Al-neelain University Investigation of the Chemical ...repository.neelain.edu.sd:8080/jspui/bitstream/123456789...of Haplophyllum tuberculatum and it includes extensive chemical study

Al-neelain University

Graduate College

Investigation of the Chemical Constituents and Antioxidant Activity

of Aerial Parts of Haplophyllum tuberculatum

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

M.Sc. (in Chemistry)

By

Fadwa Hassan Atallah Ahmed

B .Sc. Honors (in Chemistry) in Al-neelain University 2014

Supervisor

Dr. Raga Satti Mohamed Abadi

Sudan

OCT. 2017

http://neelain.edu.sd/colleges/15

بسم الله الرحمن الرحيم

الآية قال تعالي:

الأرض ثم يحرج به زرعا مختلفا الوانه ثم )الم تري ان الله انزل من السماء ماء فسلكه ينابيع في

يهيج فتراه مصفرا ثم يجعله حطاما ان في ذلك لذكري لاولي الالباب(

صدق الله العظيم

الآية الزمرسورة {12}

Dedication

I dedicate this work

To my lovely parents

To my brothers and sisters

To all whom I love

With my deepest love and

Respect.

Acknowledgment

Firstly thanks to Allah

Secondly, I would like to express my special appreciation and

thanks to my supervisor

Dr: Raga Satti Mohammed Abadi who helped me in completing

my research.

Thirdly, I would like to thanks my parents, my family, my

sisters and brothers for encouraging me.

Finally, I would like to thank my friends for helped me a lot

in finishing this study.

ABSTRACT

This study was conducted to evaluate the antioxidant activity of different extracts of aerial parts

of Haplophyllum tuberculatum and it includes extensive chemical study to identify the chemical

composition of the active extract(s) by chromatographic and spectroscopic methods.

Phytochemical screening of aerial parts of H. tuberculatum alcohol extract revealed presence of

saturated and unsaturated sterols, triterpenoids, alkaloids and carbohydrates, 2-deoxy sugars,

flavonoids, tannins, coumarins and saponins. In the quantitative analysis secondary metabolites,

saponins(23.30%), flavonoids, (20.00%), terpenoids (13.33%), & alkaloids (9.45% are extracted

by using the standard chemical protocol.

The fine powder of the H. tuberculatum aerial parts was extracted with several solvents (pet-

ether, chloroform, ethanol) using two methods (maceration and soxhlet).Thin layer

chromatography of the petroleum ether (boiling point 60-80oC) extract demonstrated the

occurance of furocoumarins compounds in the plant isolated by PTLC an afforded six pure

fractions. The antioxidant activity of extracts was estimated and the results showed that

ethanol(mac) extract show high DPPH free radical scavenging activity(82.36%) at 100 μg/ml

as compared to that of ethanol (sox) (50%) at the same concentration.

The active ethanolic ) mac ( extract was further studied by intensive analytical investigations in

order to isolate the extract components using Column chromatography TLC and preparative TLC

chromatographic fractionation, an afforded fourteen pure fractions which were analyzed by

spectroscopic methods (Infra-red and Ultra-violet).

On the other hand the chemical composition of the volatile oil of Haplophyllum tuberculatum

was investigated using GC-MS technique four compounds were completely identified and the

major compounds was carvotanacetone ( 95.58%).

ستخلصالم

أيضا شملت للاكسدة, ادة ـالمض طتهاـة انشـودراسالهدف من الدراسة تقييم مدي فعالية مستخلصات المجموع الخضرى لنبات الحزا

ائى تم إجراء المسح الكيميالدراسة التفصيلية لمستخلصات النبات باستخدام المسح الكيميائي و طرق التحليل الكروماتوغرافى والطيفى.

النباتى للمستخلص الكحولى والذى أسفر عن وجود الصابونينات، التانينات, الإستيرولات، ليوكوايثوسيانينات

دات, القلويدات، الكيومارينات و القلويدات،الكاردولينالفلافونيدات، عليه تم استخلاص مركبات الايض الثانويه من المجموع .

(%23.30), الصابونينات المعروفة القياسية الطرق باستخدام الخضري للنبات وتقديرها كميا وذلك , الفلافونيدات

(20.00%) ) التربينات (13.33% .(%9.45) القلويدات

مسحوق باستخدام طريقة )النقع والأستخلاص المستمر(كما تم استخلاص , بواسطة كروماتوغرافيا أظهر مستخلص إيثر البترول

(TLC)العمود وكروماتوغرافيا الطبقة الرقيقة ظهور مركبات فيوران كومارين التى تم فصلها الي ستة مجزئات نقية بواسطة

(PTLC) كروماتوغرافيا التحضيرية جارب المعملية الحيوية لكل المستخلصاتومن ثم أجريت الت لاختبار فعاليتهما كمضادات

مستخلص الايثانولللاكسدة ، أوضحت النتائج أن المستخلصات لها فعالية ولكن أكثرها فعالية هو الذي أعطي أعلي تأثير )نقع ( )

82.36%( عند تركيز 100 μg/ml الايثانولبينما مستخلص توسطه سجل فعالية م )بالسوكسليت( (50%) عليه عند نفس التركيز

لإجراء الطرق التحليلية مثل مستخلص الايثانول بالنقع تمِ إختيار عملية التجزئه بواسطة كروماتوغرافيا العمود وكروماتوغرافيا

الحمراء والأشعة فوق ربعة عشر مجزئآ نقيآ تم تحليلها بإستخدام طيف الأشعة تحت أفصل نها التى نتج عوالتحضيرية الطبقة الرقيقة

ومن ناحية أخرى أجري البنفسجية. بالمطياف الملحق الغاز كروماتوغرافيا تقنية العطري بإستخدام الزيت مكونات لمعرفة التحليل

%كارفاتونستون عن المركبات عبارة واغلب الدراسة موضع الزيت في على أربعة مركبات التعرف و تحديد تم بحيث الكتلي

95.58.

