international journal of advanced pharmaceutical...

INTERNATIONAL JOURNAL

OF

ADVANCED PHARMACEUTICAL SCIENCES

ABSTRACT

In epilepsy,ones activity becomes disturbed that causes sensations, emotions,

behaviors and often times seizures with muscle spasms .It has shown increase

in the brain levels of GABA and Glutamate .It causes an inhibition of seizure

activity. Anticonvulsants mediate their action through alteration in various neu-

rotransmitter levels in various regions of brain .In this context screening of

aqueous extract of Pavetta indica for anticonvulsant activity is done. In this

study we took two standard methods namely PTZ and MES method. The pa-

rameters observed were the duration of tonic hind limb flexion, clonus, tonic

limb extension, and incidence and death. It was found that treatment with

EEPT on mice produces reduced tonic hind limb extensor stage in MES in-

duced epilepsy.

Keywords : MES,PTZ,EEPT

Secunderabad

PHARMACOLOGICAL STUDY OF PAVETTEA INDICA

FOR EPILEPSY IN RATS

Satyabrata Bhanja*, T Sarkar1 ,S Sahu 2

1,2Department of Pharmaceutics, Malla Reddy College of Pharmacy, Maisammaguda

Secunderabad.Andhra Pradesh.

Volume 01, Issue 01, 20-33.

Article Received on

Revised on

Accepted on

*Corresponding Author

Satyabrata Bhanja

Malla Reddy College of

Pharmacy, Maisammaguda

B.B. Karkara

* JRF,Central Drug Research Institute, Lucknow

*BB Karkara,CDRI,Lucknow,Uttar Pradesh

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Introduction

Disease of CNS are major threat of the present and future .It is as a result of increase in mental stress

and strain .So a correct remedy for every CNS disorder should be found before it’s too late. we tend

to use medicines of herbal source that are having heterogeneous uses are invariably an alternate

choice to the artificial medicine .Synthetic medicine are standard for his or her adverse effects during

which the requirement isn't met.1, 2, 6, 19, 23

Now and so the prices of treating brain disorder are probably to extend given the new trends in

prescribing patterns towards newer and pricey AEDs.14 one among the newest studies within the

literature twenty five calculable that prices of prescribing costs within the community has up three-

fold within the last ten years. The prevailing medicament medicine encounter several adverse effects

and would like on prolonged treatment together with questionable effectualness within the

treatment, impairment of memory.9, 11, 15, 16 This forces the world of analysis to search out improved

treatments which is able to counteract the adverse effects and draw backs of the present treatment.37,

38

Herbal formulations still looked upon as there ar several ailments touching sizable amount of

populations left while not definite medicines. during this work I actually have tried to bring a brand

new plant for the treatment of brain disorder. during this work the medicine result of Pavetta indica,

against MES, PTZ iatrogenic convulsions were evaluated and beside in-vitro study.

The result of this plant on aminoalkane is additionally calculable to predict the potential

mechanism concerned within the treatment of those ailments. As brain monoamines except for

aminoalkanoic acid additionally plays a regulative role within the maintenance of traditional state

(McQuayet al., 1995)32

Collection and Authentication of material

Inner bark of the complete plants were dried in shade, separated and created to dry powder. it

absolutely was then more matured the 40mesh sieve for extraction purpose(Khandalwal K R

Niraliprakash1996)30

Ethanolic extracted products of Pavetta indica

A weighed amount (70gm) of the(Trease GE EVANS 1983) powder was subjected to hot solvent

extraction during a soxlet equipment mistreatment ethyl alcohol, at a temperature vary of 60-70°C.

Before and when each extraction the brandy was fully dried and weighed. The extract was targeted to

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status at 40°C underneath reduced pressure during a rotary vacuum evaporator. The ethanolic

extracted productof Pavetta indica Linn yielded thick brown semi-solid residues.

Percentage Yield:

Percentage yield of ethanolic extracted product of Pavetta indica Linn was found to be

17.5%w/w.

