Biological activities of Tylophora
hirsuta.
Niaz Ali
Department of Pharmacy,
University of Malakand.
1
Why to work on plants
??
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).
Import
Export
US $ 31.0 million
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
3
Objectives
1. Biological / Pharmacological investigations
2. Phytochemical investigations
4
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
5
Collection & extraction of the aerial parts
Collection
Shade drying
Grinding
Soaked in methanol (15days, thrice)
Filtration
concentrated under reduced pressure
6
Fractionation
Crude Methanolic extract
1000.0 g
Suspended in Water & fractionated with n-hexane
n-Hexane insoluble fraction
Chloroform soluble
fraction 45.0 g
n-Butanol fraction
5.0 g
Ethyl acetate soluble
fraction 20.0 g
n-Hexane soluble fraction
500.0 g
Insoluble fraction
Aqueous fraction
250.0 g
Crude extract of 100 g was reserved for biological/pharmacological screenings.
7
Isolation
Crude Methanolic extract
1000.0 g
(Fractionation)
Chloroform
n-hexane
fraction 500 g
fraction 45.0 g
Ethyl acetate
Butanol
Aqueous
fraction 20.0 g
fraction 5.0 g
fraction 250.0 g
(Column chromatography)
n-hex : EtOAc
(96.5 : 3.5)
Compound (1)
n-hex:CHCl3
(8.5 : 1.5)
Compound (2)
&
n-hex:CHCl3
(7.5:2.5)
Compound (3)
n-hex : EtOAc
(11.5:1)
Compound (4)
8
PHYTOCHEMICAL INVESTIGATIONS
Table 1: Results of preliminary phytochemical screening
S.
Test
Results
Remarks
No.
1
Alkaloids
++
Positive
2
Flavonoids
+
Positive
3
Saponin
+
Positive
4
Tannin
-
Negative
5
Terpenes and
terpenoids
+++
Strongly Positive
+: Weak, ++: Moderate and +++ : Strong
9
Biological activities
Cholinomimetic Activity (in vitro)
30
*
%of Ach Max.
25
20
15
10
5
0
0.01
0.03
0.1
0.3
1
3
5
10
[Th.Cr] mg/ml
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 ).
10
(contd.)
Spontaneous (with out atropine)
Spontaneous (with atropine)
K+(80mM) (with atropine)
180
*
% Control % Ach Max.
160
140
120
100
80
Left shift in
dose
60
40
20
0
0.01
0.03
0.1
0.3
1
[Th.Cr] mg/ml
3
5
10
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.
11
All values are Mean  SEM (n=7, *P ≤ 0.05 ).
Calcium Channel Blocking Activity
(contd.)
(right shift)
Control
(Th.cr) 0.3m g/m l
Control
0.1m icro M (Verapam il)
0.3m icro M (Verapam il)
120
110
100
90
80
70
60
50
40
30
20
10
0
A
*
-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 Max.
(Th.cr) 1m g/m l
120
110
100
90
80
70
60
50
40
30
20
10
0
B
*
-4.3 -4
-3.7 -3.4 -3.1 -2.8 -2.5 -2.2 -1.9 -1.6
Log [Ca++]M
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 ).
12
(contd.)
Spasmolytic activity of alpha amyrin acetate
Figure 7: Effects of alpha-amyrin acetate on rabbit’s jejunum preparations.
13
(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
20.0
69.1 ×10-5
38.3 ± 2.5
0.0
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
100.0
80.0
60.0
EC50=58.4 X 10-5
40.0
9.
1
2.
3
X1
05
X1
017
5
.1
X1
027
5
.1
X1
039
5
.1
X1
053
5
.1
X1
069
5
.1
X1
087
5
.1
X1
10
07.
5
1
X1
19 0- 5
3
X1
021
5
7
X1
024
5
3
X1
05
% response of control max.
120.0
Dose (Molar Concentration)
Figure 8: Spasmolytic activity of alpha amyrin acetate
14
Antibacterial activity
B. subitilis
(contd.)
S. flexenari
100
90
80
% inhibition
70
60
50
40
30
20
10
0
Figure 9: Antibacterial activity of crude extract and various fractions of Tylophora hirsuta.
15
Antifungal activity
120
(contd.)
A. flavus
F. solani
% Inhibition
100
80
60
40
20
0
Figure 10: Antifungal activity of crude extract and various
Fractions of Tylophora hirsuta.
16
(contd.)
Antileishmanial activity.
120
IC 50 values (µg/ml )
100
80
60
40
20
0
Cr
e
ud
tr a
Ex
ct
n
n
xa
e
-H
e
lo
Ch
fo
ro
e
at
et
rm
l
hy
Et
ac
ta
Bu
l
no
Aq
o
ue
us
St
d
an
d
ar
ug
Dr
Figure 11: Antileishmanial activity of crude and fractions
of Tylophora hirsuta against Leishmania major.
17
Brine shrimp (Artemia salina) lethality assay
(Contd.)
Brine shrimp (Artemia salina) Bioassay for
Tylophora hirsuta
Cotrol
n-Hexane
Ethyl acetate
Aqueos
Crude
Chloroform
n-Butanol
% shrimp killed vs
control
120
100
80
EC50
60
40
20
0
1
2
3
Etoposide (7.462 µg/ml) was used as
positive control and the number of
survivors
were measured .
Figure 14: Brine shrimp lethality assay of crude
extract and various fractions of Tylophora hirsuta.
Log 10 Dose (microgram/ml)
18
Cardiovascular effects in anaesthetized rats
Table 4: Cardiovascular effects of crude methanolic extract in
anaesthetized rats (with out Atropine).
Dose
No. of
(mg/kg)
observations
Fall in
Mean Arterial
Heart Rate
Blood Pressure
(beats/min)
(mm Hg ± SEM)
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
19
Contd.
Table 5: Cardiovascular effects in anaesthetized rats (with Atropine)
Fall in
Dose
No. of
Mean Arterial Blood
Heart Rate
(mg/kg)
observations
Pressure (mm Hg ±
(beats/min)
SEM)
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
20
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.
Thanks

For your presence and patience.
23
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).
3´
4´
2´
8
9
7
1´
O
2
C
A
6
3
10
4
5
O
B
5´
6´
Flavonoids
types
5
6
5'
4'
4
1
3
6'

1'
2

3'
'
2'
O
OH
O
HO
-Chalcanol
O
-Chalcanone
O
8
2
1
4
6
5
6'
3
10
Aurone
2'
OH
O
5'
1'
O
Chalcan-1,3-dione
O
H
O
9
7
'-Chalcanone
'-Chalcanol
Chalcone
O
H
4'
O
Aurononol
O
3'
5'
O
6'
O
8
7
O
3'
2
4
5
Flavanone
O
OH
O
Flavanonol
O
O
O
O
2'
3
10
Isoaurone
4'
1'
1
6
H
9
Auronol
O
Flavonol
OH
Flavone
O
O
O
O
Flavan
O
O
Isoflavanone
OCH3
O
3-Methoxyflavone
O
Flavan-3-ol
O
O
Isoflavone
OH
Anthocynanidin
O
Isoflavan