Table of Contents

الأية

Dedication i

Acknowledgments ii

English Abstract iii

Arabic Abstract V

Table of Contents vi

List of Table xi

List of Figures xii

List of plates xiii

List of Abbreviations xiv

List of Appendices Xv

Chapter one

1 Introduction and literature review 1

1.1 General Introduction 1

1.2 Literature review 3

1.2.1 Family Rutacaeae of H. tuberculatum 3

1.2.2 The genus Ruta 3

1.2.3 Botanical description 3

1.2.4 Taxonomy 5

1.2.5 Ecology 5

1.2.6 Geographic distribution 5

1.3 Chemistry of the H. tuberculatum 6

1.3.1 The Chemical Compositions of essential oils of H.

tuberculatum

6

1.3.2 Others Compositions 8

1.4 Traditional Uses 9

1.5 Pharmacological Aspects 9

1.5.1 Anti cancer effect 9

1.5.2 Anti-HIV effect 9

1.5.3 Anti -microbial activity 10

1.5.4 Insecticidal effect 10

1.5.5 Anti plasmodial 10

1.5.6 Anti -inflammatory activity 10

1.5.7 Anti-oxidant activity 11

1.5.8 Cardiovascular affect 11

Chapter two

2.1 Materials and Methods 12

2.1.1 Plant materials 12

2.1.2 Instrumentation 12

2.1.3 Chemicals and reagent 12

2.1.3.1 Chemicals 12

2.1.3.2 Reagents 13

2.1.4 Chromatographic materials 14

2.1.4.1 Solvents 14

2.1.4.2 Adsorbents 14

2.1.4.3 Mobile phases 14

2.1.4.4 Detection 15

2.2 Methods 15

2.2.1. Extraction methods 15

2.2.1.1 Preparation of ethanol extracts 15

2.2.1.2 Preparation of water extracts 15

2.2.1.3 Preparation of hexane(mac) extracts 15

2.2.1.4 Preparation of furocuomarins extracts 16

2.2.1.5 Successive extraction 16

2.2.2 Phytochemical Screening of extracts 16

2.3 Anti-oxidant method 16

2.4 Quantitative determinations of the chemical constituency 17

2.4.1 Alkaloids content 17

2.4.2 Saponins content 17

2.4.3 Flavonoids content 17

2.4.4 Terpenes content 18

2.5 Chromatographic methods 18

2.5.1 Examination of extracts using TLC 18

2.5.1.1 Spotting, development and visualization 18

2.5.1.2 Column Chromatography 18

2.5.1.3 Preparative Thin Layer Chromatography (PTLC) 19

2.6 Spectroscopic methods 19

2.6.1 GC-MS Parameters 19

Chapter three

3 Results and Discussion 21

3.1 Extraction and phytochemical screening of H. tuberculatum

3.2 Bioassay of Crude Extracts 23

3.3 Thin-layer Chromatography of Ethanolic Extract 27

3.4 Characterization and Analysis of isolated fractions 34

Chapter four

4 Conclusion and Recommendations 38

4.1 Conclusion 38

4.2 Recommendations 39

References

List of Tables

NO. Name of the table Page

1.1 Classification of H. tuberculatum 5

2.1 Oven temperature program 20

3.1 Percentage Yields and Properties of Different Solvents using

various methods

21

3.2 General Phytochemical Screening of H. tuberculatum Extract 22

3.3 Quantitative analysis of flavonoids, terpenes, saponins and

alkaloids

23

3.4 Evaluation of Anti-oxidant activity of ethanol

(mac),ethanol(sox), hexane(mac) of H.tuberculatum

24

3.5 GC-MS analysis of essential oil 25

3.6 TLC fractions of ethanol(mac) 28

3.7 TLC fractions of petroleum ether extract 29

3.8 TLC fractions of hexane(mac) 30

3.9 TLC fractions of furocumarins extract 31

3.10 TLC wet column fractions of ethanol(mac) extract 33

3.11 IR spectra of isolated compounds 35

3.12 IR spectra of furocumarins 36

3.13 The maximum absorption of isolated fractions 37

3.14 The maximum absorption of the isolated furocumarins 37

List of Figures

NO. Subjects Page

1.1 Leaves and flowers of H. tuberculatum 4

1.2 Distribution of H. tuberculatum 6

3.1 Anti – oxidant activity of extraxts 24

3.2 Column chromatography 34

List of Plates

NO. Name of plate Page

3.1 Plates of ethanol(mac) extract 29

3.2 Plates of petroleum ether extract 30

3.3 Plates of hexane(mac) extract 31

3.4 Plates of furocumarins 32

3.5 Plates of column extract 34

List of Abbreviations

Abbreviation Meaning and concept

Mac Maceration

Sox Soxhlet

+++ Strong

++ Medium

+ Weak

- Negative

IR Infra-red

UV Ultra violat

Rf Retardation Factor

N E Nake Eye

PTLC Preparative Thin Layer Chromatography

TLC Preparative Thin Layer Chromatography

DPPH

2,2-diphenyl-1-picrylhydrazyl

List of Appendices

No Name of Appendix

1 IR Spectrum of fraction (1)

2 UV Spectrum of fraction (1)










12 UV Spectrum offraction(1)





17 IR Spectrum offraction(4)



20 UV Spectrum of fraction(5)



1. Introduction and literature review

1.1 General introduction:

Haplophyllum tuberculatum is a herb indigenous to the northern part of Sudan where it is known

locally El-HAZA. The herb is utilized in Sudan traditionally for several indications including its use

as antispasmodic anti flatulent and to treat allergic rhinitis. (1)

A decoction of the leaves and stems is externally applied for ear and eye problems, to relieve

toothache and pustules on the head.)1( A decoction of the aerial parts is taken as a carminative and as

a decongestant. A leafy steam extract is rubbed onto the skin to protect livestock from biting insects

and flies. In Egypt the flowering aerial parts of H. tuberculatum are used as a drink to relieve fever,

for abdominal upset, anemia, gastric pains, intestinal worms, malaria, as aphrodisiac, and the

decoction is used for rheumatic pains.(3) while in Sudan the herb is used as an antispasmodic, to treat

allergic rhinitis and breathing difficulties.(1)

In Oman, the leaves are used as a remedy for headaches and arthritis and also used for treatment of

skin infections, discoloration and parasitic diseases, In Saudi Arabia, H. tuberculatum is used to treat

malaria, rheumatoid arthritis and gynecological disorders.(3)

Total extracts of the aerial parts showed significant in vitro cytotoxicity against a range of tumors cell

lines and normal peripheral blood mononuclear cells (PBMCs). Extracts from the aerial parts showed

slight hepatoprotective effect on paracetamol-induced liver damage in mice. (1)

The ethanol extract of H. tuberculatum aerial parts, rich in phenolic compounds which was found to

be active as anti-oxidant and radical scavenger ameliorating ROS-related processes and diseases as

neurodegenerative disorders.)3(

The aqueous extract of the aerial parts of plants collected from Sudan significantly decreased the

contractility and the heart rate but did not affect the flow rate of isolated perfused rabbit heart, The

extract also showed a significant relaxation of the isolated rabbit jejunum, guinea-pig ileum, rat

uterus, rat stomach strip and rat colon, thus demonstrating its antispasmodic potential.(1)

Objectives:

1- To evaluate antioxidant activity of different extracts from H. tuberculatum.

2- To investigate the best solvent that can be give the active anti-oxidant ingredients.

3- To fractionate the active anti-oxidant extract by using chromatographic methods.

4- To analyze the isolated pure fractions using UV and IR spectroscopic techniques.

5- To analyze the extracted oil using GC-MS spectroscopy.

