بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم وقل اعملوا فسيرى الله عملكم ورسوله والمؤمنين صدق الله العظيم

2. Faculty of Science Al Azhar University

3. عرض ملخص لرسالة الماجستير فى العلوم المقدمة من الطالب / سيد عبد الفتاح محمد تحت عنوان: دراسات بيئية وفيتوكيميائية على نبات صفراء (إيزاتس ميكروكاربا) Ecological and Phytochemical Studies On Isatis microcarpa J.Gay ex Boiss.

4. تحت إشراف كل من: أستاذ دكتور/نادى أحمد البسيونى غانم أستاذ البيئة والنباتات اللازهرية بكلية العلوم – جامعة الأزهر أستاذ دكتور/ عادل كامل يوسف أستاذ البيئة وكيمياء النبات بمركز بحوث الصحراء دكتور/شلبية شحات امام أستاذ كيمياء النبات المساعد بمركز بحوث الصحراء

5. تمت الدراسة بمنطقة وادي أم شيحان بأبو عجيلة- محافظة شمال سيناء. • وتعتبر منطقة شمال سيناء من أغنى مناطق جمهورية مصر العربيه من حيث إنتشار النباتات الطبيه الطبيعيه.

6. 1 PREFACE The Egyptian desert is very rich with medicinal plants belonging to many families. Cruciferae is represented in Egypt by 53 genera and 107 species (Täckholm, 1974 and Boulos, 1999). It is mostly annual, biennial or perennial herbs (Boulos, 1999). It resembled one of the largest families in the plant kingdom that is rich in medicinal plants, it iscomposed internationally of 375 genera comprising about 3200 species (Trease and Evans, 1989).

7. 2 Crucifereainclude many important plants that are grown asornamentals, which resembled about 50 genera, e.g.Alyssum, Arabis, Erysimum, Hesperis, Iberis, Lobularia, Lunaria, Malcolmiaand Matthiola, (James, 1995).

8. 3 • The most common genera, which present in Egypt, areAnastatica, Arabidopsis, Arabis, Alyssum, Brassica, Capsella, Isatis, Diplotaxis,Farsetia,Neotorularia, Malcolmia, Lepidium, Zilla, MatthiolaandSisymbrium(Täckholm, 1974 and Boulos, 1999).

9. 4 The plants of this family are used asantibacterial, antifungal, anticancer, and insecticidal (Rizk, 1986). Leaves of Isatis tinctoria contain a blue dye called woad. While seeds contain a fatty oil. Plant used for ulcers. The roots of Lepidium sativum L. are bitter and acrid, and are useful in secondary syphilis and tenesmus.

10. 5 • Isatis microcarpais annual, somewhat glaucous, glabrous or almost glabrous, up to 40 cm. Stems are fairly erect, and branched. Flowers are yellow, extended from March to April (Zohary, 1966). • The recent review of literature as far as I know has no previous chemical investigation onIsatis microcarpa.

11. 6 The present study aimed to throw some light upon the condition under which Isatis microcarpa can survive in its natural habitat and its main chemical constituents as a wild plant. Aim of the Thesis

12. 7 This studies includes: • 1- Ecological condition under which the plant was survived at North Sinai. • 2- Phytochemical studies to investigate the active materials, which presented in the plant. • 3- Microbiological activities against bacteria and fungi to determine the plant activities and its biological importance.

13. 8 Results and Discussion 1. Ecological Studies It includes the range of ecological conditions, under which Isatis microcarpacan grow and adapt themselves at Wadi Om Sheihan, Abo-Ogeila, North Sinai, where Isatis microcarpa is dominates and flourishes.

14. 9 Isatis microcarpa Leaves & Flowers

15. 10 Ecological Studies 1.Climatic Factors2. Edaphic Factors3. Vegetation Analysis

16. 11 Climatic particulars of El-Arish

17. 12 ٍSoil analysis

18. 13 soil moisture content

19. 14 Chemical Composition of the soil

20. 15 Vegetation analysis

21. 16 Vegetation analysis • The analysis of the plant transect of Wadi Om Sheihan, habitat revealed that most of the present species were perennial. • The first dominant species was Isatis microcarpa, while Asphodelus microcarpus was the second dominant species. • 3. Zilla spinosa,Euphorbia retusa, Achillea santolina, Thymelaea hirsute, Verbascum eremobiumandScrophularia deserti were among the important associate species in the community.

22. 17 __ _____________ ______

23. 18 Plant water content

24. 19 Total ash, organic matter, acid soluble &insoluble ash, water ash and crude fibres.

