Tylophora hirsuta - University of Peshawar

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20 Φεβ 2013 (πριν από 5 χρόνια και 7 μήνες)

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1

Biological activities of
Tylophora
hirsuta.

Niaz Ali


Department of Pharmacy,

University of Malakand.

Why to work on plants

??

3

Importance

of

medicinal

plants


In

the

world,

30

%

of

the

pharmaceutical

preparations

are

manufactured

from

plants
.



Global

market





US

$

60
.
0

billion



Expected

growth





US

$

5
.
0

trillion

(year

2050
)
.





Medicinal

species

exist

in

Pakistan


2000
/

8000

medicinal

plants

in

Asia
.



In

Pakistan

(
1999
)
.


I
mport





US

$

31
.
0

million



E
xport





US

$

6
.
0

million
.



Interestingly
:



1
:

Only

6
%

have

been

explored

for

their

biological

activities
.


2
:

Only

15
%

have

been

explored

phytochemically

for

their




constituents
.


On

the

analogy

of

the

above

facts,

we

set

our

objectives

for

current

work
.


References


Olayiowola

A

(
1984
)
.
WHO’s

traditional

medicine

programme
:

Progress

and

Perspective
.

WHO

Chronicle

38

(
2
)
:
76
-
81
.




Karki

M

(
2002
)
.

Medicinal

and

aromatic

Plants

Programme

in

Asia
.

Benefits

and

Challenges
.




IDRC/SARO
.

New

Delhi,

India
.

mappa@idrc
.
org
.
in


4

Objectives

1
.

Biological

/

Pharmacological

investigations



2
.

Phytochemical

investigations









5

Traditional

uses

and

pharmacological

Activities

Traditional uses

GIT

disorders

Allergic

conditions

Rheumatism

Asthma

High

blood

pressure

Pharmacological activities reported

Immunomodulatory / anti
-
inflammatory

Anticancer

Antiamoebic


Our Targets


To screen the plant Phytochemically and explore the traditional uses

on scientific grounds

6
















Collection & extraction of the aerial parts


Collection


Shade drying


Grinding


Soaked in methanol (15days, thrice)


Filtration


concentrated under reduced pressure



7













Fractionation


Crude Methanolic extract

1000.0 g

n
-
Hexane soluble fraction

500.0 g

n
-
Hexane insoluble fraction



Chloroform soluble

fraction 45.0 g


Ethyl acetate soluble

fraction 20.0 g

Insoluble fraction

n
-
Butanol fraction

5.0 g

Aqueous fraction

250.0 g

Suspended in Water & fractionated with n
-
hexane

Crude extract of 100 g was reserved for biological/pharmacological screenings.

8













Isolation


Crude Methanolic extract

1000.0 g


n
-
hexane

fraction 500 g



Chloroform

fraction 45.0 g



Ethyl acetate

fraction 20.0 g



Butanol

fraction 5.0 g


Aqueous

fraction 250.0 g


n
-
hex : EtOAc

(96.5 : 3.5)

Compound (1)




n
-
hex:CHCl
3


(8.5 : 1.5)

Compound (2)

&

n
-
hex:CHCl
3


(7.5:2.5)

Compound (3)




n
-
hex : EtOAc


(11.5:1)

Compound (4)

(Fractionation)

(Column chromatography)

9

PHYTOCHEMICAL INVESTIGATIONS


S.
No.

T
est

Results

Remarks

1

Alkaloids

++

Positive

2

Flavonoids

+

Positive

3

Saponin

+

Positive

4

Tannin

-

Negative

5

Terpenes and
t
erpenoids

++
+

Strongly
Positive

Table 1
: Results of preliminary phytochemical screening


+: Weak, ++: Moderate and +++ : Strong

10



Biological activities



Cholinomimetic Activity (in vitro)

Figure 4
: Cholinomimetic effects of the crude extract of
Tylophora hirsuta
on isolated rabbit’s jejunum preparations.

All values are Mean


SEM (n = 7,
*
P ≤ 0.05 ).