6-To investigate the quality and quantity of Furocoumarin derivatives of H. tuberculatum.

1.2 Literature review

1.2.1 Family Rutaceae of H. tuberculatum:

The 70 species which mainly distributed around the Mediterranean region of Europe and through

genus Haplophyllum A. Juss. Belongs to Rutaceae family and encompasses approximately western

Asia up to Siberia. (4) Eighteen species of Haplophyllum A. Juss. genus are found in Iran which among

them nine species are endemic and Haplophyllum buhsei Boiss is one of them, Plants of

Haplophyllum A. Juss. Genus have long been used in folk medicine for toothache, stomach and skin

diseases, and in the treatment of some types of cancer. The extracts of some Haplophyllum species

exhibit cytotoxic activity. (4)

1.2.2 The genus Ruta:

Ruta species are sources of diverse classes of natural products such as flavonoids, alkaloids, essential

oils, coumarins, phenols, saponins, lignans, and triterpenes, with biological activities including

antifungal, antioxidant, phototoxic, abortive, depressant, antidotal and anti-inflammatory.)5(

1.2.3 Botanical description:

Perennial herb, up to 40 cm tall, glabrous to short-hairy; stem usually much branched from the base,

yellowish green to almost white; glands numerous on all parts; and very variable. Leaves alternate,

strong smelling, variable in shape, from narrowly linear to short in size. Flowers are yellow and

variable in size. Petiole short below, absent above; blade very variable, shortly obovate, elliptical,

lanceolate or linear, sometimes deeply cut into 3 lobes. Inflorescence a lax corymbose cyme, upper

leaf axils, 2–10 (–15) cm in diameter, many-flowered, but flowers well-separated; bracts small, green.

The flowers are in loose corymbose terminal panicles, with five free ovate sepals. The stamens 10 are

filamentous and hairy. The petals are five and bright yellow in color, Flowers bisexual, 5-merous,

regular; sepals deltoid-ovate to broadly lancelet, c. 1 mm long, free; petals elliptical-oblong, 3–5.5

mm long, boat-shaped, narrowed into a claw, bright yellow, glabrous; anthers twice as many as the

petals; ovary almost round, 5-lobed, style 1.5–2.5 mm long. Fruit a 3–5-lobed capsule, 2.5–4.5 mm

× 1.5–2 mm, hairy, with a lot of inconspicuous to warty glands, segments apically opening, 5–10-

seeded. Seeds are kidney-shaped, dark brown or brownish-black, densely ridged. (6) Figure (1:1) show

the leaves and flowers of H. tuberculatum.

Figure (1.1): Leaves and Flowers of H. tuberculatum

1.2.4 Taxonomy:

Table (1.1): Shows the classification of H. tuberculatum

Class Magnolipsida

Order Sapindales

Family Rutaceae

Genus Haplophyllum

Species Tuberculatum

Common name EL-Haza

Scientific Name (s) Haplophylum tuberculatum

1.2.4 Taxonomy:

Table (1.1): Shows the classification of H. tuberculatum

Class Magnolipsida

Order Sapindales

Family Rutaceae

Genus Haplophyllum

Species Tuberculatum

Common name EL-Haza

Scientific Name (s) Haplophylum tuberculatum

1.2.5 Ecology:

H. tuberculatum occurs in sandy or stony desert or degraded steppe, on a variety of soils, often on silt

deposits, and also in dried watercourses, coastal plains, cultivated or fallow land and ruderal localities,

from sea-level up to 1330 m altitude.(5)

1.2.6 Geographic distribution:

Haplophyllum comprises 67 species in temperate and subtropical Eurasia and Northern Africa,

extending to tropical East Africa. In tropical East Africa only 3 species occur, figure (1:2) show the

distribution of H. tuberculatum.

Fig (1.2) distribution of H. tuberculatum

1.3 Chemistry of H.tuberculatum:

1.3.1 Chemical compositions of essential oil of H. tuberculatum:

The chemical components of the H.tuberculatum essential oil was analyzed by gas chromatography–

mass spectral (GC–MS) as well as 13C NMR spectroscopy. More than 30 compounds, constituting

about 99.7% of the total oil, were identified. The most abundant oil components were β-phellandrene

(23.3%) (4) , limonene (12.6%).(1), (Z)-β-ocimene (12.3%), β-caryophyllene (11.6%).(2), myrcene

(11.3%).(3), and α-phellandrene (10.9%), the major compounds of H. tuberculatumin essential oil of

the plant were cis-p-menth-2-en-1-ol as well as trans-p-menth-2-en-1-ol (22.9 and 16.1 %

respectively).(6)

In an investigation in Larestan, Iran, main components of H. tuberculatum was borneol (25.73%).

Other major compounds were α-Pinene (14%), Bornyl acetate (18.07%) and β-caryophyllene

(7.43%).(6)

The oil of this species from Oman revealed that the most abundant components were limonene

(12.6%), α-phellandrene (23.3%), (Z)-β-ocimene (12.3%), β-caryophyllene (11.6%), myrcene

(11.3%) and β-phellandrene (10.9%).(7)

The air-dried aerial parts of H. tuberculatum growing in Iran and analyzed by gas chromatography

(GC) and gas chromatography-mass spectrometry (GC/MS) was poor in essential oil (yield 0.5%).

However, thirty constituents representing 99.23% of total essential oil were identified in it. The main

constituents of the oil were found to be 1,8-cineole (38.1%), myrcene (10.69%), α-pinene (8.46%),

4-terpineol (6.96%) and sabinene (6.15%),Other representative compounds were identified as methyl

geranate (4.69%), γ-terpinen (4.3%) and α-terpinene (3.43%).(3)

The essential oil of H. tuberculatum collected from Saudi Arabia, wasobtained by hydro distillation.

The oil was subsequently analyzed by GC and GC-MS and 37compounds that were identified which

accounted for 96.4% of the composition, The major components were trans-p-menth-2-en-1-ol

(19.2%), cis-p-menth-2-en-1-ol (13.2%), myrcene(10.1%), δ-3-carene (8.8%), β-phellandrene

(6.9%))4(, limonene (6.6%) and cis-piperitol (6.4%))6(.(8)

CH2

CH3

CH3

CH3

H

CH3

CH3

CH2 H

CH3CH3

CH2

CH2

(2) (1) (3)

CH3CH3

CH2 (4 )

CH3

CH3

CH3 (5) CH3

CH3CH3

OH

(6)

1.3.2 Other compositions:

Adnan (J, et al (9)) were isolated two new alkaloids, haplotubinone and haplotubine from the aerial

parts of H. tuberculatumin addition, some known constituents: flindersine (7) , kusunokinin, β-

sitosterol, cholesterol, oleanolic acid, and hexadecanoic acid, have also been obtained.

