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Biological activities of Tylophora hirsuta.

Niaz Ali

Department of Pharmacy, University of Malakand.

Why to work on plants

??

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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 US $ 31.0 million Export 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.ReferencesOlayiowola 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

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Objectives

1. Biological / Pharmacological investigations

2. Phytochemical investigations

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Traditional uses and pharmacological Activities

Traditional uses

GIT disorders

Allergic conditions

Rheumatism

AsthmaHigh 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

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Collection & extraction of the aerial parts

Collection

Shade drying

Grinding

Soaked in methanol (15days, thrice)

Filtration

concentrated under reduced pressure

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Fractionation

Crude Methanolic extract1000.0 g

n-Hexane soluble fraction 500.0 g

n-Hexane insoluble fraction

Chloroform soluble

fraction 45.0 g Ethyl acetate soluble

fraction 20.0 gInsoluble fraction

n-Butanol fraction 5.0 g

Aqueous fraction250.0 g

Suspended in Water & fractionated with n-hexane

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

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Isolation

Crude Methanolic extract1000.0 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 : EtOAc (11.5:1)

Compound (4)

(Fractionation)

(Column chromatography)

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PHYTOCHEMICAL INVESTIGATIONS

S.

No.

Test Results Remarks

1 Alkaloids ++ Positive

2 Flavonoids + Positive

3 Saponin + Positive

4 Tannin - Negative

5 Terpenes and terpenoids

+++ Strongly Positive

Table 1: Results of preliminary phytochemical screening

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

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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

%o

f A

ch

Ma

x.

*

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(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

% C

on

tro

l %

Ac

h M

ax

.

[Th.Cr] mg/ml

Spontaneous (with out atropine)Spontaneous (with atropine)K+(80mM) (with atropine)

Left shift in dose

*

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Calcium Channel Blocking Activity(contd.)

A

0102030405060708090100110120

-4.3 -4 -3.7 -3.4 -3.1 -2.8 -2.5 -2.2 -1.9 -1.6

Log [Ca++]M

%C

on

tro

l Max

.

Control

(Th.cr) 0.3mg/ml

(Th.cr) 1mg/ml

B

0102030405060708090100110120

-4.3 -4 -3.7 -3.4 -3.1 -2.8 -2.5 -2.2 -1.9 -1.6

Log [Ca++]M

% C

on

tro

l Max

Control0.1micro 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)

**

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(contd.)

Spasmolytic activity of alpha amyrin acetate

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

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(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

Dose (Molar Concentration)

% r

esp

on

se o

f co

ntr

ol

max

.

EC50=58.4 X 10-5

Figure 8: Spasmolytic activity of alpha amyrin acetate

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Antibacterial activity (contd.)

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

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Antifungal activity (contd.)

Figure 10: Antifungal activity of crude extract and various

Fractions of Tylophora hirsuta.

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(contd.) Antileishmanial activity.

0

20

40

60

80

100

120

IC 5

0 v

alu

es

(µ

g/m

l )

Figure 11: Antileishmanial activity of crude and fractions of Tylophora hirsuta against Leishmania major.

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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)

% s

hrim

p ki

lled

vs

cont

rol

Cotrol Cruden-Hexane ChloroformEthyl acetate n-ButanolAqueos

Etoposide (7.462 µg/ml) was used as positive control and the number of survivors were measured .

EC50

Figure 14: Brine shrimp lethality assay of crude extract and various fractions of Tylophora hirsuta.

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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

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Contd.

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 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 AhmadDirector

Centre of Biotechnology and MicrobiologyUniversity of Peshawar.

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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

FlavonoidsThe 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

C2

3

45

6

7

89

10

1´

2´

3´

4´

5´

6´

Flavonoids

O

O

O

OH

O

OHO

O

O

O

O

O

O

O

OH

O

O

O

O

OCH3

O

O

OH O

OO

HO

O

O

O

O

H

O

O

H

OH O

OO

OO

H Flavanone

12

34

5

6

7

89

10

1'

2'

3'

4'

5'

6'

Flavan-3-ol

Flavanonol

Anthocynanidin

Flavone

Isoflavanone

Flavonol

Isoflavone

3-Methoxyflavone

Isoflavan

Flavan

1

2

3

4

5

6

1'

2'

3'

4'

5'

6'

Chalcone

' '-Chalcanol '-Chalcanone

-Chalcanone -Chalcanol Chalcan-1,3-dione

1 2

34

5

6

7

89

10 1'

2'

3'4'

5'

6'

Aurone Aurononol Auronol

Isoaurone

types