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الطالب / من الفتاح المقدمة عبد سيد محمد

: بيئية دراسات عنوان تحتنبات على صفراء وفيتوكيميائية

) ميكروكاربا) إيزاتسEcological and Phytochemical Studies On

Isatis microcarpa J.Gay ex Boiss.

Ecological and Phytochemical Studies On Isatis microcarpa J.Gay ex Boiss.

تحت إشراف كل من:

دكتور غانم /أستاذ البسيونى أحمد نادى

جامعة – البيئة أستاذ العلوم بكلية الالزهرية والنباتاتاألزهر

دكتور يوسف /أستاذ كامل عادل

الصحراء بحوث بمركز النبات وكيمياء البيئة أستاذ

امام /دكتور شحات شلبية

النبات كيمياء الصحراء المساعد أستاذ بحوث بمركز

شيحان بمنطقة تمت الدراسة أم واديعجيلة .-بأبو سيناء شمال محافظة

يناءوLمال سLة شLبر منطقLنى تعتLمن أغ L ه منLLربيLالع LرLLة مصLLمهوريLاطق جLLمن حيث إنتشLار LالنباتLات LالطبيLه اLلطLبيعيه.

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 is composed internationally of 375 genera comprising about 3200 species (Trease and Evans, 1989).

Cruciferea include many important plants that are grown as ornamentals, which resembled about 50 genera, e.g.Alyssum, Arabis, Erysimum, Hesperis, Iberis, Lobularia, Lunaria, Malcolmia and Matthiola, (James, 1995).

The most common genera, which present in Egypt, are Anastatica, Arabidopsis, Arabis, Alyssum, Brassica, Capsella, Isatis, Diplotaxis,Farsetia,Neotorularia, Malcolmia, Lepidium, Zilla, Matthiola and Sisymbrium (Täckholm, 1974 and Boulos, 1999).

The plants of this family are used as antibacterial, 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.

Isatis microcarpa is 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 on Isatis microcarpa.

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.

• 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.

• 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.

1 .Ecological Studies It includes the range of ecological

conditions, under whichIsatis microcarpa can grow and adapt themselves at Wadi Om Sheihan, Abo-Ogeila, North Sinai, where Isatismicrocarpais dominates and flourishes .

1 .Ecological StudiesIt includes the range of ecological

conditions, under which Isatis microcarpa can grow and adapt themselves at Wadi Om Sheihan, Abo-Ogeila, North Sinai, where Isatis microcarpa is dominates and flourishes.

1. Climatic Factors 2. Edaphic Factors 3. Vegetation Analysis

TemperatureR.H.)%(

Evap)mm/day(

W.S.Km/hour

R.F)mm(Mean

Max.MeanMin.

MonthlyMean

January February March April May June July August SeptemberOctober November December

21.319.420.126.330.731.132.732.728.125.825.321.1

9.07.68.6

12.316.318.320.921.316.316.412.78.6

15.213.514.419.323.524.726.827.022.221.119.014.9

68.759.767.362.366.771.770.373.367.072.072.766.7

11.08.6

10.812.918.022.724.426.018.619.815.810.6

4.49.17.16.34.33.33.82.53.32.93.65.0

59.613.067.23.40.1

0.000.000.000.000.075.4

Total annual rainfall 168.5

Soildepth)cm(

Soil texture

Granuleometric analysis of soil fraction mm%

Finegravel2.0-1.0

Coarsesand

1.0-0.5

Medium

sand0.5-0.25

Fine sand

0.25-0.125

very fine sand

0.125- 0.063

Silt & clay

< 0.063

0-20 Sandy 9.40 11.79 13.21 27.11 11.88 26.49

20-40 Sandy 15.36 17.55 14.84 22.61 19.36 10.25

Sample depth)cm(

Soil moisture content %

March April May

0-2020-40

2.523.45

1.802.82

0.951.67

Soil depth)cm(

ds/m)1:2:5(

Analysis of the soil saturation extract

Soluble cations )me/L(

Soluble anions )me/L(

Na+ K+ Ca++ Mg++ CO3-- HCO3

- Cl- SO4--

0-2020-40

8.208.42

0.390.96

0.913.10

0.361.69

2.003.00

1.001.00

0.000.00

2.001.50

2.004.00

0.273.29

1.1. The analysis of the plant transect of Wadi Om The analysis of the plant transect of Wadi Om Sheihan, habitat revealed that most of the Sheihan, habitat revealed that most of the present species were perennial. present species were perennial.