0
5
10
15
20
25
30
0.01
0.03
0.1
0.3
1
3
5
10
[Th.Cr] mg/ml
%of Ach Max.
*

11









(contd.)

Figure

5
:

Dose

response

curves

of

crude

extract

of

Tylophora

hirsuta

on


spontaneous

contractions

of

isolated

rabbit

jejunum

in

the

absence

and


presence

of

atropine

0
.
03

micro

molar
.

Extract

effects

on

potassium

induced


contractions

in

atropinized

Preparations

are

also

shown
.


All

values

are

Mean



SEM

(n=
7
,

*
P



0
.
05

)
.

0
20
40
60
80
100
120
140
160
180
0.01
0.03
0.1
0.3
1
3
5
10
% Control % Ach Max.
[Th.Cr] mg/ml
Spontaneous (with out atropine)
Spontaneous (with atropine)
K+(80mM) (with atropine)
Left shift in
dose

*


12





Calcium Channel Blocking Activity








(contd.)

A
0
10
20
30
40
50
60
70
80
90
100
110
120
-4.3
-4
-3.7
-3.4
-3.1
-2.8
-2.5
-2.2
-1.9
-1.6
Log [Ca
++
]M
%Control Max.
Control
(Th.cr) 0.3mg/ml
(Th.cr) 1mg/ml
B
0
10
20
30
40
50
60
70
80
90
100
110
120
-4.3
-4
-3.7
-3.4
-3.1
-2.8
-2.5
-2.2
-1.9
-1.6
Log [Ca
++
]M
% Control Max
Control
0.1micro M (Verapamil)
0.3micro M (Verapamil)
Figure 6
: Dose response curves of Ca
++

in absence and presence of increasing

doses of Extract of
Tylophora hirsuta
(Th.cr) and (B) verapamil in isolated

(A) rabbit’s jejunum preparations. All values are Mean


SEM (n=6, *P ≤ 0.05 ).

(right shift)

*

*

13








(contd.)

Spasmolytic activity of alpha amyrin acetate




Figure 7
: Effects of alpha
-
amyrin acetate on rabbit’s jejunum preparations.

14








(contd.)

Dose

(M)

% Response of control
max.
±

SD

2.3
×
10
-
5

95.2
±

1.0

9.1
×
10
-
5

89.3
±

1.5

17.1
×
10
-
5

88.0
±

3.0

27.1
×
10
-
5

82
±

4.0

39.1
×
10
-
5

73
±

2.6

53.1
×
10
-
5

54.0
±

4.4

69.1
×
10
-
5

38.3
±

2.5

87.1
×
10
-
5

28.6
±

3.4

107.1
×
10
-
5

23.4
±

3.7

193
×
10
-
5

14.4
±

3.3

217
×
10
-
5

6.2
±

1.7

243
×
10
-
5

0

0.0
20.0
40.0
60.0
80.0
100.0
120.0
2.3 X10-5
9.1 X10-5
17.1 X10-5
27.1 X10-5
39.1 X10-5
53.1 X10-5
69.1 X10-5
87.1 X10-5
107.1 X10-5
193 X10-5
217 X10-5
243 X10-5
Dose (Molar Concentration)
% response of control max.
EC
50
=58.4 X 10
-
5

Figure 8
: Spasmolytic activity of alpha amyrin acetate


15

Antibacterial activity





(contd.)



Figure 9
: Antibacterial activity of crude extract and various fractions of
Tylophora hirsuta.


0
10
20
30
40
50
60
70
80
90
100
% inhibition

B. subitilis
S. flexenari
16


Antifungal activity





(contd.)


Figure 10
: Antifungal activity of crude extract and various




Fractions of
Tylophora hirsuta.



0
20
40
60
80
100
120
% Inhibition

A. flavus
F. solani
17








(contd.)


Antileishmanial activity.









0
20
40
60
80
100
120
Crude Extract
n-Hexane
Chloroform
Ethyl acetate
Butanol
Aqueous
Standard Drug
IC 50 values (µg/ml )

Figure 11
: Antileishmanial activity of crude and fractions



of
Tylophora hirsuta
against
Leishmania major.