Experimental Animals

Swiss unusual person mice (20-30g) of either sex were procured from Asterace labs Hyderabad,

A.P.The animals were maintained during a well-ventilated area with 12:12 hour light/dark cycle in

polypropene cages. Common place pellet feed (Hindustan Lever restricted., Bangalore) and drinkable

was provided spontaneously through out experimentation amount. Animals were acclimatized to

laboratory conditions one week before initiation of experiments. moral committee clearance was

obtained from Institutional Animal Ethics Committee (013/IAEC/StJCOP/2.012) of CPCSEA

(Committee for the aim of management and superintendence of Experiments on Animals).(Reg

no.12078/ac/09/CPCSEA)(30, 31, 35, 39)

CHEMICALS

All the Chemicals used in the study were of analytical grade the following chemicals

were used for the experimental study.

S.No. Name of the Chemicals Sources

1 5,5 Dithio-bis-2 nitrobenzoic

acid (DTNB)

S.d Fine Chemicals Ltd., Mumbai

2 Acetic Acid Sigma Chemical Cop., Mumbai

3 Acetic anhydride Sigma Chemical Cop., Mumbai

4 Chloroform Qualigens Fine Chem., Mumbai

5 Con H2SO4 Loba Chemical Pvt. Ltd. Mumbai

6 Con HCL S.d Fine Chemicals Ltd., Mumbai

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7 DiethyleneTriaminepenta

acetic acid


8 Ammonia S.d Fine Chemicals Ltd., Mumbai

9 Ethanol Qualigens Fine Chem., Mumbai

10 Ethylene Diamine Tetra Acetic

acid (EDTA)


11 Glacial Acetic Acid S.d Fine Chemicals Ltd., Mumbai

12 Hydrogen peroxide Qualigens Fine Chem., Mumbai

13 Methanol S.d Fine Chemicals Ltd., Mumbai

14 n-butanol Qualigens Fine Chem., Mumbai

15 Neutral ferric Choride Qualigens Fine Chem., Mumbai

16 O-pthaldehyde S.d Fine Chemicals Ltd., Mumbai

17 Pentylenetetrazole Sigma Chemical Co., USA

18 Potassium Hydrogen Phosphate Qualigens Fine Chem., Mumbai

19 Potassium Hydroxide Qualigens Fine Chem., Mumbai

20 Dopamine S.d Fine Chemicals Ltd., Mumbai

21 Pyrogallol Qualigens Fine Chem., Mumbai

22 Sodium acetate S.d Fine Chemicals Ltd., Mumbai

23 Sodium bicarbonate Qualigens Fine Chem., Mumbai

24 Sodium Caboxy Methyl

Cellulose (SCMC)

Qualigens Fine Chem., Mumbai

25 Sodium Chloride S.d Fine Chemicals Ltd., Mumbai

26 Sodium dodecyl sulphate (SDS) Qualigens Fine Chem., Mumbai

27 Sodium hydroxide S.d Fine Chemicals Ltd., Mumbai

28 Sodium Sulphate S.d Fine Chemicals Ltd., Mumbai

29 Thiobarbituric acid S.d Fine Chemicals Ltd., Mumbai

30 Tricarboxylic acid Qualigens Fine Chem., Mumbai

Table No:1 Chemicals used in formulation.

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Methods:

Preliminary Phytochemical Analysis 22

The extracts were subjected to preliminary phytochemical screening for the presence or absence of

phytoconstituents by the subsequent ways.

Test for Alkaloids

The extracts were treated with dilute acid and filtered. The filtrate is employed within the following

tests.

a) Mayer's Test. (Potassium metal Bromide solution)

When the extract was treated with Mayer's chemical agent and therefore the look of cream color

indicates the presence of alkaloid.

b) Dragendorffs Test. (Potassium metal Bromide solution)

When 1ml of extract was treated with the Dragendorffs chemical agent. The look of brownness

precipitate shows the presence of alkaloid.

c) Hager's Test. (Saturated solution of Picric acid)