25. 20 Metabolic Product 1. Carbohydrates Content

26. 21 Total nitrogen and total protein

27. 22 Total lipids

28. 23 Phytochemical Studies

29. 24 Test Observation Volatile oil Coumarins Tannins Sterol and / or terpens Flavonoids Alkaloids Glycosides and / or carbohydrates Saponins Resins - ve + ve + ve +ve + ve trace + ve -ve +ve 1. Preliminary phytochemical screening

30. 25 Free sugars

31. 26 ___ ___ _______ _____ _____

33. 28 G L C of hydrocarbons and sterols

34. 29 G L C of fatty acids

35. 30 Investigation of Flavonoids

36. 31 Conc. 70% ethanolic extract  BAW (4:1:5), for 1st way AcOH 15% for 2nd one, Examined under UV light Exposed to ammonia and re-examined Revealed the presence of phenolic constituents subjected to TDPC

37. 32 Conc. 70% ethanolic extractfractionated on polyamide columnFivemain fractionsSubjected to PC8 different bands(1 – 8)were detected

38. 33 Isolation and Identification of Band (2) • Eluted with methanol/H2O, • Purified on a sephadex LH-20 column • Using methanol/H2O • Subjected to TDPC using • Solvent systems BAW 1st way • and AcOH 15% 2nd way • Revealed one major spot • Acid hydrolysis gave aglycone part, • Beside glycogene part glucose • Confirmed that aglycone partwas linked • To glucose

39. 34 Determination of the complete chemical structure of the compound was achieved through Rf- values, UV analysis, and 1H-NMR spectrum in DMSO, which concluded that the compound S2 is

40. Rf - values of compound (S2) and color reactions.

41. 35 UV spectrum of flavonoid compound (S2) 1H-NMR spectrum of flavonoid compound (S2)

42. 36

43. 37 Flavonoids are important dietary compounds, having a capacity to inhibit DNA damage and lipid peroxidation, They also have anticarcinogenic and antiproliferative effects (Romanova and Vachalkova, 1999). Some flavonoids are particularly beneficial, acting as antioxidants and giving protection against cardiovascular disease and certain forms of cancer. (Dewick, 1998). 1. Naringenin is recognized as phyto-estrogen which could affect sex hormone, cause mediate biological response by several different mechanisms, including binding to estrogen receptors. Also Naringenin could be used as antioxidant, anticarcinogenic and blood lowering lipid activities (Brev et al., 2004).

44. 38 2. Kaempferol can be used as antioxidant. 3. Apigenin plays a role in the prevention of carcinogensis, inhibition of the proliferation, and to a lesser degree, the migration of endothelial cells, and capillary formation in vitro, (Habtemariam, 1997; Romanova and Vachalkova, 1999). 4. Luteolin is the active ingredient in lutimax, which helps to reduce oxidative stress, reduce inflammation, regulate hyperactive immune systems, inhibit cancer cell growth and promote healthy carbohydrate metabolism and antioxidant properties as illustrated in the pamphlet of Lutimax, which is fundamental to vitality luteolin compound

45. 39 Percentages of total flavonoids

46. 40 Percentages of total tannins

47. 41 Microbiological activities

48. 42 Biological activity conclutions Anti-bacterial activity: It was observed from the obtained results that ethyl alcohol (96% and 70%) and water extracts of Isatis microcarpa showed the best effect on all tested bacteria with different dilutions (250, 500, 1000 and 2000 ppm). On the other hand, it was found that ether and acetone extracts of Isatis microcarpa had a significant effect on all the tested bacteria with different dilutions (250, 500, 1000 and 2000 ppm) except acetone extract (250 ppm) which produced no effect on Bacillus subtilis. Meanwhile chloroform and petroleum ether extracts showed the lowest effect on most of the tested bacteria at different dilutions.

49. 43 Anti- fungal activity: The obtained results showed that extracts of ethyl alcohol (96% and 70%) and water followed by ethyl acetate produced the best effect on all the tested fungi in different dilutions (250, 500, 1000 and 2000 ppm) of Isatis microcarpa. On the other hand, petroleum ether and chloroform extracts in different dilutions (250, 500, 1000 and 2000 ppm) produced the lowest effect on Aspergillus niger, Aspergillus flavus, Fusarium oxysporium, Fusarium solani, Alternaria tennius and microsporum fulvum.

50. 44 All fungi tested were causes skin infection and in case of immune deficit patient there were causes fungal disease e.g: pulmonary infection in human. Meanwhile the Asperigllus nigerand Asperigllus flavus were causes rutten of citrus fruits, while Fusarium oxysporium and Fusarium solaniwere causes rutten of roots and wiltering of plant, while Alternaria tenniuswas cause dry rutten in tomato fruit and bloch of this leaves,on other hand the 70% ethyl alcohol extract cause high effect on fungi tested, so the plant extract may be uses in the treatment of skin infection and may be used anti-fungal.