18

Brine shrimp (
Artemia salina
) lethality assay

(Contd.)


Brine shrimp (Artemia salina) Bioassay for
Tylophora hirsuta
0
20
40
60
80
100
120
1
2
3
Log 10 Dose (microgram/ml)
% shrimp killed vs
control
Cotrol
Crude
n-Hexane
Chloroform
Ethyl acetate
n-Butanol
Aqueos









Etoposide (7.462 µg/ml)
was used as

positive control and the number of

survivors

were measured .



EC
50


Figure 14:

Brine shrimp lethality assay of crude

extract and various fractions of
Tylophora hirsuta.

19

Cardiovascular effects in anaesthetized rats



Table 4
: Cardiovascular effects of crude methanolic extract in


anaesthetized rats (with out Atropine).

Fall in

Dose

(mg/kg)

No. of
observations

Mean Arterial
Blood Pressure
(mm Hg ± SEM)

Heart Rate

(beats/min)

1

6

0

0

3

6

0

0

10

6

23 ± 3

5 ± 0.8

30

6

46 ± 6.9

9 ± 2

100

7

64 ± 7

30 ± 4.5

300

6

113 ± 9

218 ± 8


20








Contd.

Fall in


Dose

(mg/kg)


No. of
observa
tions

Mean Arterial Blood
Pressure (mm Hg ±
SEM)

Heart Rate

(beats/min)

1

6

0

0

3

6

0

0

10

6

0

0

30

6

0

0

100

7

0

0

300

6

34 ± 4

110± 5.6


Table 5:

Cardiovascular effects in anaesthetized rats (with Atropine)


Conclusion

Based

on

the

traditional

uses

and

experimental

work,

it

is

concluded

that

The

plant

species

exhibited

excellent

anti
-
leishmanial,

spasmolytic,

calcium

channel

blocking

and

anti
-
hypertensive

activities
.


The

phytochemical

screening

revealed

the

presence

of

Alkaloids,

flavonoids

and

saponins
.


Alpha
-
Amyrin

acetate

also

exhibited

excellent

spasmolytic

activity
.

Our

current

work

opens

a

new

window

for

researchers

to

further

work

on

activity

guided

isolation

from

the

species

so

that

we

may

standardize

the

extract,

and

snatch

share(s)

from

the

global

market
.

Acknowledgments

Thanks to my supervisor Professor Dr. Bashir Ahmad

Director

Centre of Biotechnology and Microbiology

University of Peshawar.

23

Thanks


For your presence and patience.



Back up slides


Terpenes are made up of 5
-
carbon units called isoprene. The
isoprene rule states that, “Terpenes are derived from integral
number of biological equivalents of isoprene which are joined
together in a head
-
to
-
tail or head
-
to
-
head fashion”.


Synthesis of dimethyl allyl pyrophosphate

Condensation of two isoprene units

Flavonoids

The name “flavonoid” is derived from Greek word “flavus” (yellow).

Flavonoids occur in a variety of structural forms. All contain fifteen
carbon atoms in their parent nucleus and share a common
structural feature of two phenyl rings linked by a three
-
carbon
chain (diphenyl propane derivatives).

O
O
A
B
C
2
3
4
5
6
7
8
9
1
0
1
´
2
´
3
´
4
´
5
´
6
´
Flavonoids

O
O
O
O
H
O
O
H
O
O
O
O
O
O
O
O
O
H
O
O
O
O
O
C
H
3
O
O
O
H
O
O
O
H
O
O
O
O
O
H
O
O
H
O
H
O
O
O
O
O
H
F
l
a
v
a
n
o
n
e
1
2
3
4
5
6
7
8
9
1
0
1
'
2
'
3
'
4
'
5
'
6
'
F
l
a
v
a
n
-
3
-
o
l
F
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a
v
a
n
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n
o
l
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t
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y
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a
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l
a
v
a
n
1
2
3
4
5
6
1
'
2
'
3
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4
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5
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6
'
C
h
a
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c
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