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EFFECT OF MUCUNA PRURIENS EXTRACT AND SOME OF ITS

CONSTITUENTS ON MEMORY IN MICE

PRESENTED BY- SHAIKH ARIF H.

(PHARMACOLOGY)

UNDER GUIDANCE OF DR. KASTURE S.B.

SANJIVANI COLLEGE OF PHARMACEUTICAL EDUCATION & RESEARCH

KOPARGAON

2

Content

Introduction and Review of literature Aim and Objective Plan of work Material and methods Results Discussion Conclusion Reference

3

IntroductionMemory

Memory is the ability of human brain to encode, consolidate, and retrieve information.

When we receive stimulus through any of our

senses, some characteristic changes occur at the synapse that leads to formation of memory.

Depending upon the importance of

information, that information is either stored in short term or long term memory.

4

Cont..

5

Mechanism and Structure involved in memory:i] Cellular basis for learning and memory

Once activity goes through the cells they leave some imprints at the junction [that persist after the activity is gone]

Trace of activity is left in, nerve terminal or in post synaptic cells

so the efficiency of these synaptic junctions is changing either strengthening / weakening.

6

ii] Erich R.Kandel

Erich R. Kandel studied molecular mechanisms of memory storage in Aplysia.

Aplysia are ideal to study because their nervous system has only approximately 20000 nerves seen by naked eyes.

The experiments by Kandel and his colleagues showed that the changes in the neurons of the Aplysia lasted for several weeks.

Figure: California sea hare (Aplysia californica).

7

Cont..

Figure: A shock to the tail causes serotonin release from interneurons. This activates a stimulatory G protein (G), which activates adenylyl cyclase (AC), leading to production of cAMP and PKA-dependent phosphorylation of different substrates (Centre). Repeated shocks to the tail causes a persistent increase in cAMP concentration, leading to altered gene transcription and protein synthesis. This leads to growth of new synapses (Right).Where, CREB= cAMP response element-binding protein

8

Review of Literature

Lieu et al., (2010): Demonstrated that dopaminergic anti-

Parkinsonian medications, such as levodopa (LD) cause drug-induced dyskinesias (DID) in the majority of patients with Parkinson's disease (PD). Mucuna pruriens, a legume extensively used in Ayurveda to treat PD, is reputed to provide anti-Parkinsonian benefits without inducing DID. They used water extract of M. pruriens seed powder.

9

Cont..

Kasture et al., (2009): neuroprotective efficacy of MP and its potential rewarding effects were evaluated.

Thakaran et al., (2007): Demonstrated that Mucuna pruriens cotyledon powder (MPCP) has shown anti-parkinsonism and neuroprotective effects in animal models of Parkinson's disease.

10

Cont..

Manyam et al., (2004): Mucuna pruriens possesses significantly

higher antiparkinsonism activity compared with levodopa in the 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson's disease.

11

Aim and objective:

To study effect of Mucuna pruriens

extract and some of it’s constituents

on memory in mice

12

Plan of work

Literature survey Selection of drug and dose Collection of drugs and chemicals Fractionisation of extract Animal models:• Open field test (Object recognition test)- for short term

memory• Elevated plus maze model – for long term memory• Lithium induce head twitches Data collection and statistical analysis Interpretation of results

13

Material and Methods

Plant Material:

Mucuna pruriens seeds Extract (Kapikachhu) Fractionated in Chloroform & Water.

Chemicals & Drug: Piracetam (Syrup), Scopolamine (ampoule), Chloroform, CMC (Carboxy methyl cellulose) etc.

Animals used: Species: Swiss Albino MiceWeight: 18-22 gmGender: Either sex

No. of groups: 24 (Each contain 5 animal).No. of animal used: 120

14

Extraction (Procedure):

The standardized extract of Mucuna seeds (Himalaya Drug Co.) was

purchased.

The extract was fractionated into

Polar (Water)

Non polar (Chloroform)

Memory enhancing activity of the extracts was studied in healthy Swiss Albino mice

15

Cont..

Elevated plus maze(Long term)

BY

Piracetam (100 mg/kg i.p) was used as standard drug

Scopolamine (0.4 mg/kg i. p.) antagonism was used to study the probable mode of action.

Open field model(Short term)

Lithium sulphate induced head twitches

16

Phytochemical screening

Sr. No.