2.2. The first dominant species was The first dominant species was Isatis Isatis microcarpamicrocarpa, while , while Asphodelus microcarpusAsphodelus microcarpus was was the second dominant species.the second dominant species.

3. 3. Zilla spinosaZilla spinosa ,, Euphorbia retusaEuphorbia retusa,, Achillea Achillea santolinasantolina,, Thymelaea hirsute Thymelaea hirsute,, Verbascum Verbascum eremobiumeremobium andand Scrophularia deserti Scrophularia deserti were were among the important associate species in the among the important associate species in the community.community.

1.1. The analysis of the plant transect of Wadi Om The analysis of the plant transect of Wadi Om Sheihan, habitat revealed that most of the Sheihan, habitat revealed that most of the present species were perennial. present species were perennial.

2.2. The first dominant species was The first dominant species was Isatis Isatis microcarpamicrocarpa, while , while Asphodelus microcarpusAsphodelus microcarpus was was the second dominant species.the second dominant species.

3. 3. Zilla spinosaZilla spinosa ,, Euphorbia retusaEuphorbia retusa,, Achillea Achillea santolinasantolina,, Thymelaea hirsute Thymelaea hirsute,, Verbascum Verbascum eremobiumeremobium andand Scrophularia deserti Scrophularia deserti were were among the important associate species in the among the important associate species in the community.community.

Month Plant water content %

March 39.30

April 31.60

May 28.50

Month Total ash

Organic matter

Acid soluble

Acid insoluble

Water soluble

Waterinsoluble

Crude fibres

March 9.23 90.77 5.62 3.61 4.11 5.12 20.89

April 11.43 88.57 7.63 3.80 5.87 5.56 25.10

May 14.72 85.28 9.53 5.19 7.12 7.60 30.66

1 .Carbohydrates Content 1 .Carbohydrates Content Item Month Carbohydrates content

Total carbohydrates

March 2.04

April 3.58

May 1.94

Soluble carbohydrates

March 0.87

April 1.21

May 0.69

Insoluble carbohydrates

March 1.17

April 2.37

May 1.25

Item Month Percentages )%(

Total nitrogen

March 2.65

April 3.87

May 2.36

Total protein

March 16.56

April 24.19

May 14.75

Month Total lipids )%(

March 2.61

April 3.15

May 1.82

Test Test ObservationObservation

Volatile oil Coumarins TanninsSterol and / or terpensFlavonoidsAlkaloids Glycosides and / or carbohydrates SaponinsResins

- ve+ ve + ve+ve+ ve

trace + ve -ve

- ve+ ve + ve+ve+ ve

trace + ve -ve

Item Free sugars)%(

Combined sugars )%(

2.43 Xylose - 4.1

2.65 Arabinose 24.4 18.8

2.75 Ribose 22.7 14.5

3.40 Fructose 8.6 10.4

3.65 Glucose 37.5 22.4

6.90 Sucrose 6.1 8.9

7.35 Cellobiose - 8.2

Amino acids R.T. Free a. a% . Protein a. a% .