From the aerial parts the lignans justicidin-A (8) and -B were isolated, since this plant is a member

of rutaceae family, a family reported to be rich in furocoumarin derivatives. furocoumarin derivatives

are of economic value as they are used in dermatological preparation for different skin diseases, they

have been prominent in the United States for their role in photo chemotherapy (PUVA) of vitiligo,

psoriasis, Parapsoriasis, Mycosis fungoides.(10)

Recently some other biological activities of Psoralens(9) and related compounds have been reported;

these include: having anti-inflammatory and analgesic activities, antitumor activity, during the

phytochemical investigation of H. tuberculatum, two alkaloids named haplophytin- A and B have

been obtained.(11)

O

NH

O

CH3

CH3

(7)

O

O

O

O

OH

O

OH

O

OH

O

(8 ) OO O (9)

1.4 Traditional uses:

H. tumerculatum is used in traditional medicine as a remedy for headaches and arthritis, skin

discoloration, the juice is applied as a wart removal, and against parasitic diseases and other

infections, it is also used to treat nervous system, infertility and fever, herbalists for preparations used

as carminatives for children recommend Decoctions of the plant. In the north of Oman, the juice

expressed from the leaves were used as a remedy for headaches and arthritis. (2)

In Saudi Arabia, H. tuberculatum is used to treat malaria, rheumatoid arthritis and gynecological

disorders While, in Sudan the herb is used as an antispasmodic, to treat allergic rhinitis and

gynecological disorders, asthma and breathing difficulties, It is use by women for healing after

childbirth ailments, the leafy steam infusion are taken to treat nausea, constipation, and gastric

disorders.(1)

H. tuberculatum have beneficial therapeutic potential for diseases associated with oxidative stress,

This plant have promising therapeutic option for the prevention of different neurodegenerative

diseases including Alzheimer and Parkinson’s diseases, The plant is used to cure scorpion stings. It

is usually used to strengthen the children back muscles, chest pains, flatulence, stomach problems

and has sedative effects. (2)

1.5 Pharmacological aspects:

1.5.1 Anticancer effect:

The extracts from H.tuberculatum was toxic against the seven solid cancer cell lines studied with the

highest IC50values of 31.64 mg/mL (against Hep-G2 cells). H.tuberculatum induced cell cycle arrest

in G0/G1 and S phases. H .Tuberculatum extract caused apoptosis in CCRF-CEM cells by the

alteration of the mitochondrial membrane potential. (3)

1.5.2 Anti-HIV effect:

Anti-HIV agents from natural resources are belonged to various classes including terpenoids,

coumarins, alkaloids, polyphenols, tannins and flavonoids, the buchapine, quinolonealkaloid, was

isolated from methanolic extract of the aerial part of H .tuberculatum.(3)

1.5.3 Anti-microbial activity:

The content and composition of the essential oils obtained from H. tuberculatum is very variable, and

pharmacological activity of extracts of aerial parts may therefore differ considerably. The

antimicrobial activity is very promising and deserves more attention. As the aerial parts contain a

variety of alkaloids, care should be taken by when using them internally, An essential oil originating

from Oman partially inhibited the growth of Escherichia coli, Salmonella choleraesuis, and Bacillus

subtilis, The oil also affected the mycelia growth of Curvularialunata and Fusariumoxysporum in a

dose-dependent manner but had no effect on the germination of their spores. (2)

Different extracts from the aerial parts from plants originating from Libya showed good antimicrobial

activity against Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli,

Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Candida

albicans. Saud, et al.(6) reveled that in the antifungal assay, the oil of Haplophyllum tuberculatum

showed weak fungicidal activity against Alternariaalternata, Stemphyliumsolani,Curvularialunata,

Fusariumoxysporium, and Bipolarissp, but Curvularialunataand Bipolarissp are more susceptible to

the poisoning effect of the oil at higher doses.

1.5.4 Insecticidal effect:

H. tuberculatumis used to protect livestock from biting insects and flies. The ethanol extract of the

aerial parts of Haplophyllum tuberculatum possess good insecticidal activity against

Culexquinquefasciatus. (2)

1.5.5 Anti plasmodial:

Several extracts from the aerial parts showed significant antiplasmodial activity against Plasmodium

falciparum 3D7 (chloroquine sensitive) and D2 (chloroquine resistant and pyrimethamine sensitive).

(3)

1.5.6 Anti-inflammatory activity:

(Winter et.al (12)) showed that the essential oil of the aerial parts of H. tuberculatum exhibited a

remarkable acute anti-inflammatory activity against carrageenan induced oedema in rats, when

compared to the standard drug.

1.5.7 Anti-oxidant activity:

Omer A et.al (2) showed that the ethanolic extract of the aerial parts of H. tuberculatum give significant

antioxidant activity (98%) compared to vitamin E.

1.5.8 Cardiovascular affect:

H. tuberculatum cardiovascular effect were studied by Mohamed et al.(13), who reported that its

aqueous extract significantly decreased the contractility and the heart rate, but did not affect the flow

rate of the isolated perfused rabbit heart.

2. Materials and Methods

2.1Materials:

2.1.1plant materials:

The plant H. tuberculatum (El-haza) were collected from the North part of Sudan (Dongola) in

February 2017.

2.1.2 Instrumentation:

The IR Spectra were recorded on (FT/IR8400S-Shimadzu-Japan) Fourier Transform Infrared

Spectrophotometer. The UV Spectra were recorded on (UV/1800 Spectrophotometer

Shimadzu).GC/MS technique model (GC/MS-QP2010-ultra) from japans Shimadzu company.

2.1.3Chemicals and reagents:

2.1.3.1. Chemicals:

Ethanol.

Hexane.

Chloroform.

Toluene.

Petroleum ether(60-80˚C)

Ethyl acetate.

Acetic acid.

Acetic anhydride.

Ferric chloride.

Gelatin powder.

Magnesium turning

Sodium chloride.

Potassium iodide.

Aluminum trichloride.

Hydrogen peroxide.

Sodium picrate.

Sodium hydroxide.

Ammonium hydroxide.

Iodine.

2- naphthol.

Picric acid.

2.1.3.2. Reagents:

Baljects reagent:

Solution A: 1g picric acid was dissolved in 100 ml 95% alcohol. Solution B: 19g of NaOH in 100ml

water. Solution A and B were mixed before use.

Mayer’s reagent:

Solution A: 1.36g of mercuric chloride were dissolved in 60ml of distilled water. Solution B: 5g of

potassium iodide were dissolved in 10ml of water. Solution A and B were mixed and diluted to 100

with water.

Wagner’s reagent:

1.27g of iodine and 2g of KI were dissolved in 5ml of water and the solution was diluted to 100ml

with water.

Molish reagent:

To 10g of 2- naphthol, 50 ml of ethanol were added and the volume was completed to 100 ml with

ethanol.

AlCl3solution:

1% AlCl3 w/v in water.

