Thymelaea Microphylla Andgnidia Somalensis

Home , Daphne (plant), Edgeworthia gardneri, Gnidia, Stellera, Thymelaea, Thymelaea hirsuta

People's Democratic Republic of Algeria Ministry of Higher Education and Scientific Research

Mentouri Brothers University Constantine 1 Faculty of Exact Sciences Department of Chemistry

Ranking No:……… Serial No:………….

Thesis Submitted for Doctorate Degree in Science In Organic Chemistry Option: Phytochemistry

By Labib Ali Saeed Noman

Secondary metabolic component s and biological effectiveness study on two species Thymelaea microphylla and Gnidia somalensis

Members of jury:

Prof. Akkal Salah Mentouri Brothers University Constantine President

Prof. Zellagui Amar Larbi Ben Mhidi - Oum El Bouaghi University Supervisor

Prof. Rhouati Salah Mentouri Brothers University Constantine Co-Supervisor

Prof. Gherraf Noureddine Larbi Ben Mhidi - Oum El Bouaghi University Examiner

Prof. Seddik Khennouf Ferhat Abbas setif university Examiner

2017

DEDICATION

I dedicate my thesis to my loving parent souls, whose words encourage me. Thank you both for giving me strength to reach for the stars and chase my dreams. You have successfully made me the person i am becoming. You will always be remembered. My brothers have never left my side. My dearest wife, who leads me through the valley of darkness with light of hope and support. I also dedicate this dissertation to my many friends and church family who have supported me throughout the research. My beloved kids, whom i can't force myself to stop loving and my wonderful son Raed for been there to replace me and for inspire him to complet his study. To my long-time friends; Abderrahmane Mezrag, Redoun, Mohamed Zabat, Omar, Ahmed, Farid, Amar, Seif, Abbey Andreas, Siham, Majdah, Fayroz, Hanan, Loisa for their support, friendship and encouragement. I dedicate my thesis to all researchers who have concern.

Acknowledgements

I would like to thank all the people who assisted me with their expertise, work or emotional support in the completion of this thesis. First, I would like to thank my supervisor, Professor Amar Zellagui for giving me the opportunity to undertake this study and for all his encouragement and assistance throughout the study period. I wish to express my appreciation to my “second” supervisor, Professor. Salah Rhouati, for his support throughout the course of this research. I have special thanks to jury members, Professor Salah Akkal, Professor Gherraf Noureddine and Professor Seddik Khennouf for accept to discuss and correct my thesis. I had a special word of thanks goes to Professor Ibrahim Demirtaş from Laboratory of Plant Research, Department of Chemistry, Faculty of Science, Uluyazi Campus, Karatekin University, Cankiri, Turkey and Dr. Susana M Cardoso, from Department of Chemistry & QOPNA, University of Aveiro, Portugal for excellent technical information and discussion, for being a real mentor to me and for finding the answers to every “difficult question”, they were a great source of optimism and passion of this part of chemistry. I would like also to acknowledge other postgraduate students and staff members in Chemistry Department, I would also like to thank my lab-team and my lab neighbors.

Glossary of Abbreviations AREA : Agricultural Research and Extension Authority. CC : Column Cromatography.

C : Collective. COSY : COrrelation SpectroscopY (NMR). C6 cells : rat brain tumor. 13C-NMR : 13C Nuclear Magnetic Resonance. d : doublet. dd : doublet of doublet.

DAD : Diode Array Detector, UV.

DEPT : Distorsionless Enhancement by Polarization Transfer (NMR).

DMEM : Dulbecco’s modified eagle medium. diCQA : di-O-caffeoylquinic acid.

DMSO-d6 : DiMethyl Sulf Oxyde hexa deuterated (NMR) ES : Electro Spray. F : Fraction.

FBS : Fetal bovine serum. G : Gnidia. HeLa celles : Henrietta Lacks cells. Hetcore : Heteronuclear correlation spectroscopy. HMBC : Heteronuclear Multiple Bond Correlation. HMQC : Heteronuclear Multiple Quantum Correlation. 1H-NMR : 1H Nuclear Magnetic Resonance. HPLC-TOF : Liquide Chromatography/Time-of-Flight. Mass Spectrometry. Hz : Hertz. J: Coupling constant. LC-MS: Liquid chromatography–mass spectrometry. m : multiplet. MHI : Muller Hinton medium. MS : Mass Spectrometry. MSn : Multiple stage tandem MS. m/z : mass-to-charge ratio ppm : parts per million. Rt : Retention time. s : singlet. T : Thymelaea. TLC : Thin layer chromatography. UV : Ultraviolet.

W : Waste.

δ : chemical deplacement (NMR).

Table of Contents

Chapter 1

Summary ……….……….……………………………… …………………………………………… 1. Phenolic Compounds…………….……………………………… ……………………………… 01 1.1.Flavonoids………….………………………………………………………………...... 01 1.1.2. Structures and Classification of Flavonoids………………………...………………… 01 1.1.3. Chalcons …………………………….………………………………………………... 04 1.1.4. Flavones ……………………………….……………………………………………… 05 1.1.5. Flavanones………………………...…………..………………………………………. 06 1.1.6. Flavonols……………….……………………………………………………………... 07 1.1.7. Isoflavonoids………………………..……………………………………...………..... 07 1.1.8. Anthocyanins…………………………….……………………………………………. 08 1.2. 09 Coumarins………..……………………………..……………………………………….. 1.2.1. Classification and therapeutic applications of Coumarins……………………………. 10 1.3. Lignans………………………….………………………………………………………. 12 1.4. Spiro compounds……………………………………………………….……………….. 14 1.4.1. Nomenclature of Spiro compounds………………………………….………………... 14 1.5. Phenolic acids………………………………………..………………………………….. 15 1.6. Bioassays ……………… …………..………...…………….…...... 19 1.6.1. anti-cancer activity ……………… ………….……………………………………….. 19 1.6.2 Cancer and types ……………..…………….………………………………….. 20 1.6.3. Type of cancer……...….……………….……………………………………………... 21 1.6.4 Plant Derived Anti-Cancer Drugs…………………………………………………… 21

Chapter 2

2. Thymelaeaceae family ……………………………………………………………….….. 23 2.1. Classification of Thymelaeaceae family …………………...………………..……...... 24 2.2. Thymelaeaceae family morphology ……..…………………….……………………….. 24 2.3. Phytochemical Aspects …………….…………………………………...……………… 25 2.3.1. Essential oils…………….……………………………….…………………………… 25 2.3.2. Terpenes…………....………………………………………. ………………………… 26 2.3.2.1. Monoterpenes …...... 26 2.3.2.2. Diterpenes ……….…………………………………………….……………………. 27 2.3.3. Coumarins ………………………………………………………...... 28 2.3.3.1. Simple Coumarins ……………………………...…………………………………... 29 2.3.3.2. Furanocoumarins …………………………………………………………………… 29 2.3.3.3. Bicoumarins ……………………………………..………………………………….. 29 2.3.3.4. Bicoumarin dibenzofuranic derivative ………………………………………...…… 30 2.3.3.5. Tricoumarins ……………………………………...………………………………… 31 2.3.4. Flavonoides ………………………………………….…...... 32 2.3.5. Lignans ………………………………………………………...... ………………... 33 2.3.6. Spiro lactone…………………………………………………………….…………..... 34 2.3.7. Phenolic acid………………………………………………………………...... 35 2.4. Biological Aspects ……………………………………………………………………… 36 2.5. Thymelaea genus ………………………………………...... 37 2.5.1. Thymelaea genus Uses ………………………………………………...... 38 2.5.2. phytochemical of Thymelaea genus……………………………………….………….. 38 2.5.3. Thymelaea genus in Algeria………………………………………………...………… 39 2.5.4. Classification of Thymellaea microphylla Coss. et Dur.……………………..……...... 39 2.5.5. Thymellaea microphylla Coss. et Dur morphology...…...... …... 40 2.6. Gnidia genus …………………………………….………….…………………….…….. 40 2.6.1. Gnidia genus Uses …..………………………….. ...…………………………………. 43 2.6.2 phytochemical of Gnidia genus ……………….………………………………………. 43 2.6.3 Gnidia genus in Yemen……………………………………………………………...… 44 2.6.4. Classification of Gnidia somalensis Gilg. var.sphaerocephala (Bak.)Gastald ...……. 45 2.6.5. Gnidia somalensis Gilg. var.sphaerocephala (Bak.) Gastald. Morphology …………. 45

Chapter 3

3. Material and Methods ………………………………………………………….………. 46 3.1. Plant materiel of Thymelaea microphylla Coss. et Dur……………………..…………... 46 3.1.1. collection ……………………………………………………...…...... 46 3.1.2. Preparation of extract ……...... ………………………...…... 46 3.1.3. Separation and compounds purification …...…………...... …... 46 3.2. Plant material and methods of Gnidia somalensis Gilg. var.sphaerocephala (Bak.)Gastald. Extract…………………………………………………………………...... 51 3.2.1. collection ……………...…………………………...... ….….…...... 51 3.2.2. Preparation of extract ……………...………….....……..………….……………….…. 51 3.2.3. Separation and HPLC-DAD-MS analyses ….....…...……...... 51 3.3. NMR measurements ……………...…….………………………………………..……... 52 3.4. HPLC-TOF-MS spectroscopy…………………………………………………………... 52 3.5. Bioassays …………...……….....………………..……………………………...……..... 54 3.5.1. Antiproliferative assay ………………..……………….……………………..……..... 53 3.5.1.1. Preparation the stock solutions ………..……………………………………..…...... 53 3.5.1.2. Cell lines and cell culture …….……..……………………………………..…...... 53 3.5.1.3. Cell proliferation assay ……...………………………………..………………..….... 53 3.5.1.4. Statistical analyses ……...………………………………….……...………..……..... 54 3.5.1.5. xCELLigence assay…………...……………………………………………………. 54

Chapter 4

4. Results and Discussion…………………………………………………………………... 56 4.1. Identification of compounds isolated from Thymelaea microphylla Coss. et Dur. …...... 56 4.1.1. Identification of Compound 1 ……...…………….....…..……...... 56 4.1.2. Identification of Compound 2 ……...……………...... …………………………….. 63 4.1.3. Identification of Compound 3 ……...……………...... ……….…………………..... 72 4.1.4. Identification of Compound 4 ……...……………...... ……………….…………….. 80 4.1.5. Identification of Compound 5 ……...…………………………………………..……... 90 4.1.6. Identification of Compound 6 ……...……………...... 101 4.1.7. Identification of Compound 7 …...... 106 4.1.8. Identification of Compound 8 …...... 115 4.1.9. Identification of Compound 9 …...... 119 4.1.10 Identification of Compound 10……………………………………………………….. 128 4.2. Structure Identification of Gnidia somalensis Gilg. var.sphaerocephala (Bak.)Gastald compounds by HPLC-DAD-MS analysi;……………………………………. 138 4.2.1 Structural identification of hydrocinnamic acid derivatives separated from G. somalensis MeOH: H2O (7:3) extract……………………………………………………….. 139 4.2.1.1 Structural identification of compounds G1, G2 and G3………………..…………… 139 4.2.1.2 Structural identification of compound G4…………………………………………… 156 4.2.1.3 Structural identification of compound G5…………………………………………… 159 4.2.1.4 Structural identification of compound G6…..……………………..………………… 161 4.2.2 Structural identification of flavonoid derivatives separated from G. somalensis MeOH: H2O (7:3) extract …………………………………………………………………… 163 4. 2.2.1 Quercetin derivatives ……………………………………………………..………… 164 4.2.2.1.1 Structural identification of compound G7…………………………………………. 164 4.2.2.1.2 Structural identification of compound G8…………………………………………. 167 4.2.2.1.3 Structural identification of compound G9…………………………………………. 170 4.2.2.1.4 Structural identification of compound G10………………………………………... 172 4.2.2.1.5 Structural identification of compound G11………………………………………... 174 4.2.2.1.6 Structural identification of compound G12………...……….………………..….... 177 4.3. Bioassays results and discussion ……...………………………………………...... …. 180 4.3.1. Antiproliferative activity ……...…………………………………………...... 180 Conclusion…………………………………………………………………………………… 191 References………………………...……………...... ……………………...…...... 197 Abstract Résumé الملخص

Summary Allah created humans and made the earth to provide them with their needs and help them in their daily life. Humans have to make the earth a suitable environment that they can live easily. The plants are one of the most important sources that Allah created for human health. As they have a large reservoir of chemical compounds which are used in the treatment of many human diseases. Human beings have used plants for the treatment of diverse ailments for thousands of years [1]. According to the World Health Organization, most populations still rely on traditional medicines for their psychological and physical health requirements [2], since they cannot afford the products of pharmaceutical industries [3], together with their side effects and lack of healthcare facilities [4]. Rural areas of many developing countries still rely on traditional medicine for their primary health care needs and have found a place in day-to-day life. These medicines are relatively safer and cheaper than synthetic or modern medicine [5]. People living in rural areas from their personal experience know that these traditional remedies are valuable source of natural products to maintain human health, but they may not understand the science behind these medicines, but knew that some medicinal plants are highly effective only when used at therapeutic doses [6]. Plants have unique of chemical and biological substances for discovering new therapeutic benefits and they are looked to as a main source of medicinal purposes and also products in modern science. Medicinal plants maintain the health and vitality of individual and also cure various diseasesincluding cancer. Natural products discovered from medicinal plants have played an important role in treatment of cancer [7]. Taxol one of the most effective antitumor agent developed in the past three decades, it was originally isolated from the bark of Taxus brevifolia, it has been used for effective treatment of a variety of cancers including refractory ovarian cancer, breast cancer, lung cancer [8, 9, and 10]. Catharanthus roseus L. (G.) Don., is an important medicinal plant belonging to the Apocynaceae family; this plant is a dicotyledonous angiosperm and synthesizes two terpene indole alkaloids: vinblastine and vincristine that are used to fight cancer [11]. Algeria, a North African country with a large variety of soils (littoral, steppe, mountains and desert) and climates, possesses a rich flora (more than 3.000 species and 1.000 genders) [12], and many of medicinal plants studied have shown the uses in folk medicine and appears activity. The flora of Yemen is a mixture of the tropical African, Sudanian plant geographical region and Saharo-Arabian region. Yemen's flora is very rich, and the plants which identified about 2838 species, belong to 1068 genera and 179 families [13]. Many plants used in folk medicine by Yemeni people for treat several diseases [16]. for this reason we have chosen from Thymelaeaceae family the species Thymelaea microphylla Coss. et Dur. from Algeria, which has showed antioxidant and antibacterial activities in pervious study [14], and Gnidia somalensis Gilg. var.sphaerocephala (Bak.) Gastald from Yemen; the T. microphylla species has been used in folk medicine for the treatment of wounds and various cutaneous conditions such as erysipelas, skin cancer, abscess and pimples [15], and the another species Gnidia somalensis known as toxic plant causes diarrhea [16], which encouraged us to study more on this research.

Research aims:

 Extraction of active compounds from the plants under study.  Separation, purification and identification of chemical structures of the isolated compounds.  Study the effectiveness against anticancer.

CHAPTER 1 Secondary metabolites and anticancer activity

1. Phenolic compounds: Phenolic compounds form one of the main classes of secondary metabolites. They display a large range of structures and are responsible for the major organoleptic characteristics of plant-derived foods and beverages, particularly color and taste properties. They also contribute to the nutritional qualities of fruits and vegetables. Among these compounds, flavonoids constitute one of the important plant phenolics. Owing to their importance in food organoleptic properties and human health, a better understanding of their structures and biological activities indicates their potentials as therapeutic agents and also for predicting and controlling food quality. Due to the variety of pharmacological activities in the mammalian body, flavonoids are more correctly referred as “nutraceuticals” [17].

1.1 Flavonoids: Flavonoids are an important class of natural products. They are generally known to be present in plants. These include various fruits, vegetables, herbs and beverages. Flavonoids are associated with a broad spectrum of health promoting effects. They are an indispensable component in a variety of nutraceutical, pharmaceutical, medicinal and cosmetic applications. This is attributed to their anti-oxidative, anti-inflammatory, anti-mutagenic and anti-carcinogenic properties coupled with their capacity to modulate key cellular enzyme function [18].

1.1.2 Structures and Classification of Flavonoids: Flavonoids are the largest class of polyphenols. Chemically, they may be defined as a group of polyphenolic compounds consisting of substances that have two substituted benzene rings connected by the chain of three carbon atoms and an oxygen bridge [19,20], as shown in Figure1.

Figure 1: Basic flavonoid skeleton

CHAPTER 1 Secondary metabolites and anticancer activity

Flavonoids encompass a number of subclasses, and play a beneficial and sometimes a key role in a number of physiological processes. Flavonoids can be classified into six major subgroups, based on their molecular structure. Figure 2 displays the major subgroups of flavonoids with their general structures, sources and general health benefits [18].

CHAPTER 1 Secondary metabolites and anticancer activity

Scheme 1: Major subgroups of flavonoids with their general structures, sources and general health benefits [18].

CHAPTER 1 Secondary metabolites and anticancer activity

A brief description of each of these subgroups is given below:

1.1.3 Chalcones: Chalcones are a subclass of flavonoids. They are characterized by the absence of “ring C” of the basic flavonoid skeleton structure shown in Figure 2. Hence, they can also be referred to as open chain Flavonoids. Major examples of chalcones include Phloridzin, Arbutin, Phloretin and Chalconaringenin. Chalcones occur in significant amounts in tomatoes, pears, strawberries, bearberries and certain wheat products. Chalcones and their derivatives have considerable attention because of their nutritional and biological benefits [21- 23]. Figure 3, below, displays the structures of some chalcones.

Butein Okanin

Isobavachalcone Xanthohumol

Figure 3: Structures of some commonly studied chalcones.

CHAPTER 1 Secondary metabolites and anticancer activity

1.1.4 Flavones: Flavones are one of the important subgroups of flavonoids. Flavones are widely present in leaves, flowers and fruits as glycosides. Celery, parsley, red peppers, chamomile, mint and ginkgo biloba are among the major sources of flavones. Luteolin, apigenin and tangeritin belongs to this subclass of flavonoids. The peels of citrus fruits are rich in the polymethoxylated flavones tangeretin, nobiletin and sinensetin [24]. Flavones display various biological functions [25]. Figure 4.

Figure 4: Structures of some commonly studied flavones.

CHAPTER 1 Secondary metabolites and anticancer activity

1.1.5 Flavanones: Flavanone is another important class of flavonoids which are generally present in the plants. Hesperitin, Naringenin and Eriodictyol are examples of this flavonoids class. Flavanone have a pharmacological effects as antioxidant, anti-inflammatory, and ulcer protective [26, 27] Figure 5.

Naringenin Hesperetin

Hesperidin Eriodictoyl

Figure 5: Structures of some commonly studied flavanones.

CHAPTER 1 Secondary metabolites and anticancer activity

1.1.6 Flavonols: Flavonols occur widely in a variety of fruits and vegetables. Flavonols are found to be associated with wide range of health benefits which includes antioxidant, antidiabetic and reduced risk of vascular disease [28, 29]. Figure 6.

Quercetin Myricetin

Rutin Kaempferol

Figure 6: Structures of some commonly studied flavonols.

1.1.7 Isoflavonoids:

Isoflavonoids have a limited distribution in the plant kingdom and are predominantly found in soyabeans and other leguminous plants. Some Isoflavonoids have also been reported to occur in microbial organisms [30]. Isoflavonoids exhibit a wide range of biological activities; they have anti-inflammatory, antithrombotic, antihypertensive, antiarrhythmic, spasmolytic, and cancer chemopreventive properties [31]. The beneficial health effects of isoflavonoids are due to their antioxidative and phytoestrogenic properties [32], Isoflavones such as Genistein and Daidzein are commonly regarded to be phytoestrogens. Figure 7.

CHAPTER 1 Secondary metabolites and anticancer activity

Genistein Daidzein

Genistin Glycitein

Daidzin Figure 7: Structures of some commonly studied isoflavonoids.

1.1.8 Anthocyanins: Anthocyanins are pigments responsible for colors in plants, flowers and fruits [33]. Cyanidin, Delphinidin, Malvidin, Pelargonidin and Peonidin are the most commonly studied anthocyanins. They occur predominantly in the outer cell layers of various fruits such as cranberry, black currant, red grape, merlot, raspberry, strawberry, blueberry, bilberry and blackberry. Stability coupled with health benefits of these compounds enable them to be used in the food industry in a variety of

CHAPTER 1 Secondary metabolites and anticancer activity applications [34]. Anthocyanins display wide range of biological activities including anti-oxidant [35, 36], anti-inflammatory [37, 38], and anti-microbial including anti-candida activities [39, 40]. Figure 8.