Phytochemical tests Aqueous fraction Chloroform fraction

1 Test for Carbohydrate Molisch's test Fehling's test Barfoed's test Benedict's test

----

++++

2 Test for Alkaloids Hager's test Mayer's test Dragendroff's test Wagner's test

----

++++

3 Test for for proteins and amino acids Biuret test Millons’s test Ninhydrin test

+++

---

4 Test for Steroids Liebermann-Burchard test Salkowski test

++

--

17

Treatement schedule

Sr.no

Treatment EPMIR = (L1/L0) /L0

OFT(N-F)/(N+F)

1 Vehicle (SWFI)

2 Piracetam 100

3 Chloroform fraction of Mp extract 100



6 Aqueous fraction of Mp extract 100



9 Scopolamine 0.4

10 Chloroform fraction of Mp extract 100 + scopolamine 0.4 (on 7th day).

11 Aqueous fraction of Mp extract 100 + scopolamine 0.4 (on 7th day).

12 Piracetam 100 + Scopolamine 0.4 (on 7th day).

On the day before test Mice were allowed to explore box without any

object for 2 min.

18

On the day of test, In 1st Trial (T1), two identical objects were

placed in the box.

Animal Models: (For short term)i] Open field apparatus (Object Recognition Test)

The amount of time taken by each animal to complete 20 sec. exploration was measured

In 2nd Trial (T2, 90 min after T1), one old object was placed with newer one & time spent for exploring new and familiar

object was recorded.

19

Discrimination index (D) was calculated as

D = (N – F) (N + F)

Where, N- time spent for exploring new object.

F- time spent for exploring familiar object.

Cont..

20

(For short term)i] Open field apparatus (Object Recognition Test)

Results

21

Data was expressed as mean ± S.E.M. (n=5); ***p <0.001 as compared to control (one way ANOVA followed by Dunnett’s test); #p <0.05, ## p <0.05,###p <0.05

as compared to Scopolamine 0.4 group (Student’s t - test).

Sr. No

Treatment Discrimination Index Mean ± SEM

1 Vehicle (SWFI) 0.067±0.01299

2 Piracetam 100 0.12±0.0195***

3 Chloroform fraction of Mp extract 100 0.0825±0.0129***

4 Chloroform fraction of Mp extract 200 0.0356±0.0098

5 Chloroform fraction of Mp extract 400 0.00137±0.00045

6 Aq. Fraction of Mp extract 100 0.0962±0.0134***

7 Aq. Fraction of Mp extract 200 0.03211±0.0114

8 Aq. Fraction of Mp extract 400 -0.04376± 0.00235

9 Scopolamine 0.4 -0.09206 ±0.0325

10 Chloroform fraction of Mp extract 100 + scopolamine 0.4 -0.0331 ±0.0026 #

11 Aq. Fraction of Mp extract 100+ scopolamine 0.4 0.046 ±0.0081 ##

12 Piracetam 100 + Scopolamine 0.4 0.05707± 0.0026 ###

The discrimination Index for Open field apparatus

22

Vehicle

Pira

ceta

m

Chlor

ofor

m fr

actio

n 10

0

Chlor

ofor

m fr

actio

n 20

0

Chlor

ofor

m fr

actio

n 40

0

Aqueo

us fr

actio

n 10

0

Aqueo

us fr

actio

n 20

0

Aqueo

us fr

actio

n 40

0

Scop

olam

ine

Pira

ceta

m 1

00 +

Scop

o 0.

4

Chlor

ofor

m fr

actio

n 10

0 + s

copo

0.4

Aqueo

us fr

actio

n 10

0 + s

copo

0.4

-0.15

-0.1

-0.05

0

0.05

0.1

0.15

Dis

crim

inat

ion

In

dex

***

*** ***

###

#

###

The discrimination Index for open field apparatus

23

Data express in Mean ± SEM for completion of 20 sec exploration time of Novel (N) and Familiar (F) object in sec

0

50

100

150

200

250

Mean N

Mean F

Tim

e sp

ent

in e

xplo

rin

g ob

ject

s in

sec

Mea

n ±

SE

M

24

Object recognition test in neonatal and adult mice

Swiss albino mice weighing 5 gm were divided in 2 groups (n =5) i.e. Group A and Group B

Animal A was subjected to object recognition test at the body weight of 5 gm (20 sec exploration)

These test is repeated again for Animal A and Animal B when these animals reached body weight of 25 gm

No drug treatment was given to these animals.