Ammonia test solution:

375 ml of strong ammonia were diluted to 1000ml with water.

FeCl3 solution:

5% w/v of anhydrous Ferric chloride in water.

Gelatin solution:

50 ml of water were added to 1g of gelatin and allowed to stand for one hour, the solution frequently

shaking then the water was decanted. Fresh portion of 60ml of water were added to the gelatin, and

the last was dissolved with shaking and warming to 60˚C. 10g of Nacl were then added to the resulting

solution, mixed, cooled, filtered and completed to 100 ml with water.

2NHcl:

Was prepared by diluting 129 ml of conc. Hcl to 1000 ml with water.

2.1.4Chromatographic materials:

2.1.4.1. Solvents:

Petroleum ether (60-80˚C), chloroform, ethanol, ethyl acetate, toluene, and formic acid.

2.1.4.2. Adsorbents:

Silica gel for thin layer chromatography type 60 GF254 with fluorescent indicator (BDH); U.K Silica

gel for column chromatography (Kiesel gel 69 korngrobe 100-200 mesh) were obtained from Merck

(Darmstadt, Germany). Precoated silica gel aluminum sheets. Kieselgel 60 F254 Merck. Darmstadt

Germany.

2.1.4.3. Mobile phases:

Toluene: ethyl acetate: formic acid (5:4:1)

Chloroform: Ethyl acetate (4:1)

Toluene: Ethyl acetate (7:3)

2.1.4.4. Detection:

Detection of compounds was achieved using short (254 nm) and long (365 nm) UV radiation, then

spraying with vanillin / Sulphuric acid followed by heating in an oven at 110 C for 10 minutes.

2.2Methods:

2.2.1. Extraction methods:

2.2.1.1. Preparation of ethanol extracts:

The aerial parts of H. tuberculatum were collected from the plant and washed thoroughly in distilled

water to remove the contaminants; it was chopped into small pieces and dried under shade, coarsely

powdered and separately subjected to extraction by maceration (25g plant powder in 250 ml of

ethanol). The contents of the beaker were left at room temperature for three days with frequent

shaking. The extract was filtered using a funnel. The clear solution was evaporated, and the residual

extract was dried and the% yield was calculated.

2.2.1.2. Preparation of water extracts:

The aerial parts of H. tuberculatum were collected from the plants and washed thoroughly in distilled

water to remove the contaminants, it was chopped into small pieces and dried under shade, coarsely

powdered and separately subjected to extraction by maceration (25g plant powder in 250 ml water),

the contents of the beaker were left at room temperature for three days with frequent shaking. The

extract was filtered using a funnel, the clear solution was evaporated, and the residual extract was

dried and the % yield was calculated.

2.2.1.3. Preparation of hexane (mac) extracts

The aerial parts of H. tuberculatum (21g) was transferred into beaker and a solution of hexane (150ml)

was added. The contents of beaker were left at room temperature for three days with frequent shaking.

The extract was filtered using a funnel, the clear solution was evaporated, the residue had a

characteristic pleasant odor, and thus the plant product contains volatile oils. The residual extract was

dried and the % yield was calculated.

2.2.1.4. Preparation of furocoumarins extracts

Forty grams of moderately coarse powder of H.tuberculatum (aerial parts) was extracted with (500ml)

of petroleum ether (b.p. 60-80oC) using flask fitted with a reflux condenser, heated for three hours.

The extract then filtered with suction and the marc was re extracted several times until complete

exhaustion. The extracts were combined and allowed to separate from the mother liquor.

The greenish resinous residue was dissolved in 25ml of 5% sodium hydroxide solution and partitioned

with 25ml of chloroform in a reparatory funnel and left for 15 minutes. Two layers were observed,

the upper layer (the aqueous alkali layer) and the lower layer (chloroform layer).The upper layer was

separated, neutralized with 5% Hcl solution, and extracted with chloroform. The chloroform solution

was evaporated to dryness. It was found that plant material (Marc) left after petroleum ether extraction

contained a good quantity of psoralen so it was dried thoroughly and re-extracted to be determined

quantitatively twice with 100ml. of ethanol each time for 3 hours, or until complete exhaustion. The

ethanol extracts then were combined together and evaporated to dryness under reduced pressure at a

temperature not exceeding 40°C to give orange residue.)22(

2.2.1.5. Successive extraction

50g aerial part powder was extracted successively with petroleum ether (60-80 C), chloroform, and

ethanol using the Soxhlet extractor. The solvent was carefully evaporated form each extract and the

extractability of each solvent was determined.

2.2.2. Phytochemical Screening of extracts:

The ethanol extract was tested using standard method described by Harbone.(24)

2.3. Anti-oxidant method

About 0.1ml solution of DPPH was prepared in ethanol and 0.5 ml of this solution was added to 1.5

ml of H. tuberculatum hexane(mac), ethanol(mac) and ethanol(sox) in ethanol(1mg/ml) these

solutions were vortexed thoroughly and incubated in dark . Half an hour later the absorbance was

measured at 517 nm against blank samples. And the activity was calculated by anti-oxidant

activity.(15)

2.4. Quantitative determination of the chemical constituency:

2.4.1. Alkaloids content:

About 5 gram of plant sample were weighed into 250 ml beaker and 100 ml of 10% acetic acid in

ethanol were added and the beaker was covered and allowed to stand for 4 hrs. This was filtered and

the extract was concentrated on water bath to one quarter of the original volume, concentrated

ammonium hydroxide was added drop wise to the extract until the precipitation was completed. The

whole solution was allowed to settle and the precipitate was collected and washed with dilute

ammonium hydroxide and then filtered. The residual alkaloid was dried and weighed as black oily

precipitate.

2.4.2. Saponins content:

About 5 g of the plant sample were put into a conical flask and 100 ml of 20% aqueous ethanol were

added. The sample were heated over a hot water bath for 4 hrs. with continuous stirring at about 55

C0. The mixture was filtered and the residue re extracted with another 200 ml of 20% ethanol. The

combined extracts were reduced to 40 ml over water bath about at 90 C0. The concentrate was

transferred into a 250 ml separatory funnel and 20 ml ethyl ether were added and shaken vigorously.

The aqueous layer was recovered while the ether layer was discarded. The purification process was

repeated. 60 ml of n- butanol were added and the combined n- butanol extracts drop wise with 10 ml

of 5% aqueous sodium chloride. The remaining solution was heated in water bath after evaporation,

the sample was dried in the oven and weighed to a constant weight and the saponin content was

calculated as percentage yield.

2.4.3. Flavonoid content:

About 10 g of plant sample was extracted repeatedly with 100 ml of 80% aqueous methanol at room

temperature. The whole solution was filtered. The filtrate was later transferred into crucible and

evaporated to dryness over water bath and weighed to a constant weight and the flavonoids content

was calculated as percentage yield.