Figure 8: Structures of some commonly studied anthocyanin. 1.2 Coumarins: Coumarins are a group of polyphenolic compounds they belong to the family of benzopyrones, which consists of benzene ring joined by a pyrone ring, (1-benzopyran-2-one) [41]. Figure 9. More than 1300 coumarins have been identified as secondary metabolites from plants, bacteria, and fungi [42]. Coumarins were initially found in tonka bean (Dipteryxodorata Wild) in 1820 and are reported in about 150 different species distributed over nearly 30 different families, of which a few important ones are Rutaceae, Clusiaceae, Guttiferae, Caprifoliaceae, Oleaceae, Nyctaginaceae, and Apiaceae [43].

Figure 9: (1-benzopyran-2-one)

CHAPTER 1 Secondary metabolites and anticancer activity

1.2.1 Classification and therapeutic applications of Coumarins: Natural coumarins are mainly classified into six types based on the chemical structure of the compounds. Properties and therapeutic applications of natural coumarins depend upon the pattern of substitution [44]. Table 1.

Table 1. Different coumarin types and their therapeutic applications [44]:

No. Type of Chemical structure Example Pharmacological coumarin activity Antiadipogenic Esculetin Antioxidant Neuroprotective Ammoresinol Antibacterial

Antibacterial

Ostruthin Antifungal

Antibacterial 1 Simple Antifungal coumarins Anticancer Osthole Anticonvulsant Antioxidant Novobiocin Antibacterial Coumermycin Antibacterial Chartreusin Antibacterial Anticancer Fraxin Antiadipogenic Antioxidant Umbelliferone Antitubercular Antiadipogenic Fraxidin Antihyperglycemic Phellodenol A Antitubercular Anti-inflammatory Antibacterial Imperatorin Antifungal Antiviral 2 Furano Anticancer coumarins Anticonvulsant Psoralen Antifungal Anti-TB

Bergapten Anti-TB

CHAPTER 1 Secondary metabolites and anticancer activity

Anthogenol Antibacterial 3 Dihydrofurano coumarins Felamidin Antibacterial

4 Pyrano coumarins are of two types 4a Grandivittin Antibacterial Linear type Agasyllin Antibacterial Aegelinol Antibacterial benzoate Xanthyletin Anti-TB Inophyllum A, B, Antiviral C, E, P, G1, and G2 Calanolide A, B, Antiviral 4b Angular type and F (+)- Antiviral Dihydrocalanolide A and B Pseudocordatolide Antiviral C Disparinol A Antiviral

Phenyl 5 coumarins Isodispar B Antiviral

Dicoumarol Anticoagulant 6 Bicoumarins

CHAPTER 1 Secondary metabolites and anticancer activity

1.3 Lignans: The term “Lignan” was first introduced by Haworth (1948) to describe a group of phenylpropanoids attached by central carbon (C8), as shown in Figure 10, [45]. lignans can be found in more than 60 families of plants and have been isolated from different plant parts.

Figure 10: Phenylpropanoid unit and lignan structure

Most of the known natural lignans are oxidized at C9 and C9´ and, based upon the way in which oxygen is incorporated into the skeleton and on the cyclization patterns, a wide range of lignans of very different structural types can be formed. Due to this fact, lignans are classified in eight subgroups [46, 47], among these subgroups, the furan, dibenzyl butane and dibenzocyclooctadiene lignans can be further classified in “lignans with C9 (9´)-oxygen” and “lignans without C9 (9´)- oxygen”. Figure 11.

CHAPTER 1 Secondary metabolites and anticancer activity

With C9(9’)- oxygen Without C9(9’)- oxygen Furofuran Furan

With C9(9’)- oxygen Without C9(9’)- oxygen Dibenzylbutyrolactol Dibenzylbutyrolactone Dibenzylbutane

Aryltetralin arylnaphtaline With C9(9’)- oxygen Without C9(9’)- oxygen Dibenzocylooctadienes

Figure 11: Lignans derivatives

CHAPTER 1 Secondary metabolites and anticancer activity

1.4 Spiro compounds: Spiro compounds are cyclic or polycyclic organic molecules. This type of compounds consists of identical cycles (same type) or different and are connected by a single atom called spiro atom. The connecting atom or spiro atom is a quaternary carbon atom. The rings may contain heteroatoms suchas: oxygen, nitrogen or sulfur.

1.4.1 Nomenclature of Spiro compounds: The nomenclature of spiro compounds was proposed in 1900 by the Adolf von Baeyer [48], for example a spiro molecule consisting of a cyclohexane ring and other cyclopentane is called spiro [4.5] decane. Figure 12:

Spiro [4.5] decane Spiro [4.4] nonane

Figure 12: Nomenclature of spiro compounds

The spiro natural substances form a rare chemical class of natural products and are found in plants in small amounts. These molecules can be exist as a lactone compounds Figure 13 and in the various parts of the plant such as stems, leaves, flowers, fruits, bark, seeds and roots. These compounds can be biosynthesized by fungi and algae. This class of natural compounds have diverse interests therapeutic and are known as: antibiotic [49], cytotoxicity [50], antibacterial antipalludique [51], anti-inflammatory [52] and HIV [53].

CHAPTER 1 Secondary metabolites and anticancer activity

Hyperolactone A Hyperolactone B

Hyperolactone C Curcumanolide A

Figure 13: Structures of some commonly spirolactone compounds

1.5 Phenolic acids: Among the wide diversity of naturally occurring phenolic acids, at least 31 hydroxy and polyhydroxybenzoic acids have been reported in the last 10 years to have biological activities. The chemical structures, natural occurrence throughout the plant, algal, bacterial, fungal and animal kingdoms, and recently described bioactivities of these phenolic and polyphenolic acids are reviewed to illustrate their wide distribution, biological and ecological importance, and potential as new leads for the development of pharmaceutical and agricultural products to improve human health and nutrition. Figure 14 [54]. 3-Hydroxybenzoic acid is found in common plants such as grapefruit (Citrus paradisi), olive oil (Olea europaea) [55], and medlar fruit (Mespilus germanica) [56]. Gentisic acid inhibits low- density lipoprotein oxidation in human plasma [57]. In addition to being an analgesic, anti- inflammatory, antirheumatic, antiarthritic, and cytostatic agent. Salicylic acid has keratolytic, anti- inflammatory, antipyretic, analgesic, antiseptic, and antifungal properties for several skin conditions such as dandruff and seborrhoeicdermatitis, ichthyosis, psoriasis, acne, etc. [58].

CHAPTER 1 Secondary metabolites and anticancer activity

Dicaffeoylquinic acid activity concerns the inhibition of the growth of the tumoral HL-60 cells by induction of apoptose [59].

3-Hydroxybenzoic acid 4-Hydroxybenzoic acid

Pyrocatechuic acid (R1=OH, R2=R3=H) Salicylic acid (R1=R2=H) Gentisic acid (R1=R3=H, R2=OH). 6-Methylsalicylic acid (R1=CH3, R2=H) α-Resorcylic acid (R1=R2=H, R3=OH). β-Resorcylic acid (R1=H, R2=OH) Orsellinic acid (R1=CH3, R2=OH)

Protocatechuic acid (R1=R2=H) Gallic acid (R1=R2=H) Vanillic acid (R1=CH3, R2=H) Syringic acid (R1=R2=CH3) Isovanillic acid (R1=H, R2=CH3) Digallic acid (R1=H, R2=gallate)

CHAPTER 1 Secondary metabolites and anticancer activity

Lunularic acid Pinosylvic acid (R=H) Hydrangeic acid (1',2'-E-didehydro) 4-O-Methylpinosylvic acid (R=CH3) Gaylussacin (R=β-D-glucopyranoside)

Anacardic acid. Turgorin A Ginkgolic acid

Merulinic acid A

CHAPTER 1 Secondary metabolites and anticancer activity

Lasalocid Cannabidiolic acid

Cajaninstilbene acid (R1=H, R2=prenyl ) Isocajaninstilbene acid (R1=prenyl, R2=H)

Dicaffeoylquinic acid

Figure 14: Phenolic acids and polyphenolic acids.

CHAPTER 1 Secondary metabolites and anticancer activity

1.6 Bioassays: 1.6.1 anti-cancer activity: Natural products especially plants have been used for the treatment of various diseases for thousands of years. Terrestrial plants have been used as medicines in Egypt, China, India and Greece from ancient times and an impressive number of modern drugs have been developed from them. The first written records on the medicinal uses of plants appeared in about 2600 BC from the Sumerians and Acadians [60]. Among the human diseases cancer is one, probably the most important genetic disease which can be treated with medicinal plants. Every year, millions of people are diagnosed with cancer, leading to death in a majority of the cases [61]. Cancer is the abnormal growth of cells in our bodies that can lead to death. Cancer cells usually invade and destroy normal cells. These cells are born due to imbalance in the body and by correcting this imbalance, the cancer may be treated. Every year, millions of people are diagnosed with cancer, leading to death. According to the American Cancer Society deaths arising from cancer constitute 2–3% of the annual deaths recorded worldwide [62]. Thus cancer kills about 3500 million people annually all over the world. Several chemo preventive agents are used to treat cancer, but they cause toxicity that prevents their usage [63]. The increasing costs of conventional treatments (chemotherapy and radiation) and the lack of effective drugs to cure solid tumors encouraged people in different countries to depend more on folk medicine which is rooted in medicinal plants use. Such plants have an almost unlimited capacity to produce substances that attract researchers in the quest for new and novel chemotherapeutics [61]. Plants have a long history of use in the treatment of cancer [64]. In his review, Hartwell lists more than 3000 plant species that have reportedly been used in the treatment of cancer, but in many instances, the “cancer” is undefined, or reference is made to conditions such as “hard swellings”, abscesses, calluses, corns, warts, polyps, or tumors, to name a few [65]. Such symptoms would generally apply to skin, “tangible”, or visible conditions, and may indeed sometimes correspond to a cancerous condition, but many of the claims for efficacy should be viewed with some skepticism because cancer, as a specific disease entity, is likely to be poorly defined in terms of folklore and traditional medicine [65]. This is in contrast to other plant-based therapies used in traditional medicine for the treatment of afflictions such as malaria and pain, which are more easily defined, and where the diseases are often prevalent in the regions where traditional medicine systems are extensively used. Nevertheless, despite these observations, plants have played an

CHAPTER 1 Secondary metabolites and anticancer activity important role as a source of effective anticancer agents, and it is significant that over 60% of currently used anti-cancer agents are derived in one way or another from natural sources, including plants, marine organisms and micro-organisms [66, 67]. Daphne mezereum is a plant belongs to Thymelaeaceae family used as afolklore remedy for treating cancer like symptoms. A hydro alcohol extract of Daphne mezereum has exhibited a potent antileukemic activity against lymphocytic leukemia in mice. Further fractionation studies on the extract resulted in the isolation and characterization of mezerein as a potent antileukemic compound [68].

1.6.2 Cancer and types:

Cancer is a general term applied of series of malignant diseases that may affect different parts of body. These diseases are characterized by a rapid and uncontrolled formation of abnormal cells, which may mass together to form a growth or tumor, or proliferate throughout the body, initiating abnormal growth at other sites. If the process is not arrested, it may progress until it causes the death of the organism. The main forms of treatment for advance stage cancer in humans are surgery, radiation and drugs (cancer chemotherapeutic agents). Cancer chemotherapeutic agents can often provide temporary relief of symptoms, prolongation of life, and occasionally cures [69]. In recent years, a lot of effort has been applied to the synthesis of potential anticancer drugs. Many hundreds of chemical variants of known class of cancer chemotherapeutic agents have been synthesized but have a more side effects. A successful anticancer drug should kill or incapacitate cancer cells without causing excessive damage to normal cells. This ideal is difficult, or perhaps impossible, to attain and is why cancer patients frequently suffer unpleasant side effects when under-going treatment [70]. However, a waste amount of synthetic work has given relatively small improvements over the prototype drugs. There is a continued need for new prototype-new templates to use in the design of potential chemotherapeutic agents, natural products are providing such templates. Recent studies of tumor-inhibiting compound of plant origin have yielded an impressive array of novel structures [71].

CHAPTER 1 Secondary metabolites and anticancer activity

1.6.3 Types of Cancers [72]:

 Cancers of Blood and Lymphatic Systems: a) Hodgkin’s disease. b) Leukemia’s. c) Lymphomas. d) Multiple myeloma. e) Waldenstrom's disease.  Skin Cancers: a) Malignant Melanoma.

 Cancers of Digestive Systems: a) Esophageal cancer. b) Stomach cancer. c) Cancer of pancreas. d) Liver cancer. e) Colon and Rectal cancer. f) Anal cancer.  Cancers of Urinary system: a) Kidney cancer. b) Bladder cancer. c) Testis cancer. d) Prostate cancer.

 Cancers in women: a) Breast cancer. b) Ovarian cancer. c) Gynecological. Cancer. d) Choriocarcinoma.

 Miscellaneous cancers: a) Brain cancer. b) Bone cancer. c) Characinoid cancer. d) Nasopharyngeal cancer. e) Retroperitoneal sarcomas. f) Soft tissue cancer. g) Thyroid cancer.

1.6.4 Plant Derived Anti-Cancer Drugs:

 Vinca Alkaloids: The first agents introduced in clinical use were vinca alkaloids, vinblastine (VLB) and vincristine (VCR), isolated from the Catharanthus roseus. (Apocynaceae).These drugs were discovered during an investigation for oral hypoglycemic agents [73].  Andrographis Paniculata: Phytochemical investigation of the ethanol extract of the aerial parts of Andographis paniculata has been reported the isolation of 14 compounds; a majority of them are flavonoids and labdane diterpenoids. The cytotoxic activities of these compounds have been evaluated against various cell lines and found that these isolates have a potent tumour inhibitory activity against all investigated cell lines [74].

CHAPTER 1 Secondary metabolites and anticancer activity

 Cannabis sativa: In vitro studies of components of marijuana (Cannabis sativa) indicate a potential to inhibit human breast cancer cells and to produce tumor eradications. In experiments introducing marijuana to malignant brain tumors, it was found that survival of animals was increased significantly .The active components of Cannabis sativaare cannabinoids [75].  Salvia miltiorrhiza: Tanshinone-I was isolated from traditional herb Salvia miltiorrhizae, and the study revealed a potential anticancer effect of tanshinone-I on breast cancer cells, suggesting that tanshinone-I may serve as an effective drug for the treatment of breast cancer [76].  Terminalia chebula: Terminalia chebulais a source of hydrolysable tannis and its antimutagenic activity in Salmonella typhimuriumhas been documented [74]. Phenols like chebulinic acid, tannic acid, ellagic acid are the cancer growth inhibitors found in the fruits of Terminalia chebula [78].

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

2. Thymelaeaceae family: The choice of Thymelaeaceae family as general topic of this work has been guided by the many traditional uses that are identified. The Thymelaeaceae are a small family of Dicotyledons consist of 1200 species distributed into 67 genera. The members of this family are widespread in the tropics and temperate climate of the planet, particularly in Africa, and lacking in cold regions [79] Figure 15. Although this family is a heterogeneous and relatively small taxon, it has very varied uses, giving them a significant economic importance in the regions where they grow [80]. The bark of several genres particularly Wikstroemia, Daphne, Edgeworthia, and Thymelaea is used for the local manufacture of paper. It has considering as a source of incense in some mediterranean regions, the incense collected after-incision of the trunk of some species of Wikstroemia. Some plants of this family are considered toxic because they contain of tigliane or daphnane ester types which have remarkable biological activities, such as antineoplastic and cytotoxic [81, 82].

Figure 15: Distribution map of Thymelaeaceae family in world.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

2.1 Classification of Thymelaeaceae family: Thymelaeaceae family is classified as follow [83, 84]:

 Kingdom : Plants

 Superdivision : Spermatophyta

 Class : Magnoliopsida

 Division : Magnoliophyta

 Subclass : Rosidae

 Order : Myrtales

 Family : Thymelaeaceae

2.2 Thymelaeaceae family morphology: The Thymelaeaceae are mostly shrubs and their main morphological characters of the aerial part is presented as follows [85]:  Leaves:  Alternately and rarely opposite.  Flowers:  Regularly, bisexual, floral pieces normally 4 or 5.  Grouped in racemes in heads or fascicles.  Cup-shaped, the receptacle forming a deep hollow tube which the edge usually carry the floral parts.  Sepals petaloid, appearing as a continuation of the tube, stamens inserted into the tube and corolla insignificant or absent.  Plants which have ovary superior simple style, attached to the base of the receptacle, with 1 or 2 (rarely 3-8) carpelles welded.  Stamens inside the tube and the crown is almost nonexistent.  Plants with high ovarian and installed with a simple pattern to the base of the flower tablet has 1 or 2 (rarely 3-8) carpels conjunctivitis.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

 Fruits:  Achene, berry, drupe or sometimes capsule.  Seeds with little or no albumin.

2.3 Phytochemical Aspects: Phytochemical studies on the Thymelaeaceae family plants due to their widespread uses in medicine reported, and there are reports on the toxicity of these plants [79]. In the last 70 years, several species of Thymelaeaceae family have been subjected to a numerous phytochemical studies. Initially, interest may have been due to the marked toxicity of these plants, but the widespread use of some species medicinally has certainly played a part in sustaining this interest [77]. Several genera such as Daphne, Thymelaea, Pimelea, Wikstroemia and Gnidia have been researched upon extensively. The Daphne genus is of prime importance owing to its richness in a variety of different classes of natural products, especially, coumarins, lignans, flavonoids, daphnane-type, diterpene esters, steroids, guianolides and spiro lactones [86-95].

2.3.1 Essential oils: Some old trees of these family infected by some fungi to become rich in essential oils, which can manufacture perfumes and incense in India, Pakistan and Indonesia. In Pakistan identified Cytosphaera mangiferae fungi responsible for the infection of these trees and the production of aromatic odor. It is worth mentioning that these trees infected is working to increase the amount of essential oils, as is the case in A. agallocha where the infected samples by fungi is rich in oxygenated sesquiterpenes by 0.4% of essential oils, while the non-infected by 0.08%. Some constituents of essential oils from Thymelaeaceae family shown in Figure 16. [82].

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

Figure 16: Some constituents of essential oils from Thymelaeaceae family.

2.3.2 Terpenes: 2.3.2.1 Monoterpenes: Attribute used to this family plants as a perfume or incense because they contain many volatile monoterpenes [96, 97], Figure 17.

Citronellol Nerol (-)-linalol β-phellandrene

Figure 17: Some monoterpenes from Thymelaeaceae family.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

2.3.2.2 Diterpenes: Several species of Thymelaeaceae family are well known for their interesting physiological and toxic effects. The chemical nature of toxic diterpenes of Thymelaeaceae has been known only for about thirty years. Although there exists large structural variations among these classes, but the toxicity is derived from their basic skelton tigliane, ingenane and daphnane type [98] Figure 18. The tigliane, daphnane, and ingenane diterpenes esters are noted for their skin irritant effects [98].

Tigliane-type Ingenane-type

Daphnane-type

Figure 18: Diterpene types from Thymelaeaceae family.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

Many groups, particularly orthoesters of the daphnane series, exhibit potent antileukemic activity. Hence, there has been marked interest in the structural features of this series that are conducive to antitumor activity in contrast to those that are responsible for irritant properties. Representative of the daphnane tumor inhibitor such as Gnididin compound from Gnidia lamprantha Gilg and Mezerein from Daphne mezereum L [68, 99- 102]. Figure 19.

Gnididin Mezerein

Figure 19: Some daphnane-type from Thymelaeaceae family.

2.3.3 Coumarins: Coumarins play a very important role in the chemotaxonomically Thymelaeaceae. More than 40 coumarins with various skeletal patterns have been reported to be isolated from several genera of Thymelaeaceae [79, 103, 104]. They are found in the form of simple coumarins, or as dimers and trimers, or as coumarin glycosides, flavone-coumarin and coumarinolignans.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

2.3.3.1 Simple Coumarins: Some simple coumarins isolated from Thymelaeaceae family [105-109]. Figure 20.

a: (daphnetine) b : (erioside) c : (esculetine) d : (scopoletol) e : (ombelliferone)

Figure 19: Some simple coumarins from Thymelaeaceae family.

2.3.3.2 Furanocoumarins: Simple coumarins with furan ring isolated from Thymelaeaceae family [110, 111]. Figure 21.

a : (isobergaptene) b : (pimpinelline) c : (sphondine)

Figure 21: Some simple furanocoumarins from Thymelaeaceae family.

2.3.3.3 Bicoumarins: Historically, the first bicoumarin separated from this family in 1936, is a compound daphnoretine from plant Daphne mezereum [112]. Some others isolated from Thymelaeaceae family [113-117], shown in Figure 22.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

a : (daphnoretine) b : (daphnorine) c : (edgeworthine) d : (acetyldaphnoretine) e : (dimethyldaphnoretine) f : (edgeworine)

Figure 22: Some bicoumarins from Thymelaeaceae family.

2.3.3.4 Bicoumarin dibenzofuranic derivative: Bicoumarin as dibenzofuranic derivatives were isolated from Gnidia lamprantha [118-120]. Figure 23.

