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). 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. References Olayiowola A (1984).WHO’s traditional medicine programme: Progress and Perspective. WHO Chronicle 38 (2): Karki M (2002). Medicinal and aromatic Plants Programme in Asia. Benefits and Challenges. IDRC/SARO. New Delhi, India.

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 g n-Hexane soluble fraction 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 g Suspended in Water & fractionated with n-hexane Crude extract of 100 g was reserved for biological/pharmacological screenings.

8 Isolation Crude Methanolic extract g n-hexane fraction 500 g Chloroform fraction 45.0 g Ethyl acetate fraction 20.0 g Butanol fraction 5.0 g Aqueous fraction g n-hex : EtOAc (96.5 : 3.5) Compound (1) n-hex : EtOAc (11.5:1) Compound (4) (Fractionation) (Column chromatography)

9 PHYTOCHEMICAL INVESTIGATIONS 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 ). *

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 ). Left shift in dose *

12 Calcium Channel Blocking Activity (contd.) 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 × ± × ± × ± × ± × ± × ± × ± × ± × ± × ± × ± ×10-50 EC 50 =58.4 X 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.

16 Antifungal activity (contd.) Figure 10: Antifungal activity of crude extract and various Fractions of Tylophora hirsuta.

17 (contd.) Antileishmanial activity. Figure 11: Antileishmanial activity of crude and fractions of Tylophora hirsuta against Leishmania major.

18 Brine shrimp (Artemia salina) lethality assay (Contd.) 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).

20 Contd. 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).

Flavonoids types