Aspartic acid 11.03 - 1.3

Therionine 14.42 6.2 1.9

Serine 15.69 - 2.2

Glutamic acid 16.90 25.5 18.4

Glycine 24.89 5.6 1.6

Alanine 26.11 - 2.7

Valine 29.95 - 13.6

Isoleucine 36.08 6.8 0.9

Argenine 62.64 14.8 1.6

Phenylalanine 44.25 7.6 -

Lysine 54.94 3.6 -

Leucine 37.31 8.4 15.7

Tyrosine 33.54 - 2.03

Cystiene 27.56 - 1.4

Methionene 28.06 2.7 1.05

Proline 18.25 18.06 20.5

Histidine 52.07 - 14.8

Item No. of Carbon atom

Hydrocarbons and sterols)%(

Dodecane 12 0.5

Tetradecane 13 7.5

Hexadecane 16 0.1

Heptadecane 17 9.0

Octadecane 18 12.6

Eicosane 20 17.8

Docosane 22 7.6

Tricosane 23 22.1

Octacosane 28 3.3

Squalene 29 5.3

Cholestrol 27 5.8

Stigmasterol 27 4.9

Fatty acids No. of Carbon atom

Fatty acids)%(

Caprylic 8 1.6

Capric 10 0.1

Lauric 12 0.4

Myristic 14 2.1

Palmitic 16 27.2

Stearic 18 : 0 5.3

Linoleic 18 : 2 8.4

Linolenic 18 : 3 19.4

Arachidic 20 34.6

Conc. 70% ethanolic extract

BAW )4:1:5(, for 1st wayAcOH 15% for 2nd one,

Examined under UV lightExposed to ammonia and re-examined

Revealed the presence of phenolic

constituents

BAW )4:1:5(, for 1st wayAcOH 15% for 2nd one,

Examined under UV lightExposed to ammonia and re-examined

Revealed the presence of phenolic

constituents

subjected to TDPCsubjected to TDPC

fractionated on polyamide column

Five main fractions

Subjected to PC 8 different bands

(1 – 8) were detected

fractionated on polyamide column

Five main fractions

Subjected to PC 8 different bands

(1 – 8) were detected

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 part was linked To glucose

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

Solvents Rf -

values

Colors

Visible UV UV+ammonia

BAW 0.61 - Deep purple

Yellowish green

AcOH 15%

0.23 - Deep purple

Yellowish green

Rf - values of compound )SRf - values of compound )S22( and color reactions.( and color reactions.Rf - values of compound )SRf - values of compound )S22( and color reactions.( and color reactions.

UV spectrum of flavonoid compound )S2( UV spectrum of flavonoid compound )S2(

1H-NMR spectrum of flavonoid compound )S2(1H-NMR spectrum of flavonoid compound )S2(

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

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

Seasons Total flavonoids)%(

Stem Leaves Flowers

March 0.87 1.05 1.17

April 1.00 1.12 1.30

May 1.95 2.21 2.64

Seasons Total tannins)%(

Stem Leaf Flower

March 2.17 3.61 1.08

April 2.81 3.95 1.08

May 3.26 4.57 1.95

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.

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.

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.

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.

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 niger and Asperigllus flavus were causes rutten of citrus fruits, while Fusarium oxysporium and Fusarium solani were causes rutten of roots and wiltering of plant, while Alternaria tennius was 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.

دكتور غانم /أستاذ البسيونى أحمد نادىالعلوم البيئة أستاذ بكلية الالزهرية والنباتات

األزهرجامعة–

دكتور غانم /أستاذ البسيونى أحمد نادىالعلوم البيئة أستاذ بكلية الالزهرية والنباتات

األزهرجامعة– دكتور يوسف /أستاذ كامل عادل

بحوث بمركز النبات وكيمياء البيئة أستاذالصحراء

دكتور يوسف /أستاذ كامل عادل

بحوث بمركز النبات وكيمياء البيئة أستاذالصحراء

امام /دكتور شحات شلبيةالنبات كيمياء بحوث المساعد أستاذ بمركز

الصحراء

امام /دكتور شحات شلبيةالنبات كيمياء بحوث المساعد أستاذ بمركز

الصحراء

/ أحمد محمد أم دكتور أستاذخفاجى

/ دكتور المعطى أستاذ عبد ايناسطلبه محمد

/ دكتور البسيونى أستاذ أحمد نادىغانم

/ يوسف كامل عادل دكتور أستاذ

/ أحمد محمد أم دكتور أستاذخفاجى

/ دكتور المعطى أستاذ عبد ايناسطلبه محمد

/ دكتور البسيونى أستاذ أحمد نادىغانم

/ يوسف كامل عادل دكتور أستاذ

الدكاترة الذين تعاونوا معى فى البحثمن مركز هبه ابراهيم عبد المعطىد.

بحوث الصحراءالسادة أعضاء هيئة البحوث ومعاونيهم

بقسم النباتات الطبية بمركز بحوث الصحراء

الدكاترة الذين تعاونوا معى فى البحثمن مركز هبه ابراهيم عبد المعطىد.

بحوث الصحراءالسادة أعضاء هيئة البحوث ومعاونيهم

بقسم النباتات الطبية بمركز بحوث الصحراء

شكر خاصشكر خاص / عبدالرحمن عبدالرزاق الدكتور األستاذ

أبوسعده والميكروبيولوجى رئيس النبات قسم – األزهر جامعة العلوم كلية التى المثمر والتشجيع التسهيالت لكافة

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بسم الله الرحمن الرحيم

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