Gnidicoumarine

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

Eriocephalosid

Lasioerine

Figure 23: Some bicoumarin dibenzofuranic derivatives from Thymelaeaceae family.

2.3.3.5 Tricoumarins:

The first example of tricoumarine isolated from Thymelaeaceae family in (1977) [121]. Some tricoumarins isolated Thymelaeaceae family [122]. Figure 24.

a : (edgeworoside A) b : (edgeworoside B) Figure 24: Some tricoumarins in Thymelaeaceae family.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

2.3.4 Flavonoids: This family is characterized contain many diverse flavonoids such as flavones, flavonols, flavanones, C-glycosyl flavones, biflavonoides and furanobiflavonoids [123,124]. The most common flavonoids in this family is contain kinds of methyl, O-glycosyl, apigenin, genkwanin, kaempferol and luteolin [79]. Figure 25.

a : (apigenine) b : (genkwanine) c : (kaempferol) d : (luteoline)

Figure 25: Some flavonoids from Thymelaeaceae family.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

2.3.5 Lignans: A variety of lignans and neolignans have been reported from Thymelaeaceae family such as furan lignans (lariciresinol, taxiresin), furofuran lignans (syringaresinol, pinoresinol), dibenzylbutyrolactone lignans (wikstromol, kusunokinin, matairesinol), coumarinolignan (daphneticin), [88, 105 and 125-130]. Figure 26.

a: R1= H R2= H

b: R1= OCH3 R2= OCH3 a: (pinoresinol) b: (syringaresinol)

a: R1= H b: R1= OCH3 a: (taxiresin) b: (lariciresinol).

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

a : (gnidifoline) b : (wikstromol) c : (matairesinol)

Figure 26: Some lignans from Thymelaeaceae family.

2.3.6 Spiro lactone: It was reported for the first time the rare spiro lactones as: 4’,6’-Diacetyl-viburnolide A, 4’,6’- Diacetyl-12- coumaroyl-viburnolide A and Tetraacetylviburnolide A, which extracted from the family of Thymeleaeceae [131]. Figure 27.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

4’,6’-diacetyl viburnolide A tetraacetylated viburnolides

4’,6’- diacetyll-12-cumaroyl- viburnolide A

Figure 27: Some spiro lactone from Thymelaeaceae family.

2.3.7 Phenolic acids: A variety of phenolic acids have been reported from Thymelaeaceae known to have various biological activities. such as m-Hydroxybenzoic acid, p-Hydroxybenzoic acid, p- Hydroxyphenylacetic acid, Hydroxycinnamic acid, Vanillic acid, Protocatechuic acid, p-Coumaric acid, Ferulic acid, Caffeic acid and Gallic acid [132]. Figure 28.

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

Gallic acid Vanillic acid p-Hydroxybenzoic acid

Caffeic acid Coumaric acid Ferulic acid

Figure 28: Some phenolic acids from Thymelaeaceae family. 2.4 Biological Aspects: Plants of Thymelaeaceae are well known for their interesting biological activities. Several African Thymelaeaceae species have been used in the traditional treatments of a variety of medicinal complaints in humans and animals. The toxicity of plants in the Thymelaeaceae is well established for humans as well as for several animal species [86]. The diterpenes esters of tigliane and daphnane type are of violent purgatives, which trigger off, by contact with the skin or mucous membranes, an intense inflammatory reaction [133]. The symptoms of systematic toxicity resulting from ingestion of the plant material include: inflammation of lips, larynx and pharynx, difficult in swallowing, thirst, rhinitis, dizziness, abdominal pain, slow respiration, rapid pulse; pale, cold and moist skin; muscular twitching, delirium and drowsiness which can last several days. These symptoms are followed by convulsion and death in 20-25% of cases [85, 134]. The use of toxic effects of these traditional medicines in these applications doses were low to promote the beneficial effect compared to the effects secondary. The basics of using these plants in the treatment of other ailments, such that snake bites, scorpion stings [79]. The toxicity of plants in the Thymelaeaceae is of considerable importance. In France, extracts of Lasiosiphon kraussiana have been patented for use in the treatment of leprosy [135, 136]. Clinical trials are being conducted in China on preparations of Daphne, Gnidia, Wikstroemia and Pimelea species that have been reported to have anticancer activity [87 and 137-142]. The abortifacient and anticancer

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family activities of these products have been shown to be due primarily to the presence of daphnane esters. Plants of this family have been included in several large-scale screening studies investigating [143- 147], various biological activities such as hemorrhoids, stimulant, rhumatismes, asthma, lumbago, laxative extract [79, 148 and 149].

2.5 Thymelaea genus: Thymelaea is one of Thymelaeaceae genus include about 30 species in the world [149]. Table 2. Table 2. Species of Thymelaea genus:

1. Thymelaea antiatlantica Maire 16. Thymelaea nitida (Vahl).

2. Thymelaea aucheri 17. Thymelaea passerina (L.) Coss. & Germ

3. Thymelaea broteriana Cout. 18. Thymelaea procumbens A.Fern. &

R.Fern. 4. Thymelaea calycina (Lapeyr.) Meisn. 19. Thymelaea pubescens (L.) Meisn.

5. Thymelaea cilicica 20. Thymelaea putorioides

6. Thymelaea coridifolia (Lam.). 21. Thymelaea ruizii Loscos ex Casav.

7. Thymelaea dioica (Gouan) All. 22. Thymelaea salsa

8. Thymelaea granatensis Pau ex 23. Thymelaea sanamunda All.

Lacaita 9. Thymelaea gussonei 24. Thymelaea sempervirens

10. Thymelaea hirsuta (L.). 25. Thymelaea subrepens

11. Thymelaea lanuginosa (Lam.) 26. Thymelaea tartonraira (L.) All.

Ceballos & C.Vicioso 12. Thymelaea lythroides 27. Thymelaea tinctoria (Pourr.)

13. Thymelaea mesopotamica 28. Thymelaea velutina Meiss.

14. Thymelaea microphylla Coss et Dur 29. Thymelaea villosa (L.) l.

15. Thymelaea myrtifolia 30. Thymelaea virescens

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

2.5.1 Thymelaea genus Uses: Some species of Thymelaea genus have been used in the traditional medicine and reported have a biological activity.The Thymelaea hirsuta plant is traditionally used in Tunisia as an antiseptic, anti-inflammatory and hypertension [150-152], as well as antimicrobial and antioxidant activity [153]. Thymelaea lythroides extract showed significant inhibition against antifungi [154,155].

2.5.2 phytochemical of Thymelaea genus: Studies have shown the Thymelaea genus contain several type of natural compounds as shown in Table 3. Table 3. Some isolated compounds from Thymelaea genus.

Compound isolated Part Species flavones, terpenes and other [156]. Leaves Thymelaea hirsuta (L.) thymelol ((C3H2O)n) [157]. stigmasterol, β-sitosterol, alcool aliphatic , l alcool aliphatique C H O, lactone C H O [158]. 12 22 19 18 6 daphnoretine, β -sitosterol-β - D-glucoside [159]. daphnorine, daphnoretine, daphnine, daphnetine, daphnetine-glucoside, ombelliferone, scopoletine and esculetine

(coumarines) [160].

2-vicenine (C-flavone) [161]. Thymelaea hirsuta (L.) tiliroside (3-p – coumaroyl glucosyl kaempferol)

(flavanol) [162]. lupeol, β-sitosterol, phytol, β-amyrine, betuline, erythrodiol, cholesterol and lanosterol

[163].

Twigs 5,12-dihydroxy-6,7-epoxy-resiniferonol [164]. gnidicine, gniditrine, genkwadaphnine, 12-O - heptadecenoyl-5-hydroxy-6,7-epoxy-

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family resiniferonol-9, 13,14-orthobenzoate (diterpenes daphnane ([165]. Tanins. [166]. Seeds proteines [167]. Roots daphnoretine (ether de dicoumaryl). [168].

1-carboxylic acid bornane, limonene, Aerial part T.microphylla Coss. et isobutyranilide, D-menthone, Pulegone, (6E)-2,5- Dur dimethyl-1,6 octadiene, Perillal, 2-Undecanone, (Z,E)-α-Farnesene, 1-(2-Bromovinyl)-adamantane,

Artemesiatriene [169]. pentacosane, triacontanol, sitosterol, stigmasterol, Thymelaea passerina β-amyrine, ombelliferone et scopoletin. [170]. (L.) Coss. & Germ. orientine, isoorientine, vitexine, 2-vicenine, Whole plant Thymelaea tartonraira kaempferol, daphnoretine, genkwanine, 5-o- (L.) All. D-genkwanine, primeverosyl (flavone- coumarine). [171]. Lipides, sucres and amidon. [172].

2.5.3 Thymelaea genus in Algeria: There are eight species of Thymelaea genus in Algeria: T. Velutina, T. Gvirgata, T. nitida,T. virescens, T. microphylla, T. Meisn, T. hirsuta, T. passerine [173]. 2.5.4 Classification of Thymellaea microphylla Coss. et Dur. [174]:  Family: Thymelaeaceae

 Sub Family: Thymelaeoideae

 Tribe : Gnidieae

 Genus : Thymelaea

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

 Species : T.microphylla Coss. et Dur.

2.5.5 Thymellaea microphylla Coss. et Dur morphology : Called “Al Methnan”, is a small shrubs and length does not exceed a meter, have dense tangled branches, young stem is arranged and white due to the soft fluffy wool which cover the outer surface. Small squamous leaves and linear, length does not exceed 7 millimeters, yellowish white flowers spread on stem [171]. Algerian people in traditional medicine used this species in the treatment of wounds, erysipelas, abscesses, and has purgative effect. [15, 175].

2.6 Gnidia genus: Gnidia genus is one of Thymelaeaceae genus include about 154 species in the world [86] shown in Table 4 below. Table 4. Species of Gnidia genus:

1. Gnidia aberrans C.H.Wright 77. Gnidia macrorrhiza Gilg

2. Gnidia albosericea (M.Moss) B.Peterson 78. Gnidia madagascariensis (Lam.) Baill.

3. Gnidia ambondrombensis (Boiteau) Z.S. 79. Gnidia meyeri Meisn. Rogers 4. Gnidia anomala Meisn. 80. Gnidia microcephala Meisn.

5. Gnidia anthylloides (L.f.) Gilg 81. Gnidia microphylla Meisn.

6. Gnidia apiculata (Oliv.) Gilg 82. Gnidia mollis C.H.Wright

7. Gnidia bambutana Gilg & Ledermann ex 83. Gnidia montana H.Pearson Engl. 8. Gnidia baumiana Gilg 84. Gnidia multiflora Bartl. ex Meisn.

9. Gnidia baurii C.H.Wright 85. Gnidia myrtifolia C.H.Wright

10. Gnidia bojeriana (Decne. ex Cambess.) 86. Gnidia nana (L.f.) Wikstr. Baill. 11. Gnidia burchellii (Meisn.) Gilg 87. Gnidia newtonii Gilg

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

12. Gnidia burmanii Eckl. & Zeyh. ex Meisn. 88. Gnidia nitida Bolus ex C.H.Wright

13. Gnidia burmanni Eckl. & Zeyh. ex Meisn. 89. Gnidia nodiflora Meisn.

14. Gnidia caffra (Meisn.) Gilg 90. Gnidia obtusissima Meisn.

15. Gnidia calocephala (C.A.Mey.) Gilg 91. Gnidia occidentalis (Leandri) Z.S. Rogers

16. Gnidia caniflora Meisn. 92. Gnidia oliveriana Engl. & Gilg

17. Gnidia canoargentea (C.H.Wright) Gilg 93. Gnidia oppositifolia L.

18. Gnidia capitata L.f. 94. Gnidia orbiculata C.H.Wright

19. Gnidia cayleyi C.H.Wright 95. Gnidia pallida Meisn.

20. Gnidia chapmanii B.Peterson 96. Gnidia parviflora Meisn.

21. Gnidia chrysantha (Solms) Gilg 97. Gnidia parvula Dod

22. Gnidia chrysophylla Meisn. 98. Gnidia pedunculata Beyers

23. Gnidia clavata Schinz 99. Gnidia penicillata Licht. ex Meisn.

24. Gnidia compacta (C.H.Wright) J.H.Ross 100. Gnidia perrieri (Leandri) Z.S. Rogers

25. Gnidia conspicua Meisn. 101. Gnidia phaeotricha Gilg

26. Gnidia coriacea Meisn. 102. Gnidia pinifolia L.

27. Gnidia cuneata Meisn. 103. Gnidia pleurocephala Gilg

28. Gnidia danguyana Leandri 104. Gnidia poggei Gilg

29. Gnidia decaryana Leandri 105. Gnidia polyantha Gilg

30. Gnidia decurrens Meisn. 106. Gnidia polycephala Gilg ex

31. Gnidia dekindtiana Gilg 107. Gnidia polystachya P.J.Bergius

32. Gnidia denudata Lindl. 108. Gnidia propinqua (Hilliard) B.Peterson 33. Gnidia deserticola Gilg 109. Gnidia pulchella Meisn.

34. Gnidia dregeana Meisn. 110. Gnidia quadrifaria C.H.Wright

35. Gnidia dumicola S.Moore 111. Gnidia quarrei A.Robyns

36. Gnidia emini Engl. & Gilg 112. Gnidia racemosa Thunb.

37. Gnidia ericoides C.H.Wright 113. Gnidia razakamalalana Z.S.Rogers

38. Gnidia fastigiata Rendle 114. Gnidia rendlei Hiern

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

39. Gnidia flanagani C.H.Wright 115. Gnidia renniana Hilliard & B.L.Burtt

40. Gnidia foliosa (H.Pearson) Engl. 116. Gnidia rivae Gilg

41. Gnidia francisci Bolus 117. Gnidia robusta B.Peterson

42. Gnidia fraterna (N.E.Br.) E.Phillips 118. Gnidia robynsiana Lisowski

43. Gnidia fruticulosa Gilg 119. Gnidia rubescens B.Peterson

44. Gnidia fulgens Welw. 120. Gnidia rubrocincta Gilg

45. Gnidia galpini C.H.Wright 121. Gnidia scabra Thunb

46. Gnidia geminiflora E.Mey. ex Meisn. 122. Gnidia scabrida Meisn.

47. Gnidia gilbertae Drake 123. Gnidia sericea L.

48. Gnidia glauca (Fresen.) Gilg 124. Gnidia sericocephala (Meisn.) Gilg ex Engl.

49. Gnidia gnidioides (Baker) Domke 125. Gnidia setosa Wikstr.

50. Gnidia goetzeana Gilg 126. Gnidia similis C.H.Wright

51. Gnidia gossweileri (S.Moore) B.Peterson 127. Gnidia singularis Hilliard

52. Gnidia gymnostachya (C.A.Mey.) Gilg 128. Gnidia squarossa

53. Gnidia harveyana Meisn. 129. Gnidia socotrana (Balf.f.) Gilg

54. Gnidia heterophylla Gilg 130. Gnidia somalensis (Franch.) Gilg

55. Gnidia hibbertioides (S. Moore) Rogers 31. Gnidia sonderiana Meisn.

56. Gnidia hirsuta (L.) Thulin 132. Gnidia sparsiflora Bartl. ex Meisn.

57. Gnidia hockii De Wild. 133. Gnidia spicata (L. f.) Gilg

58. Gnidia humbertii (Leandri) Z.S. Rogers 134. Gnidia splendens Meisn.

59. Gnidia humilis Meisn. 135. Gnidia squarrosa (L.) Druce

60. Gnidia imbricata L.f. 136. Gnidia stellatifolia Gand.

61. Gnidia inconspicua Meisn. 137. Gnidia stenophylla Gilg

62. Gnidia insignis Compton 138. Gnidia stenophylloides Gilg

63. Gnidia involucrata Steud. ex A.Rich. 139. Gnidia strigillosa Meisn.

64. Gnidia juniperifolia Lam. 140. Gnidia styphelioides Meisn.

65. Gnidia kasaiensis S.Moore 141. Gnidia suavissima Dinter

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

66. Gnidia kraussiana Meisn. 142. Gnidia subulata Lam.

67. Gnidia kundelungensis S.Moore 143. Gnidia tenella Meisn.

68. Gnidia lamprantha Gilg 144. Gnidia thesioides Meisn.

69. Gnidia latifolia (Oliv.) Gilg 145. Gnidia tomentosa L.

70. Gnidia laxa (L.f.) Gilg 146. Gnidia triplinervis Meisn.

71. Gnidia leipoldtii C.H.Wright 147. Gnidia usafuae Gilg

72. Gnidia linearifolia (Wikstr.) B.Peterson 148. Gnidia variabilis (C.H.Wright) Engl.

73. Gnidia linearis (Leandri) Z.S. Rogers 149. Gnidia variegata Gand.

74. Gnidia linoides Wikstr. 150. Gnidia welwitschii Hiern

75. Gnidia lucens Lam. 151. Gnidia wickstroemiana Meisn.

76. Gnidia macropetala Meisn. 152. Gnidia woodii C.H.Wright

2.6.1 Gnidia genus Uses: Several species of Gnidia are both of medicinal as well as economic importance. Due to the characteristic fibrous bark of Thymelaeaceae, Gnidia species are used to tie bundles of wood, thatch and clothing [176]. The flowers of several species of Gnidia are employed for dying leather [177]. Species of Gnidia have been used in the traditional treatments of a variety of medicinal complaints in humans and animals. They have been used to treat a range of conditions in humans including conception and childbirth, asthma, backache, dropsy, boils, sores, treat bruises and burns, constipation, coughs, earache, epilepsy, headache, influenza and fevers, malaria, measles, pulmonary tuberculosis, poor appetite, smallpox, snake bites, sprains and fractures, tonsillitis, stomach and chest complaints, toothache, ulcers and yellow fever and as broad-spectrum purgatives [78, 100, 178-182]. Several Gnidia extracts have also shown antileukemic properties [78, 178, 183- 185]. leaves of Gnidia gilbertae Drake are used as a purge to induce vomiting [36].

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

2.6.2 phytochemical of Gnidia genus: Studies have shown the Gnidia genus contain several type of natural compounds as shown in. Table 5:

Table 5. Some isolated compounds from Gnidia genus:

Compound isolated Part Species Isovitexin , 6-C-β-D-glucopyranosylapigenin, Aerial part Gnidia involucrata Isoorientin, Yuankanin, Mangiferin, Mahkoside A,

Vitexin, Gnidia biflavonoid, Astragalin,

Manniflavanone, 2,3,4',5,6- pentahydroxybenzophenone-4-C-glucoside,

2,4',6-trihydroxy-4-methoxybenzophenone-2-O- glucoside [139, 144,186-189].

Gnidia socotrana 7,7’-dihydroxy-3,8’-biscoumarin, 8-(6’’- Leaves and

Umbelliferyll)-apigenin, 4’,6’-Diacetyl- twigs viburnolide A, 4’,6’-Diacetyl-12-coumaroyl- viburnolide A, Tetraacetylviburnolide A [150].

Gnididin, Gnidicoumarin [118]. - Gnidia lamprantha

Umbelliferone, syringin, 2-O-beta-D-glucosyloxy- Stem Gnidia polycephala 4-methoxybenzenepropanoic acid [190,191]

Stem Gnidia latifolia Gnididione [192] 12-Hydroxydaphnetoxin, Mezerein, - Gnidia burchellii Genkwadaphnin

[193-195]

Gnidia kraussiana Maltol, 3-Hydroxy-2-methyl-4H-pyran-4-one, - Gnidilatin, Pimelea factor P2, Excoecariatoxin,

Gnidilatidin,kraussianin [78,196]

2.6.3 Gnidia genus in Yemen: There are two species of Gnidia genus in Yemen: Gnidia somalensis Gilg. var.sphaerocephala (Bak.) Gastald. and Gnidia socotrana (Balf.f.) Gilg. [13,16].

CHAPTER 2 Phytochemical Studies of Thymelaeaceae family

2.6.4 Classification of Gnidia somalensis Gilg. var.sphaerocephala (Bak.)Gastald [73, 84, 16]:  Family: Thymelaeaceae

 Sub Family: Thymelaeoideae

 Tribe : Gnidieae

 Genus : Gnidia

 Species: Gnidia somalensis Gilg.

var.sphaerocephala (Bak.) Gastald

2.6.5 Gnidia somalensis Gilg. var.sphaerocephala (Bak.) Gastald. morphology:

Called “Barehah” is a shrub, up to 80 cm tall, young branches densely to sericeous glabrous leaves sessile, blade linear-oblanceolate, 13–28 x 2–4 mm. Yellow flowers, florescence a 20– 40-flowered head. Petals lacking. Ovary usually with a few hairs at the apex. Seed 3–4 x 1–1.5 mm. Herb widespread grows on rocky land. Toxic plant and causes diarrhea [13].

