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3. Bioactivity-guided fractionation of the stem bark extract of PterocarpusdalbergiodedRoxb. Ex Dc growing in Egypt Prof. Dr. Camilia George Faculty of Pharmacy Cairo University camilia.michel@gmail.com

4. Introduction • Economically the Leguminosae (Fabaceae) is one of the most important families of the flowering plants. • Pterocarpus is a genus representing some of the most handsome large-crowned trees of the leguminous family. • Genus Pterocarpusincludes about 60-70 species.

5. Pterocarpusdalbergioides (Roxb) Flowering tree in summer

6. Pterocarpusdalbergioides (Roxb) is native to the Andaman islands in the Indian ocean.

7. It has been introduced to Egypt as an ornamental plant. • Pterocarpusdalbergioides (Roxb) is known as: Andaman padauk East Indian mahogany Andaman redwood Padauk.

8. Taxonomy

9. Stem bark Inner surface of the stem bark Outer surface of the stem bark

10. Leaf and flower Axillary panicle Compound leaf Flowering branch Flower

11. Fruiting branch Fruits Fresh Dry Seeds

12. Aim of work • Despite of the economical importance of Pterocarpusdalbergioides (Roxb) as a timber, only one singlereport was traced demonstrating the antihyperglycemic effect of the alcohol extract of the heart woodof the plant and nothing was dealt with its anti-inflammatory activity.

13. Previous research has established the genus Pterocarpus to be a rich source of polyphenolic compounds but nothing was reported concerning the chemical investigation of the studied plant excepting only one singlereport concerning the occurrence of Maackiain and 3'-hydroxy formononetin in its wood.

14. Therefore, it was deemed of interest to carry out a bioactivity-guided fractionation study on the different organs of the Egyptian plant, to choose the most active organ, which was not previously investigated.

15. Bioactivity-guided screening • A bioactivity-guided screening of the different organs: • Stem bark • Flower • Fruit • Leaf For both antihyperglycemic and anti-inflammatory activies were carried on

16. Antihyperglycemic Activity Effectof single oral administration of the alcoholic extracts of different organs of Pterocarpusdalbergioides (Roxb) on blood glucose level in Alloxan-induced diabetic rats (acute effect).

17. Antihyperglycemic Activity 0 Effect of repeated oral administration of the alcoholic extracts of different organs ofPterocarpusdalbergioides (Roxb) on blood glucose level in alloxan-induced diabetic rats (long-term effect).

18. Antiinflammatory Activity Indomethacin Acute anti-inflammatory effect of the alcoholic extracts ofdifferent organs ofPterocarpusdalbergioides (Roxb) on carrageenan-induced rat paw oedema (n=6).

19. From the previous preliminary Pharmacological screening, one could conclude that the alcoholic extracts of the bark at a dose of 200 mg/kg b.wt. was the most potent extract as antihyperglycemic and anti-inflammatory.

20. Median Lethal dose Median lethal dose (LD50) of the alcoholic extracts ofPterocarpusdalbergioides (Roxb) of the bark’s alcoholic extract was found to be 6.9 mg/Kg.b.wt.

21. Phytochemical screening

22. Fractionation of the butanol extract Were carried on and the obtained ethyl acetate and butanol extracts of the were furhtermore subjected to both antihyperglycemic and anti-inflammatory investigation.

23. Antihyperglycemic activity of the ethyl acetate and butanol extracts of the bark, flowers and fruits of Pterocarpusdalbergioides (Roxb) Effect of single oral administration of ethyl acetate and butanol extracts of different organs ofPterocarpusdalbergioides (Roxb) on blood glucose level in alloxan-induced diabetic rats (acute effect).

24. Anti-inflammatory activity of the ethyl acetate and butanol extracts of the bark, flowers and fruits of Pterocarpusdalbergioides (Roxb) Indomethacin Acute anti-inflammatory effect of the ethyl acetate and butanol extracts of different organs ofPterocarpusdalbergioides (Roxb) on carrageenan-induced rat paw oedema (n=6).

25. TLC investigation revealed that the butanol extract of the bark showed more spots compared to its corresponding ethyl acetate extract.

26. Fractionation of the butanol extract of the stem bark of Pterocarpusdalbergioides (Roxb) 10g butanol extract VLC Chloroform Chloroform: ethyl acetate Ethyl acetate : methanol Fractions Each 100 ml TLC monitoring Fraction VI (45-56) CHCl3: EtOAc 25:75 EtOAc 440 mg Fraction I (1-10) Fraction III (19-26) Fraction IV (27-36) Fraction II (11-18) CHCl3: EtOAc 97:3 85:14 780 mg Fraction V (37-44) CHCl3: EtOAc 45:55 30:70 330 mg

27. Pulled fractions (I-VI) Were screened for Antihyperglycemic activity Anti-inflammatory activity

28. Effect of single oral administration of fractions of butanol extract of the bark ofPterocarpusdalbergioides (Roxb) on blood glucose level in alloxan-induced diabetic rats (acute effect). Antihyperglycemic Activity

29. Effect of repeated oral administration of fractions of butanol extract of the bark ofPterocarpusdalbergioides (Roxb) on blood glucose level in alloxan-induced diabetic rats (long-term effect). Antihyperglycemic Activity