25

Object recognition test in neonatal and adult mice

Results

26

The discrimination Index for open field apparatus

5 gm of animal A 25 gm of animal A 25 gm of animal B0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05D

iscr

imin

atio

n I

nd

ex

27

N F N F N F5 gm of animal A 25 gm of animal A 25 gm of animal B

0

50

100

150

200

250T

ime

of E

xplo

rati

on in

sec

Mea

n ±

SE

M

Time of Exploration in sec (Mean ± SEM)

All the treatment is given for 7 successive days by

intraperitonial route(i.p)

Transfer latency was measured after 90 min of

administration on seven day and again after 24 hr i.e. on

eighth day. 28

ii] Elevated plus maze for memory

29

Cont..

TL was expressed as Inflexion Ratio and calculated as

IR = (L1/L0) /L0

Where L1 is the TL on day seven and L0 is the TL on the eight day.

30

ii] Elevated plus maze for memory:

Results

31

Data was expressed as mean ± S.E.M. (n=5); ***p <0.001 as compare to control (one way ANOVA followed by Dunnett’s test); #p <0.001, ## p<0.001,###p <001

as compare to Scopolamine 0.4 group (Student’s t - test).

Sr. no

Treatment Inflection Ratio Mean ± SEM

1 Vehicle (SWFI) 0.06375 ± 0.012

2 Piracetam 100 0.1138 ± 0.013***

3 Chloroform fraction of Mp extract 100 0.0971 ± 0.011 ***

4 Chloroform fraction of Mp extract 200 0.0404 ± 0.0092

5 Chloroform fraction of Mp extract 400 0.01895 ± 0.0012

6 Aqueous fraction of Mp extract 100 0.08524 ± 0.0124***

7 Aqueous fraction of Mp extract 200 0.04706 ± 0.00999

8 Aqueous fraction of Mp extract 400 0.019098 ± 0.000962

9 Scopolamine 0.4 0.007 ± 0.0081

10 Chloroform fraction of Mp extract 100 + scopolamine 0.4 (on 7th day). 0.02068 ± 0.00471 #

11 Aqueous fraction of Mp extract 100 + scopolamine 0.4 (on 7th day). 0.0191 ± 0.00617 ##

12 Piracetam 100 + Scopolamine 0.4 (on 7th day). 0.03676 ± 0.00564 ###

The inflexion ratio for elevated plus maze for memory

32

Vehicle

Pira

ceta

m

Chlor

ofor

m fr

actio

n 10

0

Chlor

ofor

m fr

actio

n 20

0

Chlor

ofor

m fr

actio

n 40

0

Aqueo

us fr

actio

n 10

0

Aqueo

us fr

actio

n 20

0

Aqueo

us fr

actio

n 40

0

Scop

olam

ine

Pira

ceta

m 1

00 +

Scop

o 0.

4

Chlor

ofor

m fr

actio

n 10

0 + sc

opo

0.4

Aqueo

us fr

actio

n 10

0 + s

copo

0.4

0

0.02

0.04

0.06

0.08

0.1

0.12In

flec

tion

rat

io***

***

***

# ##

###

The inflexion ratio for elevated plus maze for memory

33

Data expressed as Mean ± SEM for Transfer latency on 7th and 8th day

Vehicle

Pira

ceta

m

Chlor

ofor

m fr

actio

n 10

0

Chlor

ofor

m fr

actio

n 20

0

Chlor

ofor

m fr

actio

n 40

0

Aqueo

us fr

actio

n 10

0

Aqueo

us fr

actio

n 20

0

Aqueo

us fr

actio

n 40

0

Pira

ceta

m 1

00+ sc

opo

0.4

Chlor

ofor

m fr

actio

n 10

0+ sco

po 0

.4

Aqueo

us fr

actio

n 10

0 + s

copo

0.4

Scop

olam

ine

0.4

0

20

40

60

80

100

120

7 th day

8th day

Tim

e fo

r tr

ansf

er la

ten

cy in

sec

Mea

n ±

SE

M

34

iii] Lithium sulphate induced head twitches

Mice divided into group of 4 (Each group contain 5 animals)

Following treatment is given to respective group:

Group No.

Treatment No. of head twitches for 1 Hr.

1 Vehicle 2 Chloroform fraction of Mucuna pruriens extract 100mg/kg

3 Aqueous fraction of Mucuna pruriens extract 100mg/kg

4 Piracetam 100mg/kg

After 30 minutes, lithium sulphate 100mg/kg i.p administered

35

Lithium sulphate administered i.p. to

mice releases serotonine from the serotonergic neuron and produces head twitches.