CHAPTER 3 Material and Methods

3. Material and methods: 3.1 Plant materiel and extraction methods of Thymelaea microphylla Coss. et Dur.: 3.1.1 Collection: The aerial parts of Thymelaea microphylla Coss. et Dur. were collected in the end of March 2010 (flowering stage) from Eloued, desert of south Algeria. The plant was identified by Dr. Chahma A. M. University of Ouargla. Fresh aerial parts were dried to constant weight at room temperature.

3.1.2 Preparation of extract:

Aerial parts of Thymelaea microphylla (2200 g) were crushed and extracted with CH2Cl2: MeOH (1:1) at room temperature. The extract was concentrated in vacuo to obtain crude extract F (103.7 g). The residue was extracted with MeOH: H2O (7:3) at room temperature. The extract was concentrated in vacuo to obtain crude extract B (5.6g).

3.1.3 Separation and purification:

The crude extract F (301.7 g) of CH2Cl2–MeOH (1:1) was fractionated by column chromatography eluted with Hexane, followed by a gradient of Hexane and CH2Cl2 up to 100% CH2Cl2 and

CH2Cl2–MeOH up to 100% MeOH to obtain 9 fractions. The fraction 5 (F5; 103.9 mg) was subjected separation by TLC plates eluted with Hexane: CH2Cl2: EtOAc (0, 5: 2 :1) to afford compounds 1 (3 mg) and 2 (50.3 mg). The fraction 6 (F6; 5.5 g) was subjected to flash column chromatography eluted with Hexane, followed by a gradient of Hexane-CH2Cl2 up to 100%

CH2Cl2 and CH2Cl2–EtOAc up to 100% EtOAc and EtOAc–ethanol up to 100% ethanol to obtain 25 fractions from collective and 51 fractions from waste. F6-W42 from waste was purified by TLC to provide compound 3 (15 mg), fractions F6-C4, F6-C5 and F6-C9 from collective was purified by TLC eluted with Hexane: CH2Cl2: MeOH (1: 2: 0,5) to afford compound 4 (31.4 mg), compound 5 (71.1 mg) and compound 6 (10.6 mg). The fraction 7 (F7; 6.3 g) was applied on column chromatography eluted with Hexane: CH2Cl2: MeOH (0,5: 2 :1) to obtain 12 fractions, the precipitate from fraction 7 purified by TLC eluted with the same system to afford compounds 7 (6.4 mg) and 8 (5.1 mg). The fraction 8 (F8; 7.3 g) was applied on column chromatography eluted with Hexane : CH2Cl2 : MeOH (0,5: 1: 1,5) to obtain 8 fractions, precipitate from fraction 5 purified

CHAPTER 3 Material and Methods

by TLC eluted with the same system to afford compound 9 (11.6 mg). In turn, extract B (70%

MeOH–H2O) was fractionated by flash column using the eluting gradient of 0–100% hexane– CHCl3, followed by a gradient of 0–100% CHCl3–MeOH, to afford 16 fractions from collective (B-C1-B-C16) and 7 fractions from waste (B-W1–B-W7). Among these, fraction B-C8 from collective (154.1 mg), that was obtained by elution with 80% CHCl3–MeOH, was purified by TLC eluted with CH2Cl2: EtOAc: MeOH (1.5:1:0.5), to afford compound 10 (12.8 mg). Figure 29, 30 and 31.

CHAPTER 3 Material and Methods

Figure 29: Plan of separation and purification of compounds isolated from Thymelaea microphylla

CHAPTER 3 Material and Methods

extract F.

Figure 30: Plan of separation and purification of compound 10 isolated from Thymelaea microphylla extract B.

CHAPTER 3 Material and Methods

Thymelaea microphylla (2200g)

extracted with (70% residue extracted with (50% MeOH- H2O) 72h CH2Cl2-MeOH) 72h

concentrated concentrated

extract (5.6 g) (B) extract (103.7 g) (F)

flash column flash column chromatography chromatography

1-9 fractions (F) 1-16 fractions 1-7 fractions collection (C) waste (W) (B-C1-B-C16) (B-W1-B-W7)

F6; 5.5 g F7; 6.3 g F8; 7.3 g B-C8; 154.1 mg CC CC TLC F5; 103.9 mg 12 fractions 8 fractions compound 10 flash column (12.8 mg) TLC chromatography F7-7 F8-5 compounds 1 (3 mg) TLC and 2(50.3 mg) compounds 7(6. mg) and 8 (5. 1 mg) TLC 1-25 fractions collective (C) 1-51 fractions waste (W) compound 9 (11.6 mg)

F6-C4; 275.9 mg F6-C5; 367.4 mg F6-C9; 124.2 mg F6-W42; 76.3 mg

TLC TLC TLC TLC

compound 4 compound 5 compound 6 compound 3 (11.4 mg) (71.1 mg) (10.6 mg) (15 mg)

Figure 31: General separation and compounds purification plan of Thymelaea microphylla extracts.

CHAPTER 3 Material and Methods

3.2 Plant material and extraction methods of Gnidia somalensis Gilg. var.sphaerocephala (Bak.)Gastald.: 3.2.1 Plant collection: For Gnidia somalensis Gilg. var.sphaerocephala (Bak.)Gastald. the aerial parts were collected in May 2014 (flowering stage) from Taiz South Western of Yemen. The plant was identified by Dr.Abdul Wali Ahmed Al Khulaidi (AREA).

3.2.2 Preparation of extract: Aerial parts of Gnidia somalensis Gilg. var.sphaerocephala (Bak.) Gastald (1500 g) were crushed and extracted with CH2Cl2–Ethanol (1:1) at room temperature. The extract was concentrated in vacuo to obtain crude extract (99.4 g). The residue was extracted with MeOH: H2O (7:3) at room temperature. The extract was concentrated in vacuo to obtain crude extract (47.5 g).

3.2.3 Separation and HPLC-DAD-MS analyses: The phenolic profile of the Gnidia somalensis Gilg. var.sphaerocephala (Bak.) Gastald

MeOH:H2O (7:3) extract was analyzed by reversed-phase HPLC, based on the method of Gardana et al. [197] with some modifications, as described below. The HPLC analysis was performed on a Knauer Smartline separation module equipped with a Knauer smartline autosampler 3800, a cooling system set to 4 °C, and a Knauer UV detector 2500. Data acquisition and remote control of the HPLC system were done by Clarity Chrom® software (Knauer, Berlin, Germany). The column was a 250 mm × 4 mm id, 5 μm particle diameter, end-capped Nucleosil C18 (Macherey- Nagel), and its temperature was maintained at 30 °C. The mobile phase comprised (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile, which were previously degassed and filtrated. The solvent gradient started with 80% A and 20% B, reaching 30% B at 10 min, 40% B at 40 min, 60% B at 60 min, 90% B at 80 min, followed by the return to the initial conditions. For the HPLC analysis, the MeOH: H2O (7:3) extract (20 mg) was dissolved in 1 mL MeOH. All samples were filtered through a 0.2 μm Nylon membrane (Whatman) and 10 μL of each solution was injected. Chromatographic data were acquired at 250-500 nm and MS instrument: LTQ XL™ Linear Ion Trap Mass Spectrometre. Electrospray ionisation process in which ionised species in the gas phase are produced from a solution via highly charged fine droplets, by means of spraying

CHAPTER 3 Material and Methods

the solution from a narrow-bore needle tip at atmospheric pressure in the presence of a high electric field (1000 to 10000 eV). Ion trap of mass spectrometer analyser that confines ions using electric fields and then selectively ejects ions of different m/z by ramping the voltage. The ion trap is frequently used for high order fragmentation studies ("MSn") because it allows successive series of trapping and fragmentation. These analyses were carried out in the negative ion mode because of its higher sensitivity in the detection of the distinct classes of phenolic compounds [198]. In general, the identification of the corresponding compound was based on the search of the [M–H]− deprotonated molecule together with the interpretation of its MSn fragmentations. Still, when standards were available, the identification of phenolic compounds was determined by comparison of the ESI–MSn data to that of the standards [199].

3.3 NMR analyses:

NMR measurements were performed on a Bruker Avence III spectrometer in DMSO-d6, CD3OD and CDCl3 (1H: 400 MHz; 13C: 100 MHz). Chemical shifts were given in ppm with tetramethylsilane (TMS) as an internal standard.

3.4 HPLC-TOF-MS spectroscopy: HPLC-TOF-MS spectra were recorded in the negative ion mode on an Agilent 6210 spectrometer. Column chromatography was carried out on silica gel (Merck, 60-230 mesh) in glass columns in open atmosphere pressure. For thin-layer chromatography, silica gel F254 (Merck) precoated plates were used. Compounds were detected under UV (254 nm) and sprayed with 5% sericsulphate-H2SO4 reagent, followed by heating at 105 °C for 1-2 min. Mass analyser Time-of- flight that separates ions of different m/z by their time of travel between the ion source and detector, through a field-free region after acceleration by a constant voltage in the source. The ions will have differing velocities depending on their mass.

CHAPTER 3 Material and Methods

3.5 Bioassays: 3.5.1 Antiproliferative assay: Cell proliferation ELISA, BrdU (colorimetric) kits were obtained from Roche Diagnostics GmbH (Mannheim, Germany). The anti-tumor drug 5-florouracil was provided Sigma. Other of antiproliferative chemicals used were in analytical grade and obtained from Sigma–Aldrich, Merck and Roche [200].

3.5.1.1 Preparation of the stock solutions: The stock solution of samples and 5-FU were prepared in DMSO and diluted with Dulbecco’s modified eagle medium (DMEM). DMSO final concentration is below 1% in all tests [200].

3.5.1.2 Cell lines and cell culture: HeLa and C6 cell lines were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2% penicilin streptomycin The medium was changed twice a week [200].

3.5.1.3 Cell proliferation assay: Antiproliferative effects of the fractions and the compounds were investigated investigated on HeLa and C6 cell lines using proliferation BrdU ELISA assay. 5-Fluorouracil (5-FU) was used as positive controls. Cultured cells were grown in 96-well plates (COSTAR, Corning, USA) at a density of 3 x 104 cells/well. In each experimental set, cells were plated in triplicates and replicated twice. The cell lines were exposed to eight concentrations of compound sample and 5-FU for 24 h at 37 °C in a humidified atmosphere of 5% CO2. Cells were than incubated for overnight before applying the BrdU Cell proliferation ELISA assay reagent (Roche, Germany), according to manufacturer’s procedure. The amount of cell proliferation was assessed by determining the A450 nm of the culture media after addition of the substrate solution by using a microplate reader (Awareness Chromate, USA) [201-205]. Results were reported as percentage of the inhibition of cell proliferation, where the optical density measured from vehicle-treated cells was considered to be 100% of proliferation. All assays were

CHAPTER 3 Material and Methods

repeated at least twice using against HeLa, and C6 cells. Percentage of inhibition of cell proliferation was calculated as follows: (1- A treatments /A vehicle control) x100.

3.5.1.4 Statistical analyses: The results of investigation in vitro are means ± SD of nine measurement. Differences between groups were tested way ANOVA. p values of < 0.01 were considered significant [200].

3.5.1.5 xCELLigence assay: A real-time cell analyzer–single plate (RTCA-SP) instrument (Roche Applied Science, Basel, Switzerland) was used to analyze the ability of the compound 1 and some fractions to inhibit cell growth of HeLa cell line. A newly developed electronic cell sensor array, the xCELLigence RTCA, was used with a recently published literature method at the concentrations of 250, 100, 50 and 10 μg/mL. All the measurements were done in and triplicated. [206].

CHAPTER 3 Material and Methods

Figure 32: General plan of Thymelaea microphylla extracts antiproliferative assay.

CHAPTER 4 Results and Discussion

4. Results and discussion: 4.1 Identification of isolated compounds from Thymelaea microphylla Coss. et Dur.: 4.1.1 Identification of Compound 1:

Figure 33: Structure of compound 1. Compound 1 was isolated as a gum. It has shown violet color on TLC after being sprayed by sulfuric acid and heated at 100 °C. The mass experiment was carried out using HPLC-TOF/MS in positive mode. The mass spectrum of this compound presented an ion quasi-molecular at m/z 359 which suggested the molecular mass is 358 aum corresponding to the molecular formula C20H22O6 with 10 unsaturation number. Figure 34.

Figure 34: Mass spectrum of compound 1.

CHAPTER 4 Results and Discussion

According to 13C - NMR spectrum there are 20 carbons. Figure 35:  12 carbons belong to aromatic rings.  2 carbons belong to methoxy groups.  1 aliphatic carbon linked with oxygen.  4 aliphatic carbons.  1 carbon at 181 ppm indicates the existence of carbonyl lactone function.

13 Figure 35: C- NMR spectrum of compound 1 (100 MHz, CDCl3).

As we begin to compare the 13C- NMR spectrum with the DEPT 135- NMR spectrum, it elucidate the existence of 6 aromatic CH carbons and also 6 substituent groups, becuase there are 6 quaternary carbons. In addition, this elucidates that aliphatic carbons are (3 CH2 , 2 CH) and one of the CH2 is linked with oxygen at 71.35 ppm. Figure 36.

CHAPTER 4 Results and Discussion

Figure 36: NMR DEPT 135 spectrum of compound 1 (100 MHz, CDCl3).

From the HETCOR- NMR analysis, showed the correlation between the carbons and the protons which are directly attached are shown in Figure 37 and the chemical shift values are presented in Table 6.

Figure 37: HETCOR- NMR spectrum of compound 1(400 MHz, CDCl3).

CHAPTER 4 Results and Discussion

From the 1H- NMR analysis, the coupling constants and integrations of aromatic ring protons; (ortho, meta) systems in two rings for 6 protons. Figure 38, Table 6.

1 Figure 38: H- NMR spectrum of compound 1 (400 MHz, CDCl3).

In contrast, the signal of protons at 4.17 ppm and 3.91 ppm which belong to carbon at 71.35 ppm is linked with oxygen, therefore suggests the existence of lactone function. The integration of protons at 3.84 ppm and 3.83 ppm have shown that, there are two methoxy groups. In addition the existence of two protons at 2.97 ppm and 2.85 ppm, as well as, four protons at 2.61 ppm, 2.55 ppm and 2.48 ppm. Figure 39. Table 6.

1 Figure 39: H- NMR spectrum of compound 1 (400 MHz, CDCl3).

CHAPTER 4 Results and Discussion

From the previous NMR analysis of compound 1 which led to the formation of lignin lactone skeleton, which is confirmed by the HMBC- NMR structural analysis, and it has shown correlations between H-2 (δH 2.48) and C-6 (δC 34.57) , C-1 (δC 181.00), between H-4 (δH 3.91, 4.17) and C- 3 (δC 40.98), C-1 (δC 181.00), between H-5 (δH 2.61) and C-1' (δC 129.53), C-3 (δC 40.98), between H-6 (δH 2.97) and C-1'' (δC 129.76), has allowed to establish the attachment position of the butrolactone and benzene rings. Also the correlations between H-2' (δH 6 .42) and C-3' (δC 146.58), C-1' (δC 129.53), and H-2'' (δH 6 .63) and C-3'' (δC 146.57), C-1'' (δC 129.76), and H-7' (δH 3.83) and C-3' (δC 146.58), between H-7'' (δH 3.84) and C-3'' (δC 146.57) indicating methoxy group were all are connected to C-3' and 3''. Figure 40, Table 6.

CHAPTER 4 Results and Discussion

Figure 40: HMBC- NMR spectrum and structural correlation of compound 1 (400 MHz, CDCl3).

CHAPTER 4 Results and Discussion

Table 6. NMR spectral data of compound 1:

1H ppm) 13C ppm) HMBC

- 181 (C1) - - 146.58 (C3') - - 146.57 (C3'') - - 129.53 (C1') - - 129.76 (C1'') - - 144.52 (C4'') - - 144.37 (C4') - 6.84 (1H, d, J = 8.0, H-5') 114.37 (C5') - 6.83 (1H, s large, H-2'') 111.43(C2'') 129.76 (C1'') ; 146.57 (C3'') 6.82 (1H, d, J = 8.0, H-5'') 114.04(C5'') - 6.62 (1H, dd, J = 8.0- 1.8, H-6'') 122.07(C6'') - 6.52 (1H, dd, J = 8.0- 1.8, H-6') 121.32(C6') - 6.42 (1H, d, J = 1.8, H-2') 110.89(C2') 129.53 (C1') ; 146.58 (C3') 4.17 (1H, dd, J = 10.1- 8.1, H-4b) 71.35(C4) 181 (CO) ; 40.98(C3) 3.91(1H, dd, J = 10.1- 8.0 H-4a) 71.35(C4) 181 (CO) ; 40.98(C3) 3.84(3H, s, H-7') 55.84(C7') 146.58 (C3') 3.83(3H, s, H-7'') 55.77(C7'') 146.57 (C3'') 2.97 (1H, dd, J = 14.0- 5.3 , H-6b) 34.57(C6) 40.98 (C2) ; 129.76 (C1'') 2.85 (1H, dd, J = 14.0- 6.9, H-6a) 34.57(C6) 40.98 (C2) ; 129.76 (C1'') 2.61 (2H, m, H-5) 38.32(C5) 40.98 (C3) ; 129.53 (C1') 2.55 (1H, m, H-3) 40.98(C3) 38.32 (C5) ; 71.35 (C4) 2.48 (1H, m, H-2) 46.56(C2) 34.57 (C6) ; 181 (CO)

From the quaternary carbons C-4' and C-4'' with chemical shift values at 144.37 ppm and 144.52 ppm respectively, indicating that the substituents of these positions 4' and 4'' are hydroxyl groups, corresponding to the molecular mass 358 amu with molecular formula C20H22O6. Therefore, compound 1 was determined to be (4''-hydroxy-3''-methoxybenzyl)-3(4'-hydroxy-3'-

CHAPTER 4 Results and Discussion methoxybenzyl) butrolactone namely matairesinol compared with literature data [15, 207]. Figure 41.

Figure 41: Structure of compound 1 matairesinol.

4.1.2 Identification of Compound 2:

Figure 42: Structure of compound 2. In the process of isolating compound 2 as a crystalline solid, It showed a violet color on TLC after being sprayed by sulfuric acid and heated at 100 °C. The mass experiment was carried out using HPLC-TOF/MS in a negative mode. The mass spectrum of this compound presented an ion quasi-

CHAPTER 4 Results and Discussion molecular at m/z 357 which suggests that the molecular mass is 358 amu corresponding to the molecular formula C20H22O6 with 10 unsaturation number. Figure 43.

Figure 43: Mass spectrum of compound 2. According to the 13C- NMR spectrum there are 20 carbons. Figure 44:

 12 carbons belong to aromatic rings.  2 carbons belong to methoxy groups.  1 aliphatic carbon linked with oxygen.  4 aliphatic carbons.  1 carbon at 179.01 ppm indicate to exist carbonyl of lactone function.

CHAPTER 4 Results and Discussion

Figure 44: 13C- NMR spectrum of compound 2 (100 MHz, DMSO-d6).

In the course of comparing the 13C- NMR spectrum with the DEPT 135- NMR spectrum, it elucidate the existence of 6 aromatic CH carbons and also 6 substituent groups, becuase there are 6 quaternary carbons. In addition, this elucidates that aliphatic carbons are (3 CH2 , 2 CH) and one of the CH2 is linked with oxygen at 71.18 ppm. Figure 45.

CHAPTER 4 Results and Discussion

Figure 45: NMR DEPT 135 spectrum of compound 2 (100 MHz, DMSO-d6).

From the HETCOR- NMR analysis, showed the correlation between the carbons and the protons which are directly attached are shown in Figure 46 and the chemical shift values are presented in Table 7.

CHAPTER 4 Results and Discussion

Figure 46: HETCOR - NMR spectrum of compound 2 (400 MHz, DMSO-d6).

From the 1H- NMR analysis, the coupling constants and integrations of aromatic ring protons; (ortho, meta) systems in two rings for 6 protons. Figure 47, Table 7.

CHAPTER 4 Results and Discussion

Figure 47: 1H- NMR spectrum of compound 2 (400 MHz, DMSO-d6). On the other hand, the signal of protons at 4.07 ppm and 3.86 ppm which belong to carbon at 71.18 ppm is linked with oxygen, therefore suggest to exist a lactone function. The integration of protons at 3.74 ppm have shown that, there are two methoxy groups. There are also two protons at 2 .84 ppm, 2.77, as well as, four protons at 2.70 ppm, 2.45 ppm and 2.42 ppm. Figure 48, Table 7.

Figure 48: 1H- NMR spectrum of compound 2 (400 MHz, DMSO-d6).