2.4.4. Terpenes content:

10 g of plant sample were transferred into a beaker and it was dissolved in 100 ml chloroform. The

contents of the beaker were left at room temperature for three days with frequent shaking. The extract

was filtered under pressure and residue was dissolved in cold ethanol (95%), the produced solution

was treated by hydrous lead acetate solution (5%) (To precipitate fatty acids and phenolic

compounds). The solution was filtered and evaporated to minimum volume over water_ bath. The

concentrated solution was cooled and transferred into a 250 separatory funnel and 10 ml of

chloroform were added and shaked vigorously. The aqueous layer was discarded while the

chloroform layer was recovered. The process was repeated two times. The filtrate was collected,

dried, and weighed to constant weight and the terpenes content was calculated as percentage.

2.5. Chromatographic Methods:

2.5.1. Examination of extracts using TLC:

The ethanol (mac) was examined using thin layer chromatography.

2.5.1.1. Spotting, development and visualization:

The activated plates were placed flat on the laboratory bench and the sample was spotted carefully at

starting point 2cm from the lower edge of the plate. After evaporation of the solvent, the plate was

placed vertically on a glass tank which contained a suitable solvent to a depth of about 1.5 cm and

the chromatogram developed. The plate was removed from the jar, allowed to dry, visualized by UV-

lamp, and finally sprayed by vanillin /conc. sulphuric acid.

2.5.1.2. Column chromatography (wet packing):

A glass column (160*4cm) was well packed with (40g) silica gel (100_200 mesh) in hexane. The

ethanol extract (2g) were dissolved in ethanol and mixed with the (5g) silica gel and the ethanol was

completely evaporated. The extract was mixed with silica gel. The top of column was covered with

silica gel. Then the column was eluted with the mobile phase (Toluene: Ethyl acetate: Formic acid)

(5:4:1), about 11 fractions were collected.

2.5.1.3. Preparative Thin Layer Chromatography (PTLC):

Toluene: ethyl acetate: formic acid (5:4:1) was using as a mobile phase, plates were inspected

by day light, then examined under UV- lamp, and finally sprayed by vanillin. Retardation Factor (Rf)

values of separated fractions appeared in day light or under UV or after sprayed and heated.(26)

2.6. Spectroscopic methods:

Fractions (1 to 11) from the column were analyzed using Infra- red (IR) and Ultra- violet (UV)

spectroscopic methods. And also fractions (1, 2, 3, 4, 5 and 6) obtained from the furcoumarins extract

were analyzed using Infra- red (IR) and Ultra- violet (UV) spectroscopic methods, And the volatile

oil obtained from hexane extract was analyzed using GC-MS instrument.

2.7 GC-MS Parameters:

Detector: Mass Spectrometer.

Model Number: Shimadzu. GC-MS-QP2010 Ultra.

Company: Shimadzu.

Country: Japan.

Capillary column: RTX-5MS …Length (30 m)…Diameter (0.25 mm)…Thickness (0.25µl).

Column oven temperature: 50.0 C0.

Injection Temp: 300.00 C0.

Injection Mode: Split.

Total Flow: 50.0 ml/min.

Column Flow: 1.69 ml/min.

Purge Flow: 3.0 ml/min.

Table (2.1): Oven temperature program:

Rate Temperature(C0) Hold Time (min)

- 50.0 0.00

7.00 180 0.00

10.00 300.0 0.00

Equilibration Time: 3.0 min

Mobile Phase: Helium.

3. Results and Discussion

3.1 Extraction and Phytochemical Screening of aerial parts of H.tuberculatum

The extraction of aerial parts of H. tuberculatum performed using different solvents and various

methods) soxhlet and maceration).The lipids were completely removed by petroleum ether (60-

80°C), and the residue was then extracted with chloro formand ethanol respectively.

Table (3.1): Percentage yields and properties of different solvents extract using various

methods:

Aerial parts of sample were extracted successively using continuous soxhlet technique and

maceration, with petroleum ether, chlorofo determine which fraction was responsible for activity.

Table (3:1) show the total weight of the successive extraction of the H. tuberculatum with different

solvents, ethanol (mac) gave the highest value of total weight in percentage yield (4.400%) followed

by petroleum ether (3.618%), ethanol (sox) (3.4745%) and finally chloroform (sox) (2.440%).

The majority of the lipid soluble components of plant aerial parts would remain in the insoluble

fraction, so the relatively hydrophobic character of compounds and its chemical structure may explain

Consistency

Yield percentage

Weight (g)

Extract

No

Greenish brown

Sticky-paste

3.618%

1.809g

Petroleum

ether(sox)

1

Yellowish-brown

sticky- paste

2.440%

1.222g

Chloroform(sox)

2

Yellowish-brown

sticky- paste

3.474%

1.737g

Ethanol(sox)

3

Brown

sticky- paste

4.400%

1.100g

Ethanol(mac)

4

the low extraction yield, while ethanol is organic solvent with high intermediate polarity, which can

extract higher amount of compounds. The differences in the extraction yield can be attributed to the

different solvents used.

Table (3.2): General phytochemical screening of H. tuberculatum ethanol extracts.

Class of compounds Ethanol extract

Triterpenes ++

Unsaturated sterol +++

Saturated sterol _

Saponin ( Forth ) ++

2-Deoxy sugars ++

Flavonols ++

Flavones +++

Cyanidin _

Leucoanthocyanins _

Tannins ++

Anthraguinones +

Alkaloids _ mayer,s ++

Alkaloids _ wagners +++

Coumarins ++

Carbohydrate +++

Reducing compound _

Key: + + + Strong + + Medium + Weak -Negative

The results of the phytochemical tests summarized in table (3.2) revealed that unsaturated

sterol, alkaloids and carbohydrates 2-deoxy sugars were strongly detected in ethanol extract.

Flavonoids glycosides, unsaturated sterols, tannins, coumarins, triterpenoids and saponins

were detected, while cyanidin, leucoanthocyanin, saturated compounds and reducing

compounds were not detected.

3.2 Bioassay of Crude Extracts:

Different concentrations of the various H. tuberculatum extracts were carefully assessed for their

Anti- oxidant activity.

Table (3.3): Quantitative analysis of flavonoids, terpenes, saponinsand alkaloids

Species Yield%

Saponins 23.30

Alkaloids 09.45

Flavonoids 20.00

Terpenes 13.33

The screening of the flavonoids with primary phytochemical screening process

revealed the presence of appreciable quantity of the flavonoids, alkaloids and saponins

(Table 3.2 ). Based upon the preliminary phytochemical test Quantitative determination

phytoconstituents were carried out for the powdered plant material by various standard

methods and found that saponin 23.3% flavonoids 20% terpenes 13.33% and alkaloid

9.45%

Table (3.4): Evaluation of Anti- oxidant activity of ethanol extract (mac), ethanol (sox),

hexane (mac) of H. tuberculatum

Sample Activity%S±D ±IC50

Ethanol(mac)

82.36 0.0336

Ethanol(sox) 50

-

Hexane(mac)

Essential oil

38.95 _

Fig (3.1): antioxidant activity of extracts

0

20

40

60

80

100

ethanol mac ethanol sox oil

acti

vity

anti oxidant activity

The figure (3:1) showed the three extracts which were tested for their antioxidant activity,

ethanol (mac) exhibited highest antioxidant activity, while ethanol (sox) extract showed

moderate antioxidant activity as compared to hexane extract (oil) which gave no activity.