When 1ml of extract was treated with the Hager's chemical agent. The look of yellow color


d) Wagner's Test . (Iodine-Potassium halide solution)

When 1ml of extract was treated with the Wagner's chemical agent. the looks of brown color


Test for Carbohydrates

a) Molisch reaction

The required extract was treated with 3ml of alpha-napthol in alcohol and targeted sulphuric acid was

extra on the perimeters of the tubing carefully. Formation of violet color ring at the junction of 2

liquids indicates the presence of carbohydrates.

b) Fehling's Test . (CuSO4.7H2O+KOH+Sodium metal Tartarate)

The required extract was treated with Fehling's solution A and B and heated in boiling water for jiffy.

The looks of reddish brown color precipitate indicates the presence of reducing sugars.

c) Benedict's Test . (Sodium Citrate+Sodium Carbonate+CuSO4.7H2O)

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The required extract was treated with Benedict's chemical agent and heated during a boiling water tub

for couple of minutes. the looks of orangeness color precipitate indicates the presence of reducing

sugars.

d) Barfoed's Test . (Copper Acetate+Glacial carboxylic acid Acid)

The required extract was treated with Barfoed's chemical agent and heated during a boiling water tub

for few minutes. The looks of orangeness color precipitate indicate that non reducing sugars is

present.

Test for Steroids

Libermannburchard Test .

The required extract was treated with little amount of targeted sulphuric acid, glacial ethanoic acid

and anhydride. the looks of green color indicates the presence of steroids.

Test for Proteins

a) Biuret's Test .

The required extract was treated with copper sulfate and sodium hydroxide answer. The looks of

violet color indicates that protein is present.

b) Millon's Test . (Mercury Nitrate Solution)

The required extract was treated with Million's reagent. the looks of pink color points out that protein

is present.

Test for Tannins

a) The required extract was treated with 100% lead acetate solution. The look of white precipitate

indicates tannins.is present.

b) The required extract was treated with aqueous atomic number 35 is answer. the looks of white

precipitate indicates tannins.is present

Test for Phenols

a) The required extract was treated with neutral ferric chloride answer. the looks of violet color

indicates phenol is present

b) The required extract was treated with 100% sodium chloride solution. The look of cream color

indicates that phenols is present

Test for Flavanoids

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a) About 5ml of extract was hydrolysed with 100% v/v vitriol and cooled. Then, it's extracted with

diethylether and divided into 3 parts in 3 separate Test . tubes. 1ml of diluted sodium carbonate, 1ml

of 0.1 N caustic soda, and 1ml of robust ammonia answer were extra to the primary, second and third

Test . tubes severally. In every tubing, development of yellow color demonstrated the presence of

flavanoids.

b) Shinodas Test:

The required extract was taken and dissolved in alcohol, thereto one piece of metallic element is

added followed by targeted hydrochloric acid on the perimeters of the tubing drop wise. it's heated

during a boiling water tub for few minutes. the looks of magenta color indicates the presence of

flavanoids.

Test for Flavanones

a) The required extract was treated with 100% NaOH. look of yellow to orange color shows the

presence of Flavonones.

b) The required extract was treated with Con. H2SO4 look of orange to crimson red color, confirms

that Flavones is presence

Test for Gums and Mucilage

In this test extract was treated with 25ml of absolute alcohol then product so obtained was filtered.

The filtrate was examined if it swells.

Test for Glycosides

The required extract was dissolved within the glacial acetic acid and few drops of ferric chloride

answer was extra, followed by the addition of targeted sulphuric acid, formation of red ring at the

junction of 2 liquids indicates the presence of glycosides.

Test for Saponins Foam Test.

1ml of the extract was diluted to 20ml with distilled water and jolted well during a test tube. The

formation of froth within the higher a part of the tubing indicates the presence of saponins.

Test for Terpenes

The required extract was treated with tin and thinoyl chloride, look of pink color points out the

presence of terpenes.

Test for Sterols

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When the required extract was treated with five-hitter hydrated oxide solution, look of pink color

points out the presence of sterols.