CHAPTER 4 Results and Discussion

From all NMR analysis of compound 2, it indicates that the compound is a lignin lactone and the HMBC- NMR analysis confirms this structure. This shows the correlations between H-2 (δH 2.70) and C-6 (δC 34.16) , C-1 (δC 179.01), between H-4 (δH 3.86, 4.07) and C-3 (δC 41.37), C-1 (δC 179.01), furthermore between H-5 (δH 2.42) and C-1' (δC 129.36), C-3 (δC 41.37), between H-6 (δH 2.77, 2.84) and C-1'' (δC 129.36) allowed to establish the attachment positions of the butrolactone and benzene rings, between H-6' (δH 6 .59) and C-5' (δC 115.78), C-1' (δC 129.36), between H-5' (δH 6 .70) and C-6' (δC 121.17), C-4' (δC 147.88), between H-6'' (δH 6.48) and C-1'' (δC 129.36), C-5'' (δC 115.85), between H-5'' (δH 6 .67) and C-6'' (δC 122.31) , C-4'' (δC 147.96). The HMBC spectrum, therefore, showed cross peaks between H-7' (δH 3.74) and C-4' (δC 147.88), between H-7'' (δH 3.74) and C-4'' (δC 147.96) which definitely, confirms the difference between compound 1 and compound 2 in methoxy groups attachment positions . Figure 49, Table 7.

CHAPTER 4 Results and Discussion

Figure 49: HMBC- NMR spectrum of compound 2 (400 MHz, DMSO-d6).

CHAPTER 4 Results and Discussion

Table 7. NMR spectral data of compound 2:

1H ppm) 13C ppm) HMBC

- 179.01 (C1) - 147.96 (C4'') 147.88 (C4') - 145.58 - (C3'') - 145.42 (C3') - - 129.36 (C1') - - 129.36 - (C1'') 6 .75 (1H, d , J = 1 .76, H-2'') 113.91(C2'') - 6 .70 (1H, d, J = 8 .03, H-5') 115.78 (C5') 121.17 (C6') ; 147.88 (C4') 6 .67 (1H, d, J = 8.03, H-5'') 115.85 122.31 (C6'') ; 147.96 (C5'') (C4'') 6 .61 (1H, d, J = 1 .76, H-2') 113.12 (C2') - 6 .58 (1H, dd, J = 8 .03- 1.76, H-6') 121.17 (C6') 115.78 (C5') ; 129.36 (C1') 6 .48 (1H, dd, J = 8.03- 1.8, H-6'') 122.31 115.85 (C5'') ; 129.36 (C6'') (C1'') 4.07 (1H, dd, J = 7.28- 5.27, H-4b) 71.18 (C4) 179.01 (CO) ; 40.98(C3) 3.86 (1H, dd, J = 7.28- 5.27 H-4a) 71.18 (C4) - 3.74 (6H, s, H-7', 7'') 56 (C7', 7'') 147.88 (C4'), 147.96 (C4'') 2 .84 (1H, dd, J = 13 .80- 5.27 , H-6b) 34.16 (C6) 46.12 (C2) ; 129.36 (C1'') 2.77 (1H, dd, J = 13 .80- 5.27, H-6a) 34.16 (C6) - 2.70 (1H, m, H-2) 46.12 (C2) 34.16 (C6) ; 179.01 (CO) 2.45 (1H, m, H-3) 41.37 (C3) 37.35 (C5) ; 71.18 (C4) 2.42 (2H, m, H-5) 38.32 (C5) 41.37 (C3) ; 129.36 (C1')

From the quaternary carbons C-3' and C-3'' chemical shift values at 145.42 ppm and 145.58 ppm respectively, it indicates that the substituents of these positions 4' and 4'' are hydroxyl groups, corresponding to the molecular mass 358 amu and molecular formula C20H22O6. Therefore,

CHAPTER 4 Results and Discussion compound 2 was determined to be (3'-hydroxy-4'-methoxybenzyl)-3(3''-hydroxy-4''- methoxybenzyl) butrolactone namely Prestegane B compared with literature data [208]. Figure 50.

Figure 50: Structure of compound 2 prestegane B.

4.1.3 Identification of Compound 3:

Figure 51: Structure of compound 3. Compound 3 was isolated as a white powder. Which showed blue fluorescence color on TLC under UV at 254 and 366 nm, and therefore indicating a coumarin skeleton. The mass experiment was carried out using HPLC-TOF/MS in negative mode. The mass spectrum of this compound presented an ion quasi-molecular at m/z 351 which suggests that the molecular mass is 352 amu corresponding to the molecular formula C19H12O7 with 14 unsaturation number. Figure 52.

CHAPTER 4 Results and Discussion

Figure 52: Mass spectrum of compound 3. According to the 13C- NMR spectrum there are 19 carbons. Figure 53:

 2 carbons belong to carbonyl function groups.  12 carbons belong to aromatic rings.  4 carbons belong to olefinic groups.  1 carbon belongs to methoxy group.

Figure 53: 13C- NMR spectrum of compound 3 (100 MHz, DMSO-d6).

CHAPTER 4 Results and Discussion

As well as according to compare the 13C- NMR spectrum with the DEPT 135- NMR spectrum elucidate to exist 8 carbons of CH belong to aromatic rings and 10 quaternary carbons. Figure 54.

Figure 54: DEPT 135- NMR spectrum of compound 3 (100 MHz, DMSO-d6).

The 1H- NMR analysis elucidates the existence of 5 aromatic protons, along with 3 olefinic protons and one methoxy group according to the integration and chemical shifts of protons which confirmed by DEPT 135- NMR analysis data. Figure 55.

CHAPTER 4 Results and Discussion

Figure 55: 1H- NMR spectrum of compound 3 (400 MHz, DMSO-d6). Also, the coupling constants of protons as shown in Figure 56 and Table 8 below, indicates:  2 olefinic protons have coupling constant (2H, d, J = 9.6).  1 aromatic proton has coupling constant(1H, d, J = 8.53) of ortho system.  1 aromatic proton has coupling constant (dd, J = 8.53, 2.26) of (ortho, meta) system.  1 aromatic proton have coupling constant (d, J = 2.26) of meta system.  2 aromatic singlet protons.  1 olefinic singlet proton.  3 protons belong to methoxy group.

The coupling constants of H-3' proton at 6.39 ppm with H-4' (1H, d, J3', 4' = 9.6) and H-4' proton at

8.05 ppm with H-3' (1H, d, J4', 3' = 9.6) indicate the existence of coumarin skeleton. In contrast the number of protons and carbons from the 1H- NMR and 13C- NMR analysis indicate the presence of two coumarin skeletons A and B. The substituent groups in the moiety A illustrate the appearance of three singlet protons; 7.88 ppm, 7.21 ppm and 6.86 ppm.

CHAPTER 4 Results and Discussion

Figure 56: 1H- NMR spectrum of compound 3 (400 MHz, DMSO-d6).

From the COSY- NMR analysis, the correlation between proton H-3' at 6.39 ppm and proton H-4' at 8.05 ppm, between proton H-6' at 7.12 ppm and protons H-5', H-8' at 7.72, 7.19 ppm confirms the 1H- NMR data and corresponds to the system of protons coupling skeleton B. Figure 57.

Figure 57: COSY- NMR spectrum of compound 3 (400 MHz, DMSO-d6).

CHAPTER 4 Results and Discussion

From the HETCOR- NMR analysis, showed the correlation between carbons and protons which are directly attached are shown in Figure 58 and the chemical shift values are presented in Table 8.

Figure 58: HETCOR - NMR spectrum of compound 3 (400 MHz, DMSO-d6). From all NMR analysis of compound 3, it indicates that the compound is a bicoumarin type. The HMBC- NMR analysis confirms the previous suggestions. This shows the correlations between H- 3' (δH 6.39) and C-2' (δC 160.46), between H-4' (δH 8.05) and C-4a' (δC 114.84) which corresponds to the previous suggested structure of skeleton B. The spectrum also displayed the correlations of skeleton A which confirms the suggested structure, by observed cross peaks between H-5' (δH 7.72) and C-4a' (δC 114.84), C-6' (δC 113.88), between H-8' (δH 7.19) and C-7' (δC 160.14), between H- 4 (δH 7.88) and C-3 (δC 135.37), C-4a (δC 110.42), C-2 (δC 157.46), between H-5 (δH 7.21) and C-4a (δC 110.42), C-6 (δC 145.55), between H-8 (δH 6.86) and C-7 (δC 151.28). The correlation between H-9 (δH 3.81) and C-6 (δC 151.28) elucidate a methoxy group attachment position. Figure 59.

CHAPTER 4 Results and Discussion

Figure 59: HMBC- NMR spectrum of compound 3 (400 MHz, DMSO-d6).

CHAPTER 4 Results and Discussion

Table 8. NMR spectral data of compound 3:

1H ppm) 13C ppm) HMBC

- 160.46 (C2') - - 160.14 (C7') - - 157.46 (C2) - - 155.47(C8a') - - 151.28 (C7) - - 148.02. (C8a) - - 145.55 (C6) - 8.05 (1H, d , J = 9.6, H-4') 144.45 (C4') 114.84 (C-4a'); 114.33 (C-3') 135.37 (C3) 7.88 (1H, s, H-4) 131.45 (C4) 110.42 (C4a); 135.37 (C3);157.46 (C2) 7.72 (1H, d , J = 8.53, H-5') 130.37 (C5') 114.84 (C-4a'); 113.88 (C-6') - 114.84 (C4a') - - 110.42 (C4a) - 7.21 (1H, s, H-5) 109.89 (C5) 145.55 (C6); 110.42 (C4a) 7.19 (1H, d, J = 2.26, H-8') 104.45 (C8') 160.14 (C-7') 7.12 (1H, dd, J = 8.53, 2.26, H-6') 113.88 (C6') - 6.86 (1H, s, H-8) 103.21 (C8) 151.28 (C7) 6.39 (1H, d, J = 9.6, H-3') 114.33 (C3') 144.45 (C4'); 160.46 (C2') 3.81 (3H, s, H-9) 56.47 (C9) 145.55 (C-6)

The chemical shift values of the quaternary carbons C-7' and C-3 at 135.37 ppm and 160.14 ppm respectively, indicate the linked positions with oxygen. in contrast, the carbon C-7 at 151.28 ppm linked with hydroxyl group, corresponding to the molecular mass 352 amu and molecular formula

C19H12O7. Therefore, compound 3 was suggested to be 7-Hydroxy-6-methoxy-3-(2-oxochromen-7- yl) oxychromen-2-one namely daphnoretin compared with literature data [15]. Figure 60.

CHAPTER 4 Results and Discussion

Figure 60: Structure of compound 3 daphnoretin.

4.1.4 Identification of Compound 4:

Figure 61: Structure of compound 4.

After isolating Compound 4 as a yellowish oil, It showed a yellow color on TLC after being sprayed by sulfuric acid and heated at 100 °C. The mass experiment was carried out using HPLC-TOF/MS in a negative mode. The mass spectrum of this compound presented an ion quasi-molecular at m/z 541 which suggestes that the molecular mass is 542 amu corresponding to the molecular formula

C30H22O10 with 20 unsaturation numbers. Figure 62.

CHAPTER 4 Results and Discussion

Figure 62: Mass spectrum of compound 4.

According to the 13C- NMR spectrum there are 15 carbons. Figure 63:

 24 carbons belong to aromatic rings.  2 aliphatic carbon linked with oxygen at 83.38 ppm.  2 aliphatic carbons at 49.33 ppm.  2 carbon at 195.54 ppm indicate the existence of 2 carbonyl function groups. The number of carbons from13C- NMR spectrum of compound 4 indicates flavonoid skeleton and the chemical shifts of (aliphatic and carbonyl group) carbons indicate the skeleton is flavanon type [201].

CHAPTER 4 Results and Discussion

Figure 63: 13C- NMR spectrum of compound 4 (400 MHz, DMSO-d6). In the course of comparing the 13C- NMR spectrum with the DEPT 135- NMR spectrum, it elucidate the existence of 6 aromatic CH carbons and also 6 substituent groups, because exist 6 quaternary carbons. In addition, this elucidates to exist 2 aliphatic carbons CH, amongst which, one of them is linked with oxygen. Figure 64.

Figure 64: DEPT 135- NMR spectrum of compound 4 (100 MHz, DMSO-d6). The 1H- NMR analysis elucidates the existence of 16 protons according to the integration of protons Figure 65, this indicate the existence of symmetrical compound, in comparison with the carbons number of CH from DEPT 135- NMR analysis data. According to the HETCOR- NMR analysis

CHAPTER 4 Results and Discussion displayed, the signal of proton at 5.71 ppm belongs to distinguished aliphatic carbon at 83.38 ppm of flavanon skeleton compared with literature data [197, 201]. Figure 69.

Figure 65: 1H- NMR spectrum of compound 4 (400 MHz, DMSO-d6). In addition, the coupling constants of protons as shown in Figure 66 and Table 9 below, shows:

 8 aromatic protons have coupling constant (d, J =8.53) of ortho system belong to two benzene rings.  4 aromatic protons have coupling constant (d, J = 2.01 ) of meta system belong to two benzene rings.  4 aliphatic protons have coupling constant (d, J =12.30) of trans system (α, β) .

CHAPTER 4 Results and Discussion

Figure 66: 1H- NMR spectrum of compound 4 (400 MHz, DMSO-d6).

The coupling constants of proton H-2 at 5.71 (1H, d, J = 12.30) and proton H-3 at 2.69 (1H, d, J = 12.30) have the trans-trans geometry confirms this flavanon structure above and the absence of a proton coupling with Hα, β-3 and Hα, α-2 confirms that there exist another substituent group in C2 position [208]. The COSY- NMR analysis, showed correlation between proton H-2 at 2.69 ppm and proton H-3 at 5.71 ppm which therefore confirms all NMR spectrum data of flavanon skeleton Figure 67.

CHAPTER 4 Results and Discussion

Figure 67: COSY- NMR spectrum of compound4 (400 MHz, DMSO d6).

The correlation between proton H-2' at 6.86 ppm and proton H-3' at 6.73 ppm, between proton H-6' at 6.98 ppm and protons H-5' at 6.78 ppm confirm the 1H- NMR data and corresponding to the ortho system of cycle B (flavonoid). The correlation between proton H-6 at 5.78 ppm and proton H-8 at 5.70 ppm corresponding to the meta system of the cycle A (flavonoid). Figure 68.

CHAPTER 4 Results and Discussion

Figure 68: COSY- NMR spectrum of compound 4 (400 MHz, DMSO-d6).

From the HETCOR- NMR analysis, showed the correlation between carbons and protons which are directly attached are shown in Figure 69 and the chemical shift values are presented in Table 9.

CHAPTER 4 Results and Discussion

Figure 69: HETCOR- NMR spectrum of compound 4 (400 MHz, DMSO-d6).

From all NMR analysis of compound 4, it indicates that the compound is flavanon type and the HMBC- NMR analysis confirms this structure. It shows the correlations between H-3 (δH 2.69) and C-2 (δC 83.38), C-4 (δC 195.54), between H-2 (δH 5.71) and C-4 (δC 195.54), C-1' (δC 127.10) which confirms the flavanon type. The spectrum displayed correlations between H-2' (δH 6.86) and quaternary carbon C-1' (δC 127.10), between H-3' (δH 6.73) and quaternary carbon C-4' (δC 158.66), between H-5' (δH 6.78) and quaternary carbon C-4', between H-6' (δH 6.98) and quaternary carbon C-1' (δC 127.10) confirms the ortho system of the cycle B, between H-8 (δH 5.70) and quaternary carbons C-9 (δC 162.74), C-7 (δC 170), between H-6 (δH 5.78) and quaternary carbon C-7 (δC 170) confirms the meta system of the cycle A. Since it is a symmetrical compound, the chemical shift values and correlations is same for both linked structures (similar structures) Figure 70, Table 9.

CHAPTER 4 Results and Discussion

Figure 70: HMBC-NMR spectrum of compound 4 (400 MHz, DMSO-d6).

Table 9. NMR spectral data of compound 4:

1H ppm) 13C ppm) HMBC

- 195.54 (C4) - - 170 (C7) - - 163.96 (C5) - - 162.74 (C9) - - 158.66 (C4') - 6.98 (2H, d, J = 8.53, H-6', 6''') 129.64 (C6', 6''') 127.10 (C1', 1''') 127.10 (C1') 6.86 (2H, d, J = 8.53, H-2', 2''') 131.45 (C2', 2''') 127.10 (C1', 1''') 6.78 (2H, d, J = 8.53, H-5', 5''') 115.81 (C5', 5''') 158.66 (C4', 4''') 6.73 (2H, d, J = 8.53, H-3', 3''') 115.81 (C3', 3''') 158.66 (C4', 4''')

CHAPTER 4 Results and Discussion

- 101.18 (C10) - 5.78 (2H, d, J = 2.01, H-6, 6'') 97.04 (C6, 6'') 170.14 (C7', 7'') 5.71 (2H, d, J = 12.30, H-2, 2') 83.38 (C2, 2'') 195.54 (C4''); 127.10 (C1''') 5.70 (2H, d, J = 2.01, H-8, 8') 96.05 (C8, 8') 170.14 (C7''); 162.74 (C9'') 2.69 (2H, d, J =12.30, H-3, 3'') 49.33 (C3,3'') 195.54 (C4); 127.10 (C1')

From the quaternary carbons C-5, C-7, C-5'', C-7'' C-4' and C-4''' with chemical shift values at 163.96 ppm, 170 ppm, 163.96 ppm, 170 ppm, 158.66 ppm, and 158.66 respectively, it indicates that the substituents of these positions are hydroxyl groups which correspond to the molecular mass 542 amu and molecular formula C30H22O10. Therefore, compound 4 was determined to be 4', 4"', 5, 5", 7, 7"- hexahydroxy-3, 3"- biflavanon namely isochamaejasmin compared with literature data [208]. Figure 71.

Figure 71: Structure of compound 4 isochamaejasmin.

CHAPTER 4 Results and Discussion

4.1.5 Identification of Compound 5:

Figure 72: Structure of compound 5.

The compound 5 was isolated as a yellowish oil, it showed brown color on TLC after being sprayed by sulfuric acid and heated at 100 °C. The mass experiment was carried out using HPLC-TOF/MS in a negative mode. The mass spectrum of this compound presented an ion quasi-molecular at m/z 491 which suggested that the molecular mass was 446 amu corresponding to [M+ (HCOOH)-H]- inaccord with the molecular formula C19H26O12 with 7 unsaturation numbers. Figure 73.

Figure 73: Mass spectrum of compound 5.

CHAPTER 4 Results and Discussion

According to the 13C- NMR spectrum there are 19 carbons. As shown in Figure 74:

 16 carbons belong to aliphatic carbon.  3 carbons belong to carbonyl function groups.

13 Figure 74: C- NMR spectrum of compound 5 (100 MHz, CD3OD). As one compares the 13C- NMR spectrum with the DEPT 90- NMR spectrum, it elucidates that there are 7 alephatic carbons of CH Figure 75.

Figure 75: DEPT 90- NMR spectrum of compound 5 (100 MHz, CD3OD).

CHAPTER 4 Results and Discussion

When we compare between DEPT 135- NMR and APT- NMR analysis, it elucidate that there are 5 alephatic carbons of CH2, two of them linked with oxygen, at 68.36 ppm and 73.38 ppm respectively.

In addition there are 3 alephatic carbons of CH3 and 2 quaternary carbons at 88.10 and 107.7 ppm respectively. Furthermore 3 carbonyl function groups at 175, 170.77 and 170.41 ppm respectively. Figures 76 and 77.

Figure 76: DEPT 135- NMR spectrum of compound 5 (100 MHz, CD3OD).

Figure 77: APT- NMR spectrum of compound 5 (100 MHz, CD3OD).

CHAPTER 4 Results and Discussion

The 1H- NMR analysis, elucidates the existence of 23 protons according to the integration of protons which corresponds to the APT- NMR data. In addition there are 3 hydroxyl groups. Figure 78.

1 Figure 78: H- NMR spectrumof compound 5 (400 MHz, CD3OD).

From the HETCOR- NMR analysis, showed the correlation between carbons and protons which are directly attached are shown in Figure 79 and the chemical shift values are presented in Table 10.

CHAPTER 4 Results and Discussion

1 Figure 79: HETCOR- NMR and H- NMR spectra of compound 5 (400 MHz, CD3OD).

CHAPTER 4 Results and Discussion

From 1H- NMR analysis, the signal at 4.50 ppm (d, J = 7.2) which is attached with carbon at 97 ppm belongs to the anomeric proton of β-arabinosyle moiety according to the coupling constants and chemical shift values. In addition to the COSY- NMR analysis, the correlation between proton H-1' at 4.50 ppm and proton H-2' at 3.42 ppm, between proton H-2' and proton H-3' at 3.37 ppm, between proton H-3' and protons H-4' at 3.62 ppm and between proton H-4' and protons H-5a' and H-5b' at 3.57, 3.34 ppm confirms the 1H- NMR data and corresponds to the arabinose structure [15]. Figure 80.

Figure 80: COSY- NMR spectrum of the moiety β- arabinosyle structure correlations of compound 5 (400 MHz, CD3OD).