36

iii] Lithium sulphate induced head twitches

Results

37

Vehicle Chloroform fraction 100mg/kg

Aqueous fraction 100mg/kg Piracetam 100mg/kg0

10

20

30

40

50

60

70

80H

ead

Tw

ich

es in

1 H

r

***

***

Data was expressed as mean ± S.E.M. (n=5); ***p <0.001 as compared to control (one way ANOVA followed by Dunnett’s test).

***

38

The memory enhancing drugs reduced lithium induces head twitches. Thus, decrease in lithium induce head twitches confirms the nootropic activity in both the fractions.

39

Discussion

The pharmacological evaluation of nootropic activity of Mucuna pruriens was carried out using object recognition test, elevated plus maze model and Lithiunm sulphate induced head twiches.

The open field apparatus has been used to study object recognition, as a parameter for short term memory has been extensively used by various laboratories.

Animals differ in their ability to explore new and familiar object and therefore discrimination index and Inflection ratio was calculated. The piracetam was used as reference standard and scopolamine used as antagonist.

40

Conclusion

Increase in discrimination index in the object recognition test,

increase in inflection ratio in the elevated plus maze, inhibition of scopolamine induced amnesia and decrease in lithium induce head twitches confirms the memory enhancing activity of

Mucuna pruriens.

Further studies are necessary to understand the active ingredient responsible for the activity and also to understand the mode of action.

References

Kasture SB. A handbook of experiments in pre-clinical pharmacology. 1st ed, Career publications 2006; p. 97, 47.

Katzenschlager R, Evans A, Manson A, Patsalos PN, Ratnaraj N, Watt H, Timmermann L, Vander GR, Lees AJ. Mucuna pruriens in Parkinson's disease: a double blind clinical and pharmacological study. J Neurol Neurosurg Psychiatry; 2004; 75: 1672-77.

Lieu CA, Kunselman AR, Manyam BV, Venkiteswaran K, Subramanian T. A water extract of Mucuna pruriens provides long-term amelioration of Parkinsonism with reduced risk for dyskinesias. Dept Neurol; 2010; 16 (7): 458-65.

Manyam BV, Dhanasekaran M, Hare TA. Neuroprotective effects of the antiparkinson drug Mucuna pruriens. Phytother Res 2004;18 (9):706-12.

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Cont..

Poornachandra MN, Khanam S, Shivananda BG, Shivananda TN, Dris R. Mucuna pruriens (L.) DC - A novel drug for learning and memory retrieval. J Food Agri Envir 2005; 3 (3&4): 13-15.

Thakaran B, Dhanashekaran M, Mize Berg J, Manyam BV. Anti-Parkinson botanical Mucuna pruriens prevents levodopa induced plasmid and genomic DNA damage. Phytother Res 2007; 21 (12): 1124-26.

Kandel ER, Spencer WA. Cellular neurophysiological approaches in the study of learning. Physiol Rev 1968; 48 (1): 65–34.

Kandel ER. The molecular biology of memory storage: A dialogue between genes and synapses. Biosci Rep 2001; 21 (5): 1030-38.

Parkinson J. An essay on the shaking plasy, J Neuropsychiatry Clin Neurosci 2002; 14 (2): 223-36.

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Cont..

Rang HP, Dale MM, Ritter JM, Moore PK, Flower RJ. Pharmacology. 6th ed.Edinburgh: Churchill Livingstone; 2007. p. 562-572.

Tripathi KD. Essentials of Medical Pharmacology. 5 thed, Jaypee Brothers, Medical Publishers limited, New Delhi, 2004; p. 84-85,93-101, 381-435.

Walker R, Whittlesea C. Clinical Pharmacy and Therapeutics. 4th ed, Churchill Livingstone Elseiver, 2007; p. 138,443,463-465.

Gelperin A. Rapid food-aversion learning by a terrestrial mollusk. Sci 1975; 189 (4202): 567–70.

Geinisman Y, Disterhoft JF, Gundersen HJG, McEchron MD, Persina IS, Power JM, Van der Zee EA, West MJ. Remodeling of Hippocampal synapse following hippocampus dependent associative learning. J Comp Neurol 2000; 417 (1): 49-59.

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Cont..

https://www.youtube.com/watch?v=30X7ORyGtGM= 07/08/12.

https://www.youtube.com/watch?v=9Wv9jrk-gXc = 09/12/12.

https://www.youtube.com/watch?v=ATR0os9w2jY, = 01/12/12.

https://www.youtube.com/watch?v=dYE6zixML1o = 09/09/12.

https://www.youtube.com/watch?v=ii4CHwVhExc= 15/11/12.

https://www.youtube.com/watch?v=-KguICny3JE= 13/08/12.

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