Toxicological Evaluations (Acute Oral Toxicity Study)

The procedure was followed by mistreatment Organization of Economic Cooperation and

Development guide lines 423 (Acute toxicant category Method) The acute toxicant category

methodology could be a step wise procedure with 3 male animals per step. relying abreast of the

mortality and/or moribund standing of animals, on the common 2-3 steps could also be necessary to

permit judgment on the acute toxicity of the Test . substance. This procedure ends up in the

employment of smallest range of animals whereas giving acceptable information based mostly

scientific conclusion. the strategy uses outlined doses (5, 50,500,2000mg/kg body weight) and

therefore the results enable a substance to be graded and classified in keeping with the Globally

harmonical System for classification of chemicals that cause acute toxicity.

Experimental procedure

Swiss albino mice weighing 20-30g were used for the study. The beginning dose level of

S.No. Phytochemical Results

1 Test for Alkaloids +Ve

2 Test for Carbohydrates +Ve

3 Test for Proteins +Ve

4 Test for Steroids -Ve5 Test for Phenols +Ve6 Test for Flavonoids +Ve7 Test for Gums & Mucilage -Ve

8 Test for Glycosides +Ve

9 Test for Saponins -Ve

10 Test for Terpenes +Ve

11 Test for Tannis +Ve

+Ve: Indicates the presence of Compounds-Ve: Indicates the absence of Compounds

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Table: 2. Preliminary Phytochemical Test for EPT

S.No. Groups Dose/

kg

b.w

Weight of

Animals

Signs of

Toxicity

Onset

Of

Toxici

ty

Duratio

n

Of study

Before After

Test1 EEPT 2000mg 25g 25g No signs of

Toxicity

Nil 14 days

2 EEPT 2000mg 25g 25g No signs of

Toxicity

Nil 14 days

3 EEPT 2000mg 25g 25g No signs of

Toxicity

Nil 14 days

Table: 3. Acute Toxicity class method OECD guidelines 423

The acute oral harmfulity study was done in keeping with OECD tips 423 (acute toxic category

method). one dose of 2000mg/kg/p.o. of the EEPT was administered three|to three} feminine mice

every and ascertained for 3 days. Animals were ascertained for signs of toxicity for initial three

hours at thirty min interval. thenceforth animals were ascertained for twenty-four hours with

continuous observation. The animals were ascertained for more fourteen days period for all toxicity

signs. There was no considerable modification in weight before and when treatment and no sign of

toxicity were ascertained. LD50 discontinue dose per kg weight was categorised as X (unclassified)

and Globally harmonical system (GHS) categories conjointly comes below X (unclassified). The

results square measure shown in (Table: 3).

Effects of EEPT on MES elicited convulsions

Phenytoin (PHT) treated animals have shown 100 percent protection against MES elicited seizures

whereas EEPT two hundred mg/kg and four hundred mg/kg have shown sixty five.20% and 70.29%

protection severally against MES elicited seizures.The EEPT at each doses and normal treated

animals had shown a big modification in period in the least stages of convulsions.EEPT two hundred

mg/kg and four hundred mg/kg had shown a big decrease within the period of tonic striated muscle

part and comparable significance (p<0.01) with the management. The results were shown in Table

3& Fig half dozen

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Effect of EEPT on PTZ elicited convulsions

Diazepam treated animals have shown 100 percent protection against PTZ elicited seizures wherever

as EEPT 200 mg/kg and 400 mg/kg have shown 61.77% and 81.5% protection severally against PTZ

elicited seizures.EEPT 201 mg/kg and 440 mg/kg had shown a significant increase in onset of

convulsion convulsions and comparable (p<0.01 and p<0.01) with the control. The results were

shown in Table 4&.Mortality of EEPT 201 mg/kg and 401 mg/kg treated teams was reduced as

comparedto management cluster animals.Values are expressed as mean ±SEM of six

observationsComparison were created between: cluster I with cluster II, III and IV.Statistical

important test for comparison was done by ANOVA, followed by Dunnet’s‘t’ check.Values