CHAPTER 4 Results and Discussion

There is also a correlation between protons H-12 at 5.20 ppm (dd; 5.7; 5.5) and H-11a, H-11b at 4.55 ppm and 4.13 ppm respectively. According to the coupling constant of H-12, which confirm the β position. Figure 81.

Figure 81: COSY- NMR spectrum of compound 5 (400 MHz, CD3OD).

From the HMBC- NMR analysis, which showed proton H-12 correlated with C-8 (δC 85.16) and the protons H-8 and H-1' correlated with same quaternary carbon C-9 (δC 107.7) which indicates the arabinose moiety is linked to C-9. In addition, there is a correlation between proton H-8 and the quaternary carbon C-5 (δC 88.10) and carbonyl group C-6 (δC 170.77). The 1H- NMR signal of H- 8 in all compounds, have the same skeleton appeared as a singlet indicating that the dihedral angle between H-8 and H-12 is nearly 90° confirmed by literature [95]. Figure 82.

CHAPTER 4 Results and Discussion

Figure 82: HMBC- NMR spectrum of compound 5 (400 MHz, CD3OD).

On the other hand, the H-17 (δH 2.1) correlates with C-12 (δC 75.14) and C-16 (δC 170.41) which indicates the acetyl group is linked to the skeleton at C-12 Figure 83.

Figure 83: HMBC- NMR spectrum of compound 5 (400 MHz, CD3OD).

CHAPTER 4 Results and Discussion

The COSY- NMR analysis shows that the protons H-3a and H-3b at 2.75 ppm and 2.39 ppm are respectively correlated together and with H-4 at 3.25 ppm, and between proton H-4 and proton H- 13 at 1.45 and 1.07 ppm. As well, there is correlation between the protons H-13 and protons H-14 at 1.32 and 1.17 ppm, and between proton H-15 at 0.9 ppm and protons H-14. Figure 84.

Figure 84: COSY- NMR spectrum of compound 5 (400 MHz, CD3OD).

The HMBC- NMR analysis, shows that proton H-4 (δH 3.25) correlates with C-13 (δC 32.29), C-5 (δC 88.10), C-2 (δC 175) and C-6 (δC 170.77) and protons H-3a, H-3b (δH 2.75, 2.39) with C-2 (δC 175) which confirms the existence of two lactone rings. Figure 85.

CHAPTER 4 Results and Discussion

Figure 85: HMBC- NMR spectrum of compound 5 (400 MHz, CD3OD).

Table 10. NMR spectral data of compound 5:

1H ppm) 13C ppm) HMBC

- 175 (C2) - - 170.77 (C6) - - 170.41 (C16) - - 107.7 (C9) - - 88.10 (C5) - 5.20 (1Hβ, d , J = 7.0- 5.0, H-12) 75.14 (C12) 5.15 (1Hα, s, H-8) 85.16 (C8) 88.10 (C5); 170.77(C6); 107.7(C9) 4.55 (1Hα, dd, J = 10-7.0, H-11a) 73.42 (C 11a) -

CHAPTER 4 Results and Discussion

4.50 (1H, d, J = 7.2, H-1') 97.00 (C1') 107.7(C9) 4.13 (1Hβ, d, J = 10- 5.0, H-11b) 73.42 (C11b) - 3.62 (1H, dt, J = 3.5- 1.5, H-4') 67.24 (C4') - 3.57 (1H, dd, J = 12.5- 1.5, H-5'a) 68.36 (C5') - 3.42 (1H, dd, J = 9.2- 7.2, H-2') 70.35 (C2') - 3.37 (1H, dd, J = 9.2- 3.5, H-3') 73.38 (C3') - 3.34 (1H, dd, J = 12.5-1.5, H-5'b) 68.36 (C5') - 2.75 (1Hβ, dd, J= 17.6-9.5, H-3b) 33.26 (C3b) 175 (C2) 2.39 (1Hα, dd, J= 17.6-10, H-3a) 33.26 (C3a) 175 (C2); 88.10(C5); 170.77(C6); 32.29(C13) 3.25 (1Hβ, dtd, J= 10- 9.5- 2.7 H-4) 37.13 (C4) - 2.1 (3H, s, H-17) 21.30 (C17) 170.41(16); 75.14 (C12)

1.45, 1.23 (2H, m, H-13) 32.29 (C13) - 1.32, 1.17 (2H, m, H-14) 21.44 (C14) - 0.9 (3H, t, H-15) 14.22 (C15) -

. From all NMR data, the chemical shift values and molecular mass 446 amu of compound 5 corresponds to molecular formula C19H26O12. Therefore, compound 5 was determined to have skeleton of dilaspirolactones, and suggested to be 1, 7- dioxa-2, 6-dioxospiro [4, 4] nonane namely microphynolide A compared with literature data [15]. Figure 86.

Figure 86: Structure of compound 5 microphynolide A.

100

CHAPTER 4 Results and Discussion

4.1.6 Identification of Compound 6:

Figure 87: Structure of compound 6.

Compound 6 was isolated as a yellowish oil, It showed brown color on TLC after being sprayed by sulfuric acid and heated at 100 °C. The mass experiment was carried out using HPLC-TOF/MS in a negative mode. The mass spectrum of this compound presented an ion quasi-molecular at m/z 403 corresponding to [M-H]- and m/z = 449 corresponding to [M+ (HCOOH)-H]- which suggests that the molecular mass is 404 amu corresponding to the molecular formula C17H23O11 resulting in 6 unsaturations. Figure 88.

Figure 88: Mass pectrum of compound 6.

101

CHAPTER 4 Results and Discussion

According to the 13C- NMR spectrum there are 17 carbons. This implies; Figure 89:

 15 carbons belong to aliphatic carbons.  2 carbons belong to carbonyl function groups. From 13C - NMR chemical shift values presented in Table 11, the compound 6 which is dilaspirolactones is similar to compound 5 (microphynolide A).

Figure 89: 13C- NMR spectrum of compound 6 (100 MHz, DMSO-d6).

Now, to compare the APT- NMR spectrum with the spectra of compound 5 (microphynolide A)

Figure 77 (page 93), it elucidates to exist just one carbon of CH3 at 14.23 ppm which belongs to the protons at 0.88 ppm of n-propyle group and two carbons of carbonyl functions. Figure 90.

102

CHAPTER 4 Results and Discussion

Figure 90: APT- NMR spectrum of compound 6 (100 MHz, DMSO-d6). . From the 1H- NMR spectrum of compound 6 Figure 91, it elucidates the absence of acetyl group at 2.1 ppm in comparison with the 1H- NMR spectrum of compound 5 Figure 78 (page 94). This confirms the absence of acetyl group in the APT- NMR analysis Figure 90 and the two carbons of carbonyl functions belong to the lactone rings of dilaspirolactone.

103

CHAPTER 4 Results and Discussion

Figure 91: 1H- NMR spectrum of compound 6 (400 MHz, DMSO-d6). . Table 11. NMR spectral data of compound 6:

1H ppm) 13C ppm) HMBC

- 175.14 (C2) - - 171.04 (C6) - - 107.8 (C9) - - 88.46 (C5) - 4.83 (1Hα, s, H-8) 87.95 (C8) - 4.50 (1H, d, J = 7.2, H-1') 97.00 (C1') 4.42 (1Hβ, d , J = 5.7- 5.5, H-12) 74.00 (C12) - 4.37 (1Hα, dd, J = 8.5- 5.7, H-11a) 73.95 (C 11a) - 3.95 (1Hβ, d, J = 8.5- 5.5, H-11b) 73.95 (C11b) - 3.61 (1H, dt, J = 3.5- 1.5, H-4') 67.31 (C4') - 3.59 (1H, dd, J = 12.5-1.5, H-5' a) 68.31 (C5') - 3.44 (1H, dd, J = 9.2- 7.2, H-2') 70.60 (C2') - 3.39 (1H, dd, J = 9.2- 3.5, H-3') 73.00 (C3') - 3.37 (1H, dd, J = 12.5- 1.5, H-5' b) 68.31 (C5') -

104

CHAPTER 4 Results and Discussion

3.25 (1Hβ, dtd, J= 9.79- 9.5- 3 H-4) 37.18 (C4) - 2.75 (1Hβ, dd, J= 17.6- 9.5, H-3b) 33.33 (C3b) - 2.36 (1Hα, dd, J= 17.6- 9.79, H-3a) 33.33 (C3a) -

1.42, 1.23 (2H, m, H-13) 33.30 (C13) - 1.33, 1.08 (2H, m, H-14) 21.25 (C14) - 0.88 (3H, t, H-15) 14.23 (C15) -

The chemical shift of quaternary carbon in position 12 at (δC 74.00) indicates the presence of hydroxyl group linked in this position, which corresponds to the molecular mass 404 amu and molecular formula C17H23O11. Therefore, compound 6 was determined to be as a skeleton of dilaspirolactones, 1, 7- dioxa-2, 6-dioxospiro [4, 4] nonane namely microphynolide B compared with literature data [15]. Figure 92.

Figure 92: Structure of compound 6 microphynolide B.

105

CHAPTER 4 Results and Discussion

4.1.7 Identification of Compound 7:

Figure 93: Structure of compound 7.

The fraction 7 (page 48) was subjected to a column chromatography eluted with Hexane: CH2Cl2: MeOH (0,5: 2 :1) to obtain 12 fractions. The compounds 7 and 8 were precipitated from fraction 7 they were separated by TLC eluted with the same system to afford compound 7 as a yellow powder.

This showed violet blackish fluorescence color on TLC under UV at 366 nm indicating the flavone or flavonol substituted in C3, along with the yellow color after being sprayed by sulfuric acid and heated at 100 °C.

The mass spectrum of this compound presented an ion quasi-molecular indicating the flavone/ flavonol-glycosyled at m/z 593 which suggested the molecular mass is 594 amu corresponding to the molecular formula C30H26O13 with 18 unsaturation number. Figure 94.

Figure 94: Mass spectrum of compound 7.

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According to the 13C- NMR spectrum there are 30 carbons. Figure 95. Thus:

 18 aromatic carbons.  2 carbons belong to carbonyl function groups.  4 olefinic carbons.  6 glucosid carbons.

13 Figure 95: C- NMR spectrum of compound 7 (100 MHz, CD3OD). As we compare the 13C- NMR spectrum Figure 95 with the APT- NMR spectrum Figure 96, it elucidates to exist 12 quaternary carbons two of them belong to carbonyl function groups at 167.49 ppm and 177.32 ppm. In addition there are 17 carbons of CH two of them belong to olefinic carbons at 114.56 ppm and 145.29 ppm, 10 carbons of CH belong to aromatic carbons and 5 carbons of CH belong to the glycoside moiety, according to the data of 1H- NMR and HETCOR - NMR analysis Figures 97 and 100 (pages 110 and 113).

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Figure 96: APT- NMR spectrum of compound 7 (100 MHz, CD3OD). The 1H- NMR analysis elucidate to exist 18 protons according to the integration of protons which corresponds to the APT- NMR data. The coupling constant of H-7''' proton at 7.47 ppm (1H, d, J =15.81) and H-8''' proton at 6.18 ppm (1H, d, J =15.81) indicates the existence of olefin function group in a trans system. The configuration of glycosidic bond was β as based on the coupling constant of anomeric proton signal at 5.16 ppm (1H, d, J = 7.2). The absence of the distinguished singlet signal of H- 3 which belongs to flavone skeleton indicate to flavonol type Figures 97 and 98.

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1 Figure 97: H- NMR spectrum of compound 7 (400 MHz, CD3OD).

From the coupling constant of protons there are. Figure 98, as in Table 11 (page 114):

 8 aromatic protons have coupling constants (d, J =9.03, 8.78) of ortho system belong to two benzene rings.  2 aromatic protons have coupling constants (d, J =2.26) of meta system.  2 olefinic protons have coupling constants (d, J =15.81) of trans system.

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1 Figure 98: H- NMR spectrum of compound 7 (400 MHz, CD3OD).

The COSY- NMR analysis showed correlation between protons H-2', H-6' at 8.02 ppm and protons H-3', H-5' at 6.86 ppm corresponding to the ortho system of the flavonoid (cycle B). Also, the correlation between protons H-6 at 6.16 ppm and proton H-8 at 6.34 ppm corresponds to the meta system of the flavonoid (cycle A), As well as showed cross peaks between protons H-2''', H-6''' at 7.36 ppm and protons H-3''', H-5''' at 6.85 ppm corresponding to the ortho system of other benzene ring. The correlation between proton H-7''' at 7.47 ppm and proton H-8''' at 6.85 ppm corresponds to the trans system of olefin function group Figure 99.

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Figure 99: COSY- NMR spectrum of compound 7 (400 MHz, CD3OD).

From the HETCOR- NMR analysis, showed the correlation between carbons and protons which are directly attached are shown in Figure 100 and the chemical shift values are presented in Table 11 (page 114).

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Figure 100: HETCOR - NMR spectrum of compound 7 (400 MHz, CD3OD). From all the NMR analysis of compound 7, it indicates that the compound is flavonol type and the HMBC- NMR analysis confirms this structure which elucidates the link between the sugar position and another benzene ring. Now, in the HMBC- NMR analysis for the flavonoid moiety, the correlations were observed between H-2', H-6' (δH 8.02) and quaternary carbon C-1' (δC 121.41), between H-3', H-5' (δH 6.86) and quaternary carbon C-4' (δC 160.16) confirms the ortho system of the flavonoid (cycle B), between H-8 (δH 6.34) and quaternary carbon C-7 (δC 159.35), between H- 6 (δH 6.16) and quaternary carbon C-5 (δC 160.18) confirms the ortho system of the flavonoid (cycle A). For the sugar moiety, it has shown correlation between proton anomeric H-1'' (δH 5.16) and C- 3 (δC 133.69) which confirms linked the sugar moiety with flavonoid at position 3. In contrast for another benzene ring, the correlations between H-2''', H-6''' (δH 7.36) and quaternary carbon C-1''' (δC 125.58), between H-3''', H-5''' (δH 6.85) and quaternary carbon C-4''' (δC 160.08). It’s therefore clear to see the linkage of benzene ring with olefin functional group by the correlation between H- 7''' (δH 7.47) and C-1''' (δC 125.58).The linkage between olefin functional group and carbonyl function by correlation between H-8''' (δH 6.18) and C-9''' (δC 167.49) confirms the existence of coumaroyl moiety, which is linked with sugar by the correlations between H-6''ab (δH 4.20, 4.34) and C-9''' (δC 167.49) Figure 101, Table 11 (page 114).

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Figure 101: HMBC- NMR spectrum of compound 7 (400 MHz, CD3OD).

Table 11. NMR spectral data of compound 7:

1H ppm) 13C ppm) HMBC

- 177.32 (C4) - - 167.49 (C9''') - - 161.28 (C9) - - 160.18 (C5) - - 160.16 (C4') - - 160.08 (C4''') - - 159.35 (C7) - - 157.49 (C2) - - 133.69 (C3) -

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8.02 (2H, d, J = 9.03, H-2', 6') 130.70 (C2', 6') 121.41 (C-1') 7.47 (1H, d, J = 15.81, H-7''') 145.29 (C7''') 125.58 (C-1''') 7.36 (2H, d, J = 8.78, H-2''', 6''') 129.83 (C2''', 6''') 125.58 (C-1''') - 125.58 (C1''') - - 121.41 (C1') - 6.86 (2H, d, J = 8.78, H-3', 5') 115.51 (C3', 5') 160.16 (C-4') 6.85 (2H, d, J = 9.3, H-3''', 5''') 115.72 (C3''', 5''') 160.08 (C-4''') 6.34 (1H, d, J = 2.26, H-8) 94.76 (C8) 159.35 (C7) 6.18 (1H, d, J = 15.81, H-8''') 114.56 (C8''') 167.49 (C-9''') - 113.26 (C10) - 6.16 (1H, d, J = 2.26, H-6) 100.59 (C6) 160.18 (C5) 5.16 (1H, d, J =7.28, H-1'') 103.32 (C1'') 133.69 (C-3) 4.34 (1H, dd, J =11.8- 2.0, H-6''a) 62.91(C6''a) 167.49 (C-9''') 4.20(1H, dd, J =11.8-6.5, H-6''b) 62.91(C6''b) 167.49 (C-9''') 3.50 (1H, m, H-3'') 73.38 (C3'') - 3.48 (1H, m, H-2'') 74.41 (C2'') - 3.47 (1H, m, H-5'') 74.29 (C5'') - 3.35 (1H, m, H-4'') 70.24 (C4'') -

According to the chemical shift values of quaternary carbons in positions C5, C7, and C4''' at 160.18 ppm, 159.35 ppm and 160.08 ppm respectively, it indicates that the substituents of these positions are hydroxyl groups which corresponds to the molecular mass 594 amu and molecular formula

C30H26O13. Therefore, compound 7 was determined to be as a Kaempferol 3-O-β-6"- coumaroyl glucopyranoside namely trans-tiliroside compared with literature data [175]. Figure 102.

Figure 102: Structure of compound 7 trans-tiliroside.

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4.1.8 Identification of Compound 8:

Figure 103: Structure of compound 8. .

Compound 8 was isolated as a yellow powder. It has shown violet blackish fluorescence color on TLC under UV at 366 nm and yellow color after being sprayed by sulfuric acid and being heated at 100 °C. The mass experiment was carried out using HPLC-TOF/MS in negative mode. The mass spectrum of this compound presented an ion quasi-molecular at m/z 593 which suggested the molecular mass is 594 amu corresponding to the molecular formula C30H26O13 with 18 unsaturation number. Figure 104.

Figure 104: Mass spectrum of compound 8.

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According to the 13C- NMR spectrum compound 8 have 30 carbons Figures 102, and in comparison with the 13C- NMR spectrum of compound 7 Figure 95 (page 108), it contains the same number of carbons:  18 aromatic carbons.  2 carbons belong to carbonyl function groups.  4 olefinic carbons.  6 glucosid carbons.

13 Figure 105: C- NMR spectrum of compound 8 (100 MHz, CD3OD). The 1H- NMR analysis Figure 106, elucidate to exist 18 protons according to the integration and coupling constant of protons which corresponding to the 1H- NMR analysis data of compound 7 Figure 97 (page 110). However the coupling constant of H-7''' proton at 7.47 ppm (1H, d, J =12.3) and H-8''' proton at 6.18 ppm (1H, d, J =12.3) indicate to exist olefin function group in cis system. Figure 107, Table 12.

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1 Figure 106: H- NMR spectrum of compound 8 (100 MHz, CD3OD).

1 Figure 107: H- NMR spectrum of compound 8 (400 MHz, CD3OD).

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Table 12. NMR spectral data of compound 8:

1H ppm) 13C ppm) HMBC

- 177.04 (C4) - - 166.45 (C9''') - - 160.24 (C9) - - 159.40 (C5) - - 159.86 (C4') - - 159.21 (C4''') - - 158.37 (C7) - - 156.58 (C2) - - 132.47 (C3) - 7.97 (2H, d, J = 9.0, H-2', 6') 129.65 (C2', 6') 121.41 (C-1') 7.41 (1H, d, J = 12.3, H-7''') 144.56 (C7''') 125.58 (C-1''') 7.31 (2H, d, J = 8.76, H-2''', 6''') 128.74 (C2''', 6''') 125.58 (C-1''') - 124.23 (C1''') - - 120.61 (C1') - 6.80 (2H, d, J = 8.76, H-3', 5') 114.38 (C3', 5') 160.16 (C-4') 6.78 (2H, d, J = 9.0, H-3''', 5''') 114.73 (C3''', 5''') 160.08 (C-4''') 6.18 (1H, d, J = 2.24, H-8) 94.23 (C8) 159.35 (C7) 6.16 (1H, d, J = 12.3, H-8''') 113.83 (C8''') 167.49 (C-9''') - 112.67 (C10) - 6.00 (1H, d, J = 2.24, H-6) 99.34 (C6) 160.18 (C5) 5.15 (1H, d, J =7.28, H-1'') 102.52 (C1'') 133.69 (C-3) 4.34 (1H, dd, J =11.8- 2.0, H-6''a) 62.13 (C6''a) 167.49 (C-9''') 4.10 (1H, dd, J =11.8- 6.5, H-6''b) 62.13 (C6''b) 167.49 (C-9''') 3.46 (1H, m, H-3'') 72.45 (C3'') - 3.43 (1H, m, H-5'') 73.18 (C5'') 3.40 (1H, m, H-4'') 69.42 (C4'') 3.37 (1H, m, H-2'') 74.41 (C2'') -

According to the chemical shift values and molecular mass 594 amu the molecular formula is

C30H26O13 and the compound was determined to be as a Kaempferol 3-O-β-6"-coumaroyl glucopyranoside namely cis-tiliroside compared with literature data [175]. Figure 108.

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Figure 108: Structure of compound 8 cis-tiliroside.

4.1.9 Identification of Compound 9:

Figure 109: Structure of compound 9.