Out of all above tested extracts it was noticed that the ethanolic extract of crude powders of

plant aerial parts was more potent than the corresponding ethanolic extract of their fat free

(soxhlet).This may be due to decomposition or heat inactivation of the active constituents

during the extraction process, another possibility explaining the loss of the activity , was

that the distribution of the active components in ethanol led to loss of the synergistic action

of these components hence the activity was lost. It was clear from the antioxidant tests, the

use of alcohol as a solvent of choice increases of the antioxidant activity, due to its ability to

remove all the active compounds. Nevertheless, ethanol extraction was adopted in this study

due to the fact that recovery of active compounds was easy from ethanol, in addition to the

fact that it did not facilitate hydrolysis of certain compound.

Table (3.5): GC-MS analysis of essential oil (obtained by hexane mac)

Peak RT Area% IUPAC name Commn

name

M.

formula

M.weight

1 8.16 1.77 1.6octadien-

ol,3,7-dimethyl

linalool C10H18O 154

2 11.39 95.58 2-cyclohexane-

one,2-methyl-

5-(1-methyl)

carvotan

acetone

C10H16O 152

3 14.44 1.03 2-cyclopente-

1-one,3-

methyl-2-(2-

pentene)

Jamsone C11H16O 164

4

16.82 1.62 Ethanone,1-

(1,4-dimethyl-

3-cyclohexen-

1)

- C10H16O 152

(1)

CH3CH3

CH3

O

(2)

CH3

CH3

CH3

O

(4)

(3)

The results showed in fig (3:5) that the volatile oil of H. tuberculatum which prepared by using

hexane (mac) extract given 4 compounds when was analyzed by GC-MS while in the some

literature or other studies the essential oil compositions of numerous species of H. tuberculatum

were reported that there are thirty eight compounds or more and this depend on many factors such

as geographic distribution, phenologicalcycle, seasonal variation, plant organ and extraction method

used.

In this method the hexane extract was obtained by maceration while in the other studies used

hydrodistilation method of ethanol extract.(8) so there is difference in the compound which was

obtained difference in polarity and difference of the species however the literature showed that the

main component of the essential oil was terpenes where as in this study the component is ketones

CH3

CH3

OH

CH3

CH2

O

CH3

CH3

.Four active compounds were isolated from the fraction and their structure were determined by IR

spectral data analysis. And one of this structures which isolated was identical with the other studies

which give same formula and structure and this compound is linalool. . It is possible that the yield

of essential oil will vary with the type and age of the plant as well as water content of the material

and the method of extraction.

The essential oil normally contained higher levels of volatile components; therefore, it was

reasonable to have lowe ryields. Physiological and environmental factors as well as processing

conditions have direct influence on volatile oil content.In addition, the difference of extraction yield

can be attributed to the solvents different polarity, which influences the extraction of plant

components

3.3 Thin-layer Chromatography of Ethanolic (mac) Extract:

Based on the Anti-oxidant activity results in tables (3.4), the ethanol (mac) extract was selected and

carefully studied. The active ethanol (mac) extract was subjected to various analytical thin layer

trials using various solvent systems. The active ethanol extract was fractionated in a preparative

TLC. Finally eleven fractions were separated. The characteristic colors and RF values of these

fractions are tabulated in tables (3.6) and plates (3.1) below.

Table (3.6): TLC fractions of ethanol (mac):

Fraction

Rf value

Color

Vis UV(365גnm) UV( ג 254nm) Spray

reagent

1 0.93 Green Red _ Green

2 0.87 _ Red _ _

3 0.83 _ Light _ _

4 0.77 Light

yellow

Red _ Red

5 0.72 _ Green _ _

6 0.70 Green Brown _ Green

7 0.68 Light green Red _ Red

8 0.64 _ Light _ Blue

9 0.60 _ Light _ _

10 0.58 _ Brown Red

11 0.40 Yellow Red _ Blue

Plate (3.1): TLC plates of ethanol (mac)

Mobile phase: (Toluene: Ethyl acetate: Formic acid)

Table (3.7): TLC fractions of the petroleum ether extract:

Fractions

RF value

Color

Vis UV(365גnm) UV( ג 254nm) Spray reagent

1 0.93 Yellow Grey Yellow Violet

2 0.90 Green Grey Green Green

3 0.86 Light

green

Red _ Green

4 0.80 Light

green

Red _ Green

5 0.6 Yellow Grey _ Green

Plate (3.2): TLC plates of petroleum ether extract

Table (3.8): TLC Fractions of hexane (mac):

Fractions

RF

value

Color

Vis UV(365גnm) UV( ג 254nm) Spray

reagent

1 Yellow Red _ Violet

2 _ Yellow _ Violet

3 _ Light _ Green

4 Green Grey _ Blue

5 _ _ _ _

6 Yellow Light _ Violet

Plate (3.3): TLC plates of hexane (mac)

Mobile phase: (Toluene: ethyl acetate)

Table (3.9): TLC fractions of the furocoumarins extract:

Fractions

RF

value

Color

Vis UV(365גnm) UV( ג 254nm) Spray reagent

1 0.95 Light green Light green Red Violet

2 0.80 Light green Light green Red Violet

3 0.60 Yellow Yellow Brown Violet

4 0.50 _ _ Light blue Pink

5 0.30 Yellow Yellow Brown Pink

6 0.06 Light yellow Light yellow Light blue Pink

The identification of furocoumarin (Psoralen) derivatives was approved by TLC using the following

system: chloroform: ethyl acetate and the fractions which obtained were identified by IR spectral

and UV spectrometer. The compounds isolated gave characteristic fluorescence as shown by

coumarins and the color of these compounds was intensified on spraying with 10% KOH.

The screening of the coumarins with primary phytochemical screening process revealed the

presence of appreciable quantity of the coumarins (1.1871).The Thin Layer Chromatography

(TLC) process confirms the possible presence of coumarins by revealing the fluorescent

bands which on further derivatization gave four bands fluorescence on long wavelength

(154nm).The method of extraction of phytochemicals is the most important procedure in the

development of pharmaceutical use of any plant species which is known or reported to have

medicinal importance. Preparative TLC is one of the cheapest and yet highly reliable

procedure in the fantasy of collecting pure compound from the crude plant extract. The

crude extract revealed the red, two light blue, fluorescent band and brown band at Rf =

0.95, 0.80, 0.60, 0.50, 0.30 and 0.06 cm-1 respectively . The presence of band at 3020.cm-1

indicates the possible presence of the free alkenes (=CH) stretch having the region of 3100-

3010 wavenumbers (cm-1). The presence of band at 1620cm-1 indicates the possible presence

of aromatic compound (C=C stretch) having the region of ~1600 wavenumbers (cm-1) .