expressed in seconds

EFFECT OF EEPT ON PTZ INDUCED CONVULSIONS

Group Treatment % of

Protection

Duration of

convulsion

Onset of

clonic

convulsion

% of

Protection

mortalityI CONTROL 0 72.29 ± 8.83 177.70 ± 2.36 50

II DIAZEPAM 100 10.58 ± 2.62**

765.33 ±

9.06** 100

III EEPT(200mg) 69.66 28.21 ± 3.30**

496.83 ±

3.70** 84.44

IV EEPT(400mg) 80.48 21.87 ± 1.33**

566.83 ±

5.79** 100

Table: 4 Effect of EEPT on PTZ induced convulsions

Values are expressed as mean ±SEM of six observations

Comparison were made between: Group I with Group II, III and IV

Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s‘t’ test.

Values expressed in seconds

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Fig 1:Effect of EEPT on PTZ indused Convulsions

Discussion:

In encephalopathy, traditional pattern of somatic cell activity becomes disturbed in brief once the

nerves within the brain “fire” ad lib inflicting sensations, emotions, behaviors and sometimes times

seizures with muscle spasms similarly as loss of consciousness (Radoset al., 2005)It has been

rumored to extend the brain levels of aminoalkanoic acid associate degreed salt that causes an

inhibition of seizure activity. (Bhaduriet al., 1995)5It is understood that anticonvulsants mediate the

action through alteration in numerous neurochemical levels in numerous regions of brain. Gamma

amino butaric acid (GABA) systems have a major role with relevancy anticonvulsive properties.

Lowering of aminoalkanoic acid in brain ends up in the looks of convulsion.In the gift study for the

screening of binary compound extract of Pavetta indica for antiepileptic drug activity, 2 normal

strategies specifically MES and PTZ strategies are used. therefore here the parameters discovered

were the length of tonic limb flexion, tonic limb extension, clonus, and incidence and death.

MES methodology contains a high degree of productivity for medicine helpful within the

management of tonic convulsion seizures. And for PTZ elicited seizures showing convulsive response

were used for the experiment (Rolaet al., 2002)It is found that treatment with EEPT on mice

considerably reduces in tonic limb skeletal muscle stage in MES elicited encephalopathy. finally, we

recommend that ethanolic extracted product of Pavetta indica Linn. markedly protects

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encephalopathy elicited by MES and PTZ that are mediate by levels of monoamines and inhibitor

enzymes. more studies are needed to isolate and characterize numerous phytoconstituents to

determine the impact on numerous varieties of encephalopathy like generalized seizure, petit mal,

absence and partial seizure.

Conclusion

The preliminary phytochemical screening of EEPT shows presence of assorted chemistry constituents

like alkaloids, carbohydrates, proteins, sterols, polyphenolic compounds and gums etc. The

anticonvulsant drug activity of EEPT was assessed by MES elicited convulsion and PTZ elicited

convulsion that it showed significant attenuative.In MES elicited encephalopathy, EEPT at each

200mg/kg and 400mg/kg exhibits important anticonvulsant drug activity significantly tonic limb

skeletal muscle stage.In PTZ elicited encephalopathy, EEPT at each 200mg/kg and 400mg/kg

exhibits important anticonvulsant drug activity and delayed the onset of convulsions and cut back the

length of convulsion. Pavetta indicaLinn could be a renowned plant that is getting used in Indian

ancient Medicines for encephalopathy and central nervous system associated disorders. therefore this

analysis is expressed to form the evident impact of the complete plant inner bark on

encephalopathy.The investigation is on disbursed on each in vivo and in vitro model like MES and

PTZ elicited convulsion In MES and PTZ elicited encephalopathy, EEPT at each 200mg and 400mg

exhibited considerably anticonvulsant drug activity. finally from the observation, it may be envisaged,

the anticonvulsant drug activity of Pavetta indica Linn. more studies are needed to isolate and

charecterised numerous phytoconstituents and to determine the impact of assorted varieties of

encephalopathy like generalized seizure, petit mal, absence and partial seizure.

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