Compound 9 was isolated as a white powder. It showed violet fluorescence color on TLC under UV at 366 nm indicating flavone or flavonol substituted in C3 The mass experiment were carried out using HPLC-TOF/MS in negative mode. The mass spectrum of this compound presented to an ion quasi-molecular indicating flavone/ flavonol-glycosyled at m/z 577 which suggested the molecular mass is 578 amu corresponding to the molecular formula C27H30O14 with 13 unsaturation number. Figure 110.

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Figure 110: Mass spectrum of compound 9. According to the 13C- NMR spectrum there are 27 carbons. As shown in Figure 111:

 12 aromatic carbons.  2 carbons belong to olefinic group.  1 carbon belongs to carbonyl function group.  1 carbon belongs to methoxy group.  11 glycoside carbons.

Figure 111: 13C- NMR spectrum of compound 9 (100 MHz, DMSO-d6).

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As well as according to compare the 13C- NMR spectrum Figure 111 with the DEPT- NMR spectrum Figure 112 confirms that exist 1 carbon belongs to methoxy function group at 56.55 ppm, 8 quaternary carbons one of them belongs to carbonyl function group at 177.23 ppm, 16 carbons of CH, 6 of them belong to aromatic carbons, and one olefin carbon, and the others nine carbons belong to glycoside moiety two of them are anomeric carbons at 104.10 and 104.50 ppm, in addition to exist 2 carbons of CH2 at 66.08 ppm and 69.10 ppm to confirm the existence of two sugars.

Figure 112: DEPT- NMR spectrum of compound 9 (100 MHz, DMSO-d6).

The 1H- NMR analysis indicate 23 protons according to the integration of protons Figure 113, which corresponding to the DEPT- NMR data.

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Figure 113: 1H- NMR spectrum of compound 9 (400 MHz, DMSO-d6). The coupling constant of H-2', 6' (2H, d, J =8.80) at 7.91 and coupling constant of H-3', 5' (2H, d, J =8.80) at 6.91 ppm corresponding to the ortho system of flavonoid (cycle B). The coupling constant of H-6 (1H, d, J = 2.35) at 6.85 ppm and the coupling constant of H-8 (1H, d, J = 2.35) at 7.01ppm corresponding to the meta system of flavonoid (cycle A). The existence of distinguished singlet signal of H- 3 at 6.68 ppm led to flavone skeleton Figure 114.

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Figure 114: 1H- NMR spectrum of compound 9 (400 MHz, DMSO-d6). The chemical shift value and coupling constant of the anomeric sugar proton H-1'' at 4.76 (1H, d, J = 7.63) indicate the presence of glucose and the anomeric proton revealed the β-configuration. As well as the chemical shift value and coupling constant of the anomeric proton H-1''' at 4.18 (1H, d, J =7.73) indicate the existence of xylose sugar and the anomeric proton in β-position by comparison of their spectral data with those reported in literature [209]. In addition to chemical shift value of 3H-8' confirm the existence methoxy group at 3.83 ppm Figure 115.

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Figure 115: 1H- NMR spectrum of compound 9 (400 MHz, DMSO-d6).

From the HETCOR- NMR analysis, showed the correlation between carbons and protons which are directly attached are shown in Figure 116 and the chemical shift values are presented in Table 13.

Figure 116: HETCOR - NMR spectrum of compound 9 (400 MHz, DMSO-d6).

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From all NMR analysis of compound 9 indicate that this compound is flavone type and the HMBC- NMR analysis confirm this structure and elucidate the link positions of sugars and methoxy group. HMBC- NMR analysis for the flavonoid moiety has shown correlations between H-2', H-6' (δH 7.91) and quaternary carbon C-1' (δC 121.50), between H-3', H-5' (δH 6.91) and quaternary carbon C-4' (δC 161.32) confirm the ortho system of the flavonoid (cycle B), between H-8 (δH 7.01) and quaternary carbon C-7 (δC 164.00), between H-6 (δH 6.85) and quaternary carbons C-7 (δC 164.00), C-5 (δC 158.51) confirm the meta system of the flavonoid (cycle A). HMBC spectrum showed cross peaks between H-3 singlet proton at (δH 6.68) and neighbor carbons C-2 (δC 164.03) and C-4 carbonyl group at (δC 177.23) which confirm flavone type. For the glucose sugar moiety displayed correlation between the anomeric proton H-1'' (δH 4.76) and C-5 (δC 158.51) which confirm linked the glucose with flavonoid at position 5 and for the xylose sugar showed correlations between H-1''' (δH 4.18) and C-6'' (δC 69.10) which confirm linked the xylose with glucose in position 6'' of glucose. Furthermore HMBC correlation was observed between methoxy group 3H-8' and C-7 (δC 164.00) which confirm linked the methoxy group with flavonoid in position 7. Figure 117, Table 13.

Figure 117: HMBC- NMR spectrum of compound 9 (400 MHz,DMSO-d6).

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Table 13. NMR spectral data of compound 9:

1H ppm) 13C ppm) HMBC

- 177.23 (C4) - - 164.03 (C2) - 161.32 (C4') - 164.00 (C7) - - 158.87 (C9) - - 158.51 (C5) - - 121.50 (C1') - - 109.57 (C10) - 7.91 (2H, d, J = 8.80, H-2', 6') 128.60 (C2', 6') 121.50 (C1') 7.01 (1H, d, J = 2.35, H-8) 96.94 (C8) 164.00 (C7) - 124.23 (C1''') - - 120.61 (C1') - 6.91 (2H, d, J = 8.80, H-3', 5') 116.35 (C3', 5') 161.32 (C4') 6.85 (1H, d, J = 2.35, H-6) 103.83 (C6) 158.51(C5); 164.00 (C7) 6.68 (1H, s, H-3) 113.83 (C3) 164.03 (C2); 177.23 (C4) - 112.67 (C10) - 4.76 (1H, d, J = 7.63, H-1'') 104.10 (C1'') 158.51(C5) 4.18 (1H, d, J =7.73, H-1''') 104.50 (C1''') 69.10 (C6'') 3.96 (1H, d, J =9.98, 6''a) 69.10 (C6''a) - 3.83 (3H, s, H-8') 56.55 (C8') - 3.67 (1H, dd, J =11.15-5.2, H-5'''eq) 66.08 (C5'''eq) - 3.63 (1H, dd, J =9.98-6.4, 6''b) 62.13 (C6''b) - 3.55 (1H, dd, J =9.39-7.0, 5'') 76.30 (C5'') - 3.35 (1H, m, H-2'') 73.92 (C2'') - 3.28 (1H, m, H-3'') 75.90 (C3'') - 3.27 (1H, m, H-4''') 69.95 (C4''') - 3.19 (1H, m, H-4'') 70.08 (C4'') - 3.11 (1H, t, J =8.80, H-3''') 76.85 (C3''') - 3.01 (1H, t, J = 11.15, H-5'''ax) 66.08 (C5'''ax) - 2.97 (1H, t, J = 8.80, H-2''') 73.66 (C2''') -

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According to the chemical shift values of quaternary carbon in position C4' at 161.32 ppm linked with hydroxyl group which corresponding to the molecular mass 578 amu and molecular formula

C27H30O14. Therefore, compound 9 is determined to be as a Genkwanin 5-O-β-D-xylopyranosyl- (1→6)-β-D-glucopyranoside namely Yuankanin compared with literature data [209]. Figure 118.

Figure 118: Structure of compound 9 Yuankanin.

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4.1.10 Identification of Compound 10:

Figure 119: Structure of compound 10.

Compound 10 was obtained as a brown yellowish solid, It showed blue color on TLC after being sprayed by p-Anisaldehyde – sulfuric acid and heated at 100 °C. The compound was determined as − C19H26O7N2Na by negative HPLC-TOF/MS at m/z 417.0944 corresponding to [M − H + Na] Figure

120, Calcd for C19H26O7N2Na, 417.1637.

Figure 120: Mass spectrum of compound 10.

The ESI-MS analysis in the positive mode of this compound revealed a base molecular peak ion at m/z 395, and this in turn showed the main fragment Y on MS2 spectrum at m/z 233 [M – 162 + H]+. Other intense fragments in MS2 spectrum were observed at m/z 185 [M–180 – 30 + H]+, which corresponded to the simultaneous loss of the sugar moiety along with the two methyl groups attached to the phenolic part of the molecule Figure 121.

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Figure 121: MS/MS2 spectrum of compound 10.

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CHAPTER 4 Results and Discussion

According to the 13C- NMR spectrum there are 19 carbons. As shown in Figure 122:

 7 aromatic carbons.  2 carbons belong to olefinic group.  3 carbon belong to aliphatic carbon.  2 carbon belong to methoxy group.  5 glycoside carbons. The 13C NMR, APT and Hetcor spectra Figures 122, 123 and 124, revealed the presence of nineteen carbon signals. From these, the three signals at δ 62.2, δ 61.4 and δ 61.2 were, respectively, assigned to the aliphatic oxygenated secondary carbons (CH2–O) for C-12, C-6′ and C-5′, respectively, and the four oxymethine carbon signals at δ 104.1, δ 73.7, δ 76.5 and δ 70.3 corresponded to C-1′, C-2′, C-3′ and C-4′, respectively Table 14. The two olefinic carbon signals at δ 129.9 and δ 128.7 corresponded to C-10 and C-11, respectively, while carbon signals at δ 55.6, δ 55.9 and δ 22.3 were ascribed to the methoxy groups at C-10′ and C-11′, and methyl group at C-7′, respectively. The carbon signals at δ 103.9 and δ 104.0 were assigned to the aromatic carbon (CH) at C-4 and C-7, respectively, and the five quaternary carbon signals at δ 134.5 at C-8, C-9 and δ 152.9 at C-5, C-6 and δ 133.9 at C-2 suggested a benzimidazole skeleton with three carbons substituted Figure 119, Table 14.

13 Figure 122: C- NMR spectrum of compound 10 (100 MHz, CD3OD).

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Figure 123: APT-NMR spectrum of compound 10 (100 MHz, CD3OD).

Figure 124: Hetcor-NMR spectrum of compound 10 (100 MHz, CD3OD).

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The 1H- NMR spectrum Figure 125, confirmed the previous suggestion. It displayed two proton signals for an olefin function group in trans system at δ 6.58 (1H, d, J = 16, H-10) and δ 6.36 (1H, dt, J = 16- 6, H-11), besides two aromatic proton signals at δ 6.77 (2H, s, H-4, H-7), one singlet proton signal at δ 4.88 (1H, s, NH, H-1) of the benzimidazole skeleton [210], and two methoxy group signals at δ 3.87 (3H, s, H-10′) and δ 3.54 (3H, s, H-11′). The configuration of glycosidic bond was β as based on the coupling constant of anomeric proton signal at δ 4.18 (1H, d, J = 8, H-1′) and identified as xylose, along with the five osidic proton signals being assigned at δ 3.17 (1H, t, J = 9.0, H-2′), δ 3.32 (1H, m, H-3′), δ 3.29 (1H, m, H-4′), δ 3.90 (1H, dd, J = 12- 6, H-5′eq) and δ 3.70 (1H, dd, J = 12- 6, H-5′ax) [211]. The spectrum also indicated the presence of two oxymethylene group signals at δ 4.25 (2H, dd, J = 6- 1.2, H-12) and δ 3.62 (2H, m, H-6′), and of three protons signals at δ 1.20 (3H, t, H- 7′) for the methyl group Table 14.

1 Figure 125: H- NMR spectrum of compound 10 (400 MHz, CD3OD).

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The analysis of the HMBC spectrum Figure 126 confirmed the suggested structure since it revealed cross peaks between the protons signal at δ 3.87 (OCH3– H-10′) with the carbon signal at δ 152.9 (C-5), the proton signals at δ 3.54 (OCH3– H-11′) with the carbon signal at δ 152.9 (C-6), the proton signal at δ 4.88 (H-1) with the quaternary carbon signal at δ 133.9 (C-2) of the benzimidazole skeleton, the proton signal at δ 6.58 (H-10) and the quaternary carbon signal at δ 133.9 (C-2) indicative of an olefin function group at (C-2), the proton signal at δ 6.36 (H-11) with the carbon signal at δ 62.2 (C-12). The sugar linkage was determined on the basis of the HMBC correlations of the anomeric proton H-1′ with the carbon signal at δ 62.2 (C-12) and proton H-3′ with the carbon signal at δ 61.4 (C-6′) Table 14.

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Figure 126: HMBC - NMR spectrum of compound 10 (400 MHz, CD3OD).

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Moreover, the COSY spectrum Figure 127 displayed cross peaks confirming the suggested structure, namely H-10 (δ 6.58) with H-11 (δ 6.36) and H-11 with H-12 (δ 4.25).

Figure 127: COSY - NMR spectrum of compound 10 (400 MHz, CD3OD). Along with those observed, for the sugar moiety: H-1′ (δ 4.18) with H-2′ (δ 3.17), H-2′ with H-3′ (δ 3.32), H-4′ (δ 3.29) with H-5′ax (δ 3.70), H-4′ with H-5′eq (δ 3.90), H-6′ (δ 3.62) with H-7′ (δ 1.20) Figure 128 and Table 14.

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Figure 128: COSY - NMR spectrum of compound 10 (400 MHz, CD3OD).

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Table 14. NMR spectral data of compound 10:

1H ppm) 13C ppm) HMBC

- 152.94 (C5, 6) - - 134.46 (C8, 9) - - 133.86 (C2) - 6.77 (2H, s, H-7, 4) 104.00 (C7, 4) - 6.58 (1H, d, J = 16, H-10) 129.86 (C10) 133.86 (C2) 6.36 (1H, dt, J = 16- 6, H-11) 128.66 (C11) 62.15 (C12) 4.88 (1H, s, H-1) - 133.86 (C2) 4.25 (2H, dd, J = 6- 1.2, H-12 ) 62.15 (C12) - 4.18 (1H, d, J = 8, H-1') 104.05 (C1') 62.15 (C12) 3.90 (1H, dd, J = 12- 6, H-5'eq) 61.15 (C5') - 3.87 (3H, s, H-10') 55.64 152.94 (C5) 3.54 (3H, s, H-10') 55.92 152.94 (C6) 3.70 (1H, dd, J = 12- 6, H-5' ax) 61.15 (C5') - 3.62 (1H, m, H-6') 61.35 (C6') 61.35 (C6') 3.32 (1H, m, H-3') 76.46 (C3') - 3.29 (1H, m, H-4') 70.25 (C4') - 3.17 (1H, t, J = 9, H-2') 73.71 (C2') - 1.20 (1H, t, H-7') 22.26 (C7') -

Also note that all the structural data inferred from the NMR spectrum was in accordance with the previous ESI-MS analysis Figure 121. Compound 10 was thus found to be a new benzimidazole derivative and was named microphybenzimidazole. Its structure is similar to compound Arvense 1 which linked with glucose [212]. Thymelaeaceae family rarely alkaloids contain and for the first time reported was β-adenosine isolated from Edgeworthia gardneri [213].

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Figure 129: Structure of compound 10 microphybenzimidazole.

4.2 Structure identification of Gnidia somalensis Gilg. var.sphaerocephala (Bak.)Gastald compounds by HPLC-DAD-MS analysis:

The phenolic profile of the Gnidia somalensis Gilg. var.sphaerocephala (Bak.) Gastald MeOH: H2O (7:3) extract was analyzed by reversed-phase HPLC-DAD-MS in negative mode [197], the spectrometry chromatogram showed in Figure 130. Data acquisition was carried out with Xcalibur® data system (Thermo Finnigan, San Jose, CA, USA) [199]. The identification of phenolic compounds focused on clear compound peaks and was determined by comparison of the ESI–MSn data to that of the standards. The structure of phenolic compounds was also confirmed by the MS/MS data published in the literature. The compared literature data with our results led us to identify 12 polyphenol compounds [214- 223].

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Figure 130: HPLC-DAD-MS spectrometry chromatogram of G. somalensis extract.

4.2.1 Structural identification of hydrocinnamic acid derivatives separated from G. somalensis

MeOH: H2O (7:3) extract:

In this part of our work, the structural identification of these compounds rely on mass spectrometry and comparison with literature data, along with the typical UV absorption spectra of hydroxycinnamic acid and its derivatives (consisting of quinic acid or other polyhydroxy-aliphatic acids) which have a λ max between 305 and 330 nm (band I) and a shoulder between 290 and 300 nm (band II) [224]. These derivatives were separated from G. somalensis MeOH: H2O (7:3) extract.

4.2.1.1 Structural identification of compounds G1, G2 and G3:

The compounds G1, G2 and G3 were separated at retention time 14.35, 14.82 and 15.29 min respectively showed characteristic UV absorption spectrum at λ max 296, 324 nm consistent with that described for caffeic and caffeoylquinic acid derivatives [225, 226]. From spectrometry chromatogram of G. somalensis MeOH: H2O (7:3) extract, the peaks at retention time 14.35, 14.82 and 15.29 min with the precursor ion at m/z 515 [M – H]¯ were found to be diCQA derivatives. The mass spectrometric detection of molecular ions and the interpretation of their fragmentation pattern 2 3 in the MS and MS experiment allows to identify isomeric compounds mono and di CQA derivatives acids and to precisely distinguish between isomeric compounds since different

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CHAPTER 4 Results and Discussion fragmentation patterns can be attributed to stereo chemical relationships [227, 228]. Based on these particularities thoroughly described in the literature, the compounds were identified as 3, 4-diCQA, 3, 5-diCQA and 4, 5-diCQA, respectively.

According to spectral data of ESI-MS the G1 is identified as 3,4-di-O-[E]- caffeoylquinic acid by comparison of spectral data of the mentioned compound with the literature [229], allowed us to identify the structure of this compound. The mechanism of fragmentation of 4,5-di-O-[E]- caffeoylquinic acid is proposed as the following:

The ionization started initially with the more acidic proton of the molecule is the COOH, thus in all the quinic acid derivatives followed by migration of proton. Considering the deprotonated molecular ion (A) at m/z 515 [M – H]¯ , we propose the loss of first caffeoyl unit to form B at m/z [M – H– ¯ 2 C9H6O3] which is the major fragment in MS , this is because of the loss of caffeoyl (162 mau) Figures 134 (page 144). From this point referring to the fragmentation of compound G1 we propose the structure of compound G2 as 3,5-di-O-[E]- caffeoylquinic acid and compound G3 as 4,5-di-O- [E]- caffeoylquinic acid. We can distinguish between the two structures by mass spectra of isomers 3, 5-diCQA (x) Figure 139 (page 148) and 4, 5-diCQA (y) Figure 145 (page 153).

The elimination of the second molecule of water from the fragment at m/z 353 led to the fragment ¯ (b) at m/z 317 [M – 2H2O] . We continue in this way the loss of another molecule of water will give a complete aromatization of a benzoic acid molecules to afford the fragment (d) at m/z 299 [M – ¯ ¯ 3H2O] . Finally the loss of CO2 (- 44 amu) to give the fragment at m/z 255 [M – 3H2O– CO2] Figures 135 (page 145), 141 (page 150) and 147 (page 155).

For the compound G1 at retention time 14.35 min with the parent ion at m/z 515 [M – H] ¯ produced fragment ions at m/z 353 corresponding to [M– H– caffeyol acid]¯, with the relative intensities of fragments of MS2 experiment: 353 (100); 335 (10%); 173 (6%); 203 (5%); 299(4%); 179 (4%); 191(2%); 317(1%); 497 (1%). The daughter fragment ions m/z 353 produced fragment ions with the 3 ¯ relative intensities of fragments of MS experiment: 173 (100%) [quinic aicd– H2O–H] ; 179 (79%) [caffeic acid– H]¯; 191 (73%) [quinic acid– H]¯; 154 (1%). This pattern of fragmentation assigned to 3, 4-diCQA [214], Figure 133 (page 143). The mass spectrum of electrospray ionization of this compound at m/z 515 [M– H]¯ which suggest molecular mass of 516 corresponding to molecular formula C25H24O12 with unsaturation number 14. The mass spectrum analysis carried out on ESI(-)

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MS/MS in MS2 and MS3 experiment. Figures 131, 132 and the fragmentation behavior of compound G1 which proposed in three pathways presented in Figures 144-146.

Figure 131: Negative ion MS2 spectrum of compound G1 (3, 4-diCQA).

Figure 132: Negative ion MS3 spectrum of compound G1 (3, 4-diCQA).

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Figure 133: Structure of compound G1 (3, 4-diCQA).

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Pathway I: O O OH OH O C O O O O OH C O OH O OH T.P HO OH O-Caffeoyl O OH [3, 4-diCQA- H] m/z =515 OH (A) OH

O OH O OH O O O H C O C O HO HO O O-Caffeoyl OH O-Caffeoyl OH O H

C O OH O OH C O O HO C OH O HO O OH OH OH O O

OH O OH O [3-O-[E]-Caffeoylquinic acid- H] H m/z = 162 m/z =353 (B)

O O OH C C OH HO HO O OH O OH C OH HO O OH O O OH m/z =191 O Figure 134: Proposed fragmentation patterns of compound G1 (Pathway I).