Plate (3.4): PTLC plates of furocumarin

Mobile phase: (chloroform: ethyl acetate (

Table (3.10): TLC Wet Column fractions of ethanol (mac) extract

Color

No

RF N.E UV(365nm) UV(254nm) Spray reagent

1

F1

1.367

_

Light

_

Pink

2

3

4

5

F2

0.582 Yellow

Green

Dark

0.607 Yellow Red Yellow

0.645 Yellow _ _

0.683 Yellow

_ _

6 F3 0.708 Green Brown _ Green

7 F4 0.721 Light green Red _ Red

8

9

10

11

F5 0.772 _ Light _ Blue

0.835 _ Light _

0.873 _ Brown Red

0.936 Yellow Red Blue

Plate(3.5): TLC plate of column extract

Mobile phase:(toluene: ethylacetate:formic acid)

Fig (3.2): Column chromatography

3.4 Characterization and Analysis of isolated fractions:

The component eluted from the preparative TLC chromatography ended up with the isolation of

five pure fractions from active ethanol (mac) extract using Mobile phase (Toluene: ethyl acetate:

Formic acid) (5:4:1) Identification of fractions were based on different spectroscopic techniques

such as UV, IR spectroscopy.

Table (3.11): IR spectra of isolated fractions:

NO Fractions

Functional group(cm-1) Appendix

1

F1

2925.81(C-

H)Stretching,1724.24(C=O)stretching

(aldhyde),756.04(C-H)

bending(aromatic)

1

2

F2

3018.39(C-H)stretching

,1215.07(C=O)stretching, 756.04(C-H)

bending(aromatic)

3

3

F3

3018.39(C-H)stretching,

1215.07(C-O)stretching,757.97(C-

H)bending

Aromatic

5

4

F4

3018.39(C-H)stretching,1213.14(C-

O)stretching, 748.33(C-H) bending

aromatic

7

5

F5

3020(C-H)stretching,

1728.10(C=O)stretching, 731(C-

H)bending

Aromatic

9

Table (3.12): IR spectra of furocoumarins:

No

Fractions

Functional groups(cm_1) Appendix

1

F1

2921.96(C-H) stretching,

1724.24(C=O)stretching,750.26(C-H) bending

1

2

F2

2974.03(C-H)stretching,1049.20(C-

O)stretching,1380.94(C-H)bending,757.97(C-

H)bending aromatic

3

3

F3

3020.32(C-

H)stretching,1739.67(C=O)stretching,1215.07(C-

O)stretching,757.97(C-H)bending aromatic

5

4

F4

1049.20(C-O)stretching, 757.97(C-H)bending

aromatic

7

5

F5

3020.32(C-H)stretching,1728.10(C=O)stretching,

757.97(C-H)bending aromatic

9

6 F6 3020(C-H) stretching, 752.19(C-H) bending

aromatic

11

Table (3:13): the maximum absorption of the isolated fractions:

Table (3:14): the maximum absorption of the isolated furocuomarins:

Fractions Λmax Absorbance RF

F1 361 1.588 1.93

F2 371 1.194 1.91

F3 361 1.294 1.86

F4 341 1.245 1.81

F5 371 1.271 1.61

Fractions Λmax Absorbance RF

F1 370 0.127 0.95

F2 370 0.159 0.80

F3 370 0.144 0.60

F4 370 0.100 0.50

F5 370 0.115 0.30

F6 370 0.108 0.06

4. Conclusions and recommendations

4.1 Conclusions:

The main objectives of this work were to investigate the antioxidant property of Alhaza and

to identify and quantify active compounds in the plant aerial parts extracts .A number of

conclusions can be drawn from the work reported here:

Different extraction methods were applied on aerial parts powder. Results showed significant

differences of extraction yields among extracts, the yields were in this order ethanol mac >

petroleum ether> chloroform> ethanol (sox). Therefore, plant extracts qualities and quantities

are strongly correlated to the raw material and extraction types. Ethanol mac showed the

highest yield in all extracts.

Qualitative and quantitative chemical analysis of the composition of ethanol mac extracts

allowed to detect a wide range of chemicals. Saponins, flavonoids, terpenes tannins and

alkaloids were major components present in aerial parts. Free radical scavenging of phenolic

compounds is an important property underlying their various biological activities.

From the above procedural workout, it can be clearly concluded that the plant

Haplophyllum tuberculatum does contain the furanocoumarins. The above stated procedure

is therefore results to be very simple in the procedural workout isolation of compounds

which seems to be very difficult in the case of plant extracts. This procedure, hence

therefore, though being a very simple process, is remarkably very efficient for the

purification of the compounds from crude extracts of the plants. Also the solvent

system, standardized for the separation of coumarins, founds to be suitable for the

separation of , where the clear separation of bands is one of the outmost tasks to be

faced during the results obtained in this study are only first step in seeking substances from

natural source and biologically active.

4.2 Recommendations:

Based on the obtained results the following is strongly recommended:

Further studies are necessary in order to determine whether Haplophyllum tuberculatum

could be applied in a manner that will be antioxidant but not phototoxic. Accordingly, there

is a need to develop a formulation to improve the biological effectiveness for medical

applications.

This study to be developed in pharmacology to give anti-bacterial and anti-fungal drugs for

treatment the skin infection.

The need for modern equipments for extraction, purification, isolation and identification of

plant constituents, to enable research students to accomplish their goal successfully and to

obtain reliable results.

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0

0.1

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330 340 350 360 370 380 390 400 410

Ab

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7

Appendix (4) UV spectrum of fraction 2

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335 340 345 350 355 360 365 370 375 380

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345 350 355 360 365 370 375 380 385 390 395

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345 350 355 360 365 370 375 380 385

Ab

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wavelength


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345 350 355 360 365 370 375 380 385 390 395

Ab

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wavelength


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345 350 355 360 365 370 375 380 385

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330 340 350 360 370 380 390 400

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330 340 350 360 370 380 390 400

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wavelength


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335 340 345 350 355 360 365 370 375 380 385

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335 340 345 350 355 360 365 370 375 380 385

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335 340 345 350 355 360 365 370 375 380 385

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Documents

Al-neelain University Investigation of the Chemical ...repository.neelain.edu.sd:8080/jspui/bitstream/123456789...of Haplophyllum tuberculatum and it includes extensive chemical study