SOD from bovine liver was dissolved in 100ml of carbonate buffer.

Procedure

0.5ml of tissue homogenate was diluted with 0.5ml of distilledwater, to which 0.25 ml of ice-cold ethanol and 0.15ml of ice-coldchloroform was added. The mixture was mixed well using cyclomixer for 5 minutes and centrifuged at 2500 rpm. To 0.5ml of supernatant, 1.5ml of carbonate buffer and 0.5ml of EDTA solution


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were added. The reaction was initiated by the addition of 0.4ml of epinephrine and the change in optical density/minute was measuredat 480nm against reagent blank. SOD activity was expressed as

units/mg protein. Change in optical density per minute at 50 %inhibition of epinephrine to adrenochrome transition by the enzymeis taken as the enzyme unit. Calibration curve was prepared by using10-125 units of SOD.

3) Assay of endogenous antioxidant (Reducedglutathione i.e. GSH)

Reduced glutathione was determined by the method described byMoron et al. (1979)

Reagents

1.Trichloroacetic acid (20% w/v )20gms of trichloroacetic acid was dissolved in sufficient quantityof distilled water and the final volume was made up to 100 ml of distilled water.

2.Phosphate Buffer (0.2M, pH 8.0)0.2M sodium phosphate was prepared by dissolving 30.2gmssodium phosphate in 600ml of distilled water, the pH was adjustedto 8.0 with 0.2M sodium hydroxide solution and the final volumewas adjusted up to 1000 ml with distilled water.

3.DTNB reagent (0.6mM)60mg of 5,5-dithiobis (2-nitrobenzoic acid) was dissolved in 50 mlof buffer and the final volume was adjusted to 100 ml with buffer.


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4.Standard glutathione

10mg of reduced glutathione was dissolved in 60 ml of distilledwater and the final volume was made up to 100 ml with distilledwater

Procedure

Equal volumes of tissue homogenate (supernatant) and 20%

TCA were mixed. The precipitated fraction was centrifuged and to0.25ml of supernatant, 2ml of DTNB reagent was added. The finalvolume was made up to 3ml with phosphate buffer. The colourdeveloped was read at 412nm against reagent blank. Differentconcentrations (10-50 g) of standard glutathione were taken andprocessed as above for standard graph. The amount of reducedglutathione was expressed as g of GSH / mg protein.

4) Estimation of total protein

The method of Lowry et al. (1951) was used for the estimation of

total protein.

Reagents

1. Sodium hydroxide (0.1 M)4gms of sodium hydroxide was dissolved in 400 ml distilled waterand the final volume was made up to 1000 ml with distilled water.


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2. Lowry C reagent a) Copper sulphate in 1% sodium potassium tartarate (1 %w/v) 0.5gm of copper sulphate was dissolved in 1% sodiumpotassium tartarate (Prepared by dissolving 1gm of sodiumpotassium tartarate in 100ml of distilled water).b) Sodium carbonate in 0.1M sodium hydroxide (2% w/v)2gm of sodium carbonate was dissolved in 100ml of 0.1Msodium hydroxide. 2ml of solution (a) was mixed with 100ml of solution (b) just before use.

3. Standard protein (Bovine serum albumin)

20mg of bovine serum albumin was dissolved in 80ml of distilledwater and few drops of sodium hydroxide were added to aidcomplete dissolution of bovine serum albumin and to avoidfrothing. Final volume was made to 100ml with distilled water andstored overnight in a refrigerator.

4 . Folins phenol reagent

Folins phenol reagent was diluted with distilled water in the ratioof 1:2. (i.e. 1ml of Folins phenol reagent was mixed with 2ml of distilled water).

Procedure

Diluted membrane fraction aliquots (0.1 ml) were taken in testtubes. To this, 0.8ml of 0.1M sodium hydroxides and 5ml of Lowry C

reagent was added and the solution was allowed to stand for 15minutes. Then 0.5ml of 1N Folins phenol reagent was added and thecontents were mixed well on a vortex mixer. Colour developed wasmeasured at 640nm against reagent blank containing distilled waterinstead of sample. Different concentrations (40-200 g) of standard


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protein (Bovine serum albumin) were taken and processed as abovefor standard graph. The values were expressed as mg of protein/gmof wet tissue (mg/gm).

RESULTS

Treatment of lenses with H2O2 caused distinct morphologicchanges. Lower concentrations of H2O2 (0.2 mM) caused the lens tobe hazy after 6 hours and to worsen progressively between 12 and24 hours. Higher levels of H2O2 (0.5 mM) induced similarmorphologic changes, but sooner (within 1 hour) and more severe.Both H2O2-treated groups showed a dramatic and gradual GSHdepletion during the 24-hour incubation, but the GSH level at 50% orabove appeared to be essential in maintaining lens clarity. However,

TTase, Trx, and TR activities, protein expressions, and mRNAtranscriptions in the epithelial layers of these lenses were increased,but each enzyme had a distinct pattern. Under mild H2O2 stress, aslow and transient activation of TTase, Trx, and TR was observed.However, under stronger H2O2 stress, all three enzymes showed avery rapid increase and then a steady decline in activity. Westernblot and RT-PCR analyses revealed that this increase in activity in all

three enzymes was due to the induction of protein and mRNAexpression. In the control group (no oxidative stress) all threeenzyme activities and their respective expressions remainedconstant throughout the experimental period.

PHOTOGRAPHS


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Refrences

1. Barrett G. Haik, et al (2003), department of ophthalmology University of


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Tennessee. (1)2. Dr. C K kokate et al text book of pharmacognocy (2004). ( 16)3. Balasubramanian D. Bansal A K et al The biology of cataract (2009). (12)4. Journal of Health Encyclopedia - Diseases and Conditions. (14)


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