30. Acute anti-inflammatory effect of the alcoholic extracts ofdifferent organs ofPterocarpusdalbergioides (Roxb) on carrageenan-induced rat paw oedema (n=6). Anti-inflammatory Activity

31. Fractionation of the butanol extract of the stem bark of Pterocarpusdalbergioides (Roxb) 10g butanol extract VLC Chloroform Chloroform: ethyl acetate Ethyl acetate : methanol Fractions Each 100 ml Pulled together according to similarity Fraction VI (45-56) Active Fraction I (1-10) Fraction III (19-26) Fraction IV (27-36) Fraction II (11-18) Active Fraction V (37-44) Active CC-Sephadex LH –20 Methanol Methanol :water Increasing polarity Subfractions Further CC –Sephadex LH-20 P2 19 mg P1 23 mg F1 20 mg

32. Identificationandcharacterizationoftheisolatedcompounds Data of compound P1

33. M+- H2O M+ EI-mass spectrum of compound P1

34. 2 3 1 4 6 5 Gentisic acid H6 H3 H4 H3 H6 H4 1H-NMR spectrum of compound P1 (DMSO)

35. 2 3 1 4 6 5 Gentisic acid C6 C2 C5 C4 C3 COOH C1 13C-NMR spectrum of compound P1 (DMSO)

36. 2 3 1 4 6 5 Compound P1 = Gentisic acid On the basis of the available literature, this is the first report for isolation of gentisic acid from genus Pterocarpus.

37. Data of compound P2

38. 3 2 4 1 5 6 Gallic acid H2, H6 OH (3, 4 &5) 1H-NMR spectrum of compound P2 (DMSO)

39. 3 2 4 1 5 6 C3, C5 C2, C6 COOH C4 Gallic acid C1 13C-NMR spectrum of compound P2 (DMSO)

40. 3 2 4 1 5 6 Compound P2 = Gallic acid Gallic acid isolated for the first time from genus Pterocarpus.

41. Data of compound F1

42. H2 H3', 5' H2', 6' H8 H1’’ H6 1H-NMR spectrum of compound F1 (DMSO)

43. C2', 6' C3', 5' C3'' C5'' C8 C2'' C9 C1’ C4 C3 C2 C10 C7 C4' C5 C1' C6'' C4'' C6 13C-NMR spectrum of compound F1 (DMSO)

44. Compound F1 = Genistin This report is the first to deal with the isolation of genistin from genus Pterocarpus.

45. CorrelationbetweenthebiologicalactivitiesandtheisolatedconstituentsofPterocarpusdalbergioides(Roxb)CorrelationbetweenthebiologicalactivitiesandtheisolatedconstituentsofPterocarpusdalbergioides(Roxb) Based on these observations, the antihyperglycemic and anti-inflammatory activities of the alcoholic and butanol extracts and their active fractions of the bark of Pterocarpusdalbergioides (Roxb) could be attributed mainly to their phenolic constituents. As most of the isolated compounds were reported to possess such activities.

46. A. Antihyperglycemic action • Genistin increased basalinsulin secretion and inhibits glucose uptake into rabbit intestinal brush border membrane vesicles which may help to reduce postprandial hyperglycemia (1). • Gallic acid showed alpha-glucosidase, glycogen phosphorylase inhibitory activities and enhance insulin receptor sensitivity(2). (1)Vedavanam, K.; Srijayanta, S.; O' Reilly, J.; Raman, A.; Wiseman, H.; "Phytother Res", 13(7), 601-608,1999. (2) Hung, T.H.W.; Peng, G.; Kota, B.P.; "Toxicology and Applied Pharmacology", 207(2), 160-169, 2005.

47. B- Anti-inflammatory action • Gentisic acid inhibited both the cyclooxygenase and 12-lipoxygenase enzymes and decreased the production of the leukotrienes C4 on the stomach wall during the gastric lesion induced by ethanol. Furthermore, it has been shown to be a more inhibitor of prostaglandin synthetase activity than salicylic acid in-vitro(5). • Gallic acidbinding and antagonizing P-selection (an adhesion molecule that is critically involved in the attachment of inflammatory cells to the vessel wall)(6). • (5) Radomski, M.; Michalska, Z.; Marcinkiewicz, E.; Gryglewski, R. J.; "Pharmacological Research Communications", 18, 1015-1030, 1986. • (6) Appeldoorn, C. C.; Bonnefoy, A.; Lutters, B. C.; Daenens, K.; van Berkel, T. J.; Hoylaerts, M. F.; Biessen, E. A.; "Circulation", 111(1), 106-112, 2005.

48. Conclusion • The ethanolic extract of the bark possessed the most potent antihyperglycemic activity. This effect could be attributed to the synergistic action of the isoflavone and phenolic acid content. • The result of the anti-inflammatory activity showed promising evidence for the anti-inflammatory effect of the alcoholic extract of the bark which can be attributed mainly to the phenolic acid content.

49. Thus, it is hoped to be used as supplement in antidiabetic dosage forms and to formulate new and more potent anti-inflammatory drugs of natural origin.

50. Recommendations • From the previous findings, propagation of the plant should be encouraged due to its promising biological activities in addition to their well known economical applications. • Clinical trials should be performed in order to support all the above investigations and to facilitate their pharmaceutical formulations.