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Pathway II:

Figure 135: Proposed fragmentation patterns of compound G1 (Pathway II).

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Pathway III:

Figure 136: Proposed fragmentation patterns of compound G1 (Pathway III).

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Compound G2 at retention time 14.82 min with the parent ion at m/z 515 [M – H]¯ produced fragment ions at m/z 353 corresponding to [M– H– caffeyol acid] ¯, with the relative intensities of fragments of MS2 experiment: 353 (100%); 203 (7%); 299 (6%); 335 (4%); 179( 2%); 191 (1%). The daughter fragment ions m/z 353 produced fragment ions with different and distinguished relative intensities of fragments of MS3 experiment: 191 (100%) [quinic aicd–H] ¯; 179 (55%) [caffeic acid– H] ¯ ; 173 ¯ (48%) [quinic aicd– H2O–H] ; 135 (6%). This pattern of fragmentation assigned to 3, 5-diCQA [214], Figure 139. The mass spectrum of electrospray ionization of this compound at m/z 515 [M– ¯ H] which suggest molecular mass of 516 corresponding to molecular formula C25H24O12 with unsaturation number 14. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2 and MS3 experiment. Figures 147, 138 and the fragmentation behavior of compound G2 which proposed in three pathways presented in Figures 140-142.

Figure 137: Negative ion MS2 spectrum of compound G2 (3, 5-diCQA).

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Figure 138: Negative ion MS3 spectrum of compound G2 (3, 5-diCQA).

Figure 139: Structure of compound G2 (3, 5-diCQA).

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Pathway I: OH OH

OH OH

[3, 5-diCQA- H] m/z =515 (A)

O O OH O O O O OH OH OH C T.P C O OH O HO OH O OH O

O O

O O-Caffeoyl O O-Caffeoyl C O O O HO O H C O OH HO HO O OH OH O O

OH H

O OH

C O

O O OH C O HO O OH O-Caffeoyl O O O H OH C O O HO m/z = 162 OH HO [5-O-[E]-Caffeoylquinic acid- H] O

m/z =353 (B) OH

C O O

C OH HO OH HO O O O

OH C OH HO O OH HO Figure 140: Proposed fragmentation patterns of compound G2 (Pathway I).

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Pathway II:

Figure 141: Proposed fragmentation patterns of compound G2 (Pathway II).

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Pathway III:

Figure 142: Proposed fragmentation patterns of compound G2 (Pathway III).

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According to the different retention time at 15 min of compound G3 with the parent ion at m/z 515 [M – H]- ; it’s another isomer of diCQA. The parent ion produced at m/z 515 [M – H] ¯ fragment ions at m/z 353 corresponding to [M– H– caffeyol acid] ¯, with the relative intensities of fragments of MS2 experiment: 353 (100%); 203 (14%); 299 (12%); 317 (9%); 335 (6%); 255 (4%);173 (2%). The daughter fragment ions m/z 353 produced fragment ions with different and distinguished than other relative intensities of diCQA isomer fragments of MS3 experiment: 173 (100%) [quinic aicd– ¯ ¯ ¯ H2O–H] ; 179 (59%) [caffeic acid– H] ; 191 (25%) [quinic acid– H] ; 135 (5%). This pattern of fragmentation assigned to 4, 5-diCQA [214], Figure 145. The mass spectrum of electrospray ionization of this compound at m/z 515 [M– H]¯ which suggest molecular mass of 516 corresponding to molecular formula C25H24O12 with unsaturation number 14. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2 and MS3 experiment. Figures 143, 144 and the fragmentation behavior of compound G3 which proposed in three pathways presented in Figures 146-148.

Figure 143: Negative ion MS2 spectrum of compound G3 (4, 5-diCQA).

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Figure 144: Negative ion MS3 spectrum of compound G3 (4, 5-diCQA).

Figure 145: Structure of compound G3 (4, 5-diCQA).

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Pathway I: OH

[4, 5-diCQA- H] m/z =515 (A)

O O Caffeoyl -O O O O O OH O C O T.P C O HO OH O OH HO OH OH OH

O O-Caffeoyl O O-Caffeoyl C O O O HO O H C O OH HO HO O OH OH O O

H OH

O OH

C O

O O OH C O HO O OH O-Caffeoyl O O O H OH C O O HO m/z = 162 OH HO [5-O-[E]-Caffeoylquinic acid- H] O

m/z =353 (B) OH

C O O

C OH HO OH HO O O O m/z = 191 OH C OH HO O OH HO Figure 146: Proposed fragmentation patterns of compound G3 (Pathway I).

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Pathway II:

Figure 147: Proposed fragmentation patterns of compound G3 (Pathway II).

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Pathway III:

Figure 148: Proposed fragmentation patterns of compound G3 (Pathway III).

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4.2.1.2 Structural identification of compound G4:

The mass spectrum of electrospray ionization of this compound at m/z 353 [M– H]¯ which suggest molecular mass of 354 corresponding to molecular formula C16H18O9 with unsaturation number 8. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2 experiment. Figure 149.

The compound G4 was separated at retention time 7.65 min showed characteristic UV absorption spectrum at λ max 294, 323 nm consistent to that described for caffeic and caffeoylquinic acid derivatives [225, 226]. From spectrometry chromatogram of G. somalensis MeOH: H2O (7:3) extract, the peak at retention time 7.65 min with the precursor ion at m/z 353 [M– H]¯ produced ¯ ¯ fragment ions at m/z 191 [quinic acid–H] , m/z 179 [caffeic acid– H] and m/z 135 [caffeic acid– ¯ 2 CO2–H] in MS experiment. The fragmentation behavior of compound G4 which proposed in two pathways presented in Figures 150, 151. This pattern of fragmentation assigned to caffeoylquinic acid. By comparing our results with literature, we suggest compound G4 is caffeoylquinic acid [214].

Figure 149: Negative ion MS2 spectrum of compound G4 (4-O-[E]-Caffeoylquinic acid).

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Figure 150: Proposed fragmentation patterns of compound G4 (4-O-[E]-Caffeoylquinic acid) (Pathway I).

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Pathpway II:

O O O OH OH O O H C O Inversion HO O OH O OH HO OH m/z = 353 [4-O-[E]-Caffeoylquinic acid- H] OH OH

m/z =353 OH -H2O

O O O O O

C O 2/Inversion O HO HO OH OH OH Transfer H+ O OH m/z = 335 OH -H O 2 OH

O OH O O

C C O O O O OH

C OH m/z = 317 OH O

OH OH m/z = 173 O m/z = 162

Figure 151: Proposed fragmentation patterns of compound G4 (4-O-[E]-Caffeoylquinic acid) (Pathway II).

4.2.1.3 Structural identification of compound G5:

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CHAPTER 4 Results and Discussion

The mass spectrum of electrospray ionization of this compound at m/z 367 [M– H] ¯ which suggest molecular mass of 368 C17H20O9 with unsaturation number 8. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2 experiment. Figure 152.

The compound G5 was separated at retention time 12, 28 min showed characteristic UV absorption spectrum at λ max 290, 324 nm consistent to that described for caffeic and caffeoylquinic acid derivatives [225, 226]. From spectrometry chromatogram of G. somalensis MeOH: H2O (7:3) extract, the peak at retention time 12, 28 min with the precursor ion at m/z 367 [M– H] ¯ produced 2 ¯ ¯ fragment ions in MS experiment at m/z 193 [ferulic acid– H] , m/z 191 [quinic acid– H] , m/z 179 ¯ [caffeic acid– H] obtained by the loss of CH3 from ferulic acid . The fragmentation behavior of compound G5 which proposed in pathway presented in Figure 153. This pattern of fragmentation assigned to feruloylquinic acid. By comparing our results with literature, we suggest compound G5 is feruloylquinic acid [215].

Figure 152: Negative ion MS2 spectrum of compound G5 (feruloylquinic acid).

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Figure 153: Proposed fragmentation patterns of compound G5 (feruloylquinic acid).

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4.2.1.4 Structural identification of compound G6:

The mass spectrum of electrospray ionization of this compound at m/z 337 [M– H]¯ which suggest molecular mass of 338 C16H18O8 with unsaturation number 8. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2 experiment. Figure 154.

The compound G6 was separated at retention time 11.7 min showed characteristic UV absorption spectrum at λ max 311nm consistent to that described for caffeic and caffeoylquinic acid derivatives

[225, 226]. From spectrometry chromatogram of G. somalensis MeOH: H2O (7:3) extract, the peak at retention time 12, 28 min with the precursor ion at m/z 337 [M– H] ¯ produced a daughter ion in MS2 experiment at m/z 191[quinic acid– H]¯, and m/z 163 [Coumaric acid– H] ¯ . The fragmentation behavior of compound G6 which proposed in pathway presented in Figure 155. This pattern of fragmentation assigned to coumaroylquinic acid. By comparing our results with literature, we suggest compound G6 is coumaroylquinic acid [214].

Figure 154: Negative ion MS2 spectrum of compound G6 (Coumaroylquinic acid).

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Figure 155: Proposed fragmentation patterns of compound G6 (coumaroylquinic acid).

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4.2.2 Structural identification of flavonoid derivatives separated from G. somalensis MeOH: H2O (7:3) extract:

The flavonoids are characterized by mass spectroscopy electrospray ionization in negative mode ESI (-) MS/MS. The flavonoids nomenclature of different fragments were established according to (Domon et al., 1998) and (Ma et al., 1997) rules [230, 231], which in this figure, suggests an example of fragmentation, Figure 156. Evaluation of UV absorption spectra of flavonoid offers additional information for structural characterization. Characteristic absorption maxima of flavonols exhibit band I usually in the 300-380 nm region and band II from 240 to 280 nm. Depending on the B-ring oxidation pattern, band II can be observed as either one or two peaks, for 4’-oxygenated and for 3’,4’,(5’)-oxygenated flavonols [232].

Figure 156: Nomenclature of fragments of ESI-MS in negative mode of flavonols according to Domon and Ma rules [230, 231]. The ions contained aglycones are indicated by K, IXj, Yj, where j is the number of interglycosidic cut bond. K and I indicate the cut in the sugar cycle. The ions k, I A (or k, I B) are specific cuts of the aglycone, where k and I indicate the bond cut in the cycle C.

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4. 2.2.1 Quercetin derivatives:

The chemical structures of flavonoid Compounds are based on the UV absorption and MS spectral data [233]. The compounds presented UV maxima are in agreement with a flavonol or a 3-O- substituted flavonol and were identified as quercetin or methyl quercetin derivatives. Three flavonol glycosides and Three methyl quercetin derivatives were detected in the G. somalensis MeOH: H2O (7:3) extract. Based on comparison of their retention time, UV absorption and mass spectral data with those of available reference compounds and those from the literature [216-221, 222 and 223].

4.2.2.1.1 Structural identification of compound G7:

The mass spectrum of electrospray ionization of this compound at m/z 477 [M– H] ¯ which suggest molecular mass of 478 corresponding to molecular formula C21H18O13 with unsaturation number 12. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2 and MS3.

Assigns the principal fragment of flavonoid confirmed with MS2. The spectrum fragmentation of ¯ ¯ [M – H] ion at m/z 447 indicate the loss of hexose (-177 mau) characterized by the fragment Y0 at m/z 301 which indicates first the substitution preferentially in position 3 of the aglycone [198]. ¯ Secondly the value of fragment Y0 at m/z 301 oriented us towards the aglycone to be suggested as quercetin. The presence of other pics corresponding to a typical flavonol type [234].

Also we observed first the ion 1,3A¯ at m/z 151 in MS3 experiment comes from the fragmentation of ¯ cycle by retro-Diels-Alder reactions (RDA), on the second hand we notice that the ion Y0 is ¯ precursor of fragment at m/z 271[Y0 –CH2O] obtained by loss of neutral molecule CH2O (-30 mau), 1,2 ¯ ¯ ¯ as well as the product ion A at m/z 179 [Y0¯ – H – cycle B– CH2] , whereas the fragment Y0 [M – H–177] ¯ at m/z 301 indicate the rupture at the level of sugar of type hexose Figures 157, 158. The fragmentation behavior of compound G7 which is proposed in two pathways presented in Figure 159. By comparing our results with literature, we suggest compound G7 is quercetin-3-O- glucuronide [235, 217].

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Figure 157: Negative ion MS2 spectrum of compound G7 (quercetin-3-O-glucuronide).

Figure 158: Negative ion MS3 spectrum of compound G7 (quercetin-3-O-glucuronide).

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Figure 159: Proposed fragmentation patterns of compound G7 (Pathway I and II).

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4.2.2.1.2 Structural identification of compound G8: The mass spectrum of electrospray ionization of this compound at m/z 507 [M– H] ¯ which suggest molecular mass of 508 corresponding to molecular formula C22H20O14 with unsaturation number 12. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2, MS3 and MS4. Figures 160- 162.

The peak from spectrometry chromatogram of G. somalensis MeOH: H2O (7:3) extract at retention ¯ ¯ time 13.8 min exhibited [M – H] ion at m/z 507 which produced daughter fragment ion Y0 at m/z 331 [M –177– H]¯ in MS2 experiment, indicate that loss of hexose moiety. According to the UV absorption spectrum at λ max 257, 351 nm illustrate that flavonol compound [216], and the produced fragment ion m/z 331 is a distinguished fragment ion of laricitrin derivatives, which confirmed by 3 the base peak of fragment ion at m/z 316 in MS experiment after loss of molecule CH3 (-15 mau) [236]. As well as the peak at m/z 316 produced a base peak of fragment ion 1,3B¯ at m/z 166 in MS4 experiment. Hence this pattern of fragmentation assigned to laricitrin-hexuronide. The fragmentation behavior of compound G8 which proposed in pathway presented in Figure 163. By comparing our results with literature, we suggest compound G8 is laricitrin-hexuronide [218].

Figure 160: Negative ion MS2 spectrum of compound G8 (laricitrin-hexuronide).

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Figure 161: Negative ion MS3 spectrum of compound G8 (laricitrin-hexuronide).

Figure 162: Negative ion MS4 spectrum of compound G8 (laricitrin-hexuronide).

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Figure 163: Proposed fragmentation patterns of compound G8 (laricitrin-hexuronide).

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4.2.2.1.3 Structural identification of compound G9: The mass spectrum of electrospray ionization of this compound at m/z 639 [M– H] ¯ which suggest molecular mass of 640 corresponding to molecular formula C27H32O17 with unsaturation number 12. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2. Figure 164. The UV spectral data of compound G9 at λ max 269, 354 nm allowed us to identify the compound as flavonol derivatives [216], along with the peak at retention time 13.4 min with the precursor ion at m/z 639 [M – H]¯ led to the flavonol glycoside compound. The parent ion m/z 639 produced 2 ¯ fragment ions at m/z 493 in MS experiment comes from loss of rhmnosyl moiety [Y1 – C6H10O4– H]¯, and observe the fragment ions at m/z 331 indicate that compound G9 is a one of laricitrin ¯ ¯ derivatives, also the loss of glucoside moiety [Y0 – C6H10O5– H] , the fragment ions at 316 indicate loss of CH3 group (-15 mau) [236]. The fragmentation behavior of compound G9 which proposed in pathway presented in Figure 165. Therefore this pattern of fragmentation assigned to laricitrin- rutinoside. By comparing our results with literature, we suggest compound G9 is laricitrin-rutinoside [216].

Figure 164: Negative ion MS2 spectrum G9 (laricitrin-rutinoside).

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Figure 165: Proposed fragmentation patterns of compound G9 (laricitrin-rutinoside).

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4.2.2.1.4 Structural identification of compound G10: The mass spectrum of electrospray ionization of this compound at m/z 345 [M– H] ¯ which suggest molecular mass of 346 corresponding to molecular formula C17H14O8 with unsaturation number 10. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2. Figure 166.

From spectrometry chromatogram of G. somalensis MeOH: H2O (7:3) extract, the peak at retention time 19.96 min with the parent ion at m/z 345 [M – H] ¯ produced daughter ion at m/z 315 in MS2 ¯ spectrum corresponding to [M –C2H6– H] which elucidate that compound loss molecule (-30 mau). ¯ The presence of fragment ion at m/z 330 [M –CH3– H] due to loss molecule (-15 mau) from the parent ion m/z 345 which confirm linked the compound G10 with two methyl groups according to the daughter ion at m/z 315 [236, 237]. The MS2 experiment shows fragment ion at m/z 301 ascribed to be quercetin derivatives. This pattern of fragmentation ions in comparison with literature and UV absorption spectral data assigned to myricetin- di methyl ether. The fragmentation behavior of compound G10 which proposed in pathway presented in Figure 167. By comparing our results with literature, we suggest compound G10 is Syringetin [238, 222].

Figure 166: Negative ion MS2 spectrum of compound G10 (Myricetin- di methyl ether).

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Figure 167: Proposed fragmentation patterns of compound G10 (Myricetin- di methyl ether).

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4.2.2.1.5 Structural identification of compound G11: The mass spectrum of electrospray ionization of this compound at m/z 359 [M– H] ¯ which suggest molecular mass of 360 corresponding to molecular formula C18H16O8 with unsaturation number 10. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2, MS3 and MS4. Figures 168- 170.

The fragmentation pattern of the ion at m/z 359 [M – H]- at retention time 21 min showed a major ¯ 2 fragment ion at m/z 344 [M –CH3– H] in MS experiment [236], corresponding to the loss of the methyl group. The peak at m/z 344 produced base fragment ion at m/z 329 in MS3 spectrum which 4 display loss of other CH3 group (-15 mau). As well as in MS experiment the peak at m/z 344 produced base fragment ion at m/z 314 indicate to loss methyl group. According to the UV absorption spectral data at λ max 258, 351 and MS2 fragments at m/z 301; the compound belongs to the quercetin derivatives. In comparison with literature; this pattern of fragmentation assigned to quercetagetin-trimethyl ether. The fragmentation behavior of compound G11 which proposed in pathway presented in Figure 171. By comparing our results with literature, we suggest compound G11 is quercetagetin-trimethyl ether [239].

Figure 168: Negative ion MS2 spectrum of compound G11 (Quercetagetin-trimethyl ether).

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Figure 169: Negative ion MS3 spectrum of compound G11 (Quercetagetin-trimethyl ether).

Figure 170: Negative ion MS4 spectrum of compound G11 (Quercetagetin-trimethyl ether).

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Figure 171: Proposed fragmentation patterns of compound G11 (quercetagetin-trimethyl ether).

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4.2.2.1.6 Structural identification of compound G12: The mass spectrum of electrospray ionization of this compound at m/z 373 [M– H] ¯ which suggest molecular mass of 374 corresponding to molecular formula C19H18O8 with unsaturation number 10. The mass spectrum analysis carried out on ESI (-) MS/MS in MS2. Figure 172. The peak at retention time 23.9 min exhibited base ion at m/z 373 [M – H]¯. Compound G12 was identified as a quercetin derivatives based on MS2 fragment at m/z 301. The mass spectrometric behavior of compound G17 allowed to detect four methyl groups in this compound supported by ¯ the fragment ion at m/z 358 [M –CH3– H] [236], followed by loss other methyl group at m/z 343 ¯ [M –C2H6– H] , along with to the fragment ion at m/z 328 which elucidate loss (-15 mau), finally ¯ the fragment ion at m/z 313 [M –C4H12– H] confirm loss the fourth methyl group from compound G17. The comparison of MS2 experiment data with the literature assigned to casticin. The fragmentation behavior of compound G12 which proposed in pathway presented in Figure 173. By comparing our results with literature, we suggest compound G12 is casticin [219, 223].

Figure 172: Negative ion MS2 spectrum of compound G12 (Casticin).

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Figure 173: Proposed fragmentation patterns of compound G12 (Casticin).

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All the compounds isolated from G. somalensis extrac are identified by RT, λmax and mass spectra, as summarized in the Table 14.

Table 14. Compounds of G. somalensis extrac Identified by HPLC-DAD- ESI-MSn:

G RT λmax m/z MS2 Information obtained from direct Identiﬁcation (min) [M-H]- injection 1 7.65 294, 323 353 353= 191, 179, - Caffeoylquinic acid 173, 135 2 11.7 311 337 337= 191, 163 - Coumaroylquinic acid 3 12,28 290, 324 367 367= 193, 191, 179 - feruloylquinic acid

4 13.4 269, 354 639 639= 493, 331, 316 - Laricitrin rutinoside

5 13.8 257, 351 477 477= 301 477= 301 quercetin-3-O- Ms3(301)=179; 151 glucuronide 6 13.8 257, 351 507 507= 331, 316, 241 507= 331; 316; 241 laricitrin-hexuronide Ms3(331)=316; 209; 166 Ms4(316)=166;287;271;243;138 7 14.35 296, 324 515 515= 353 515=353(100); 335(10); 173(6); 3,4-Di-O- 203(5); 299(4); 179(4); 191(2); caffeoylquinic aci 317(1);497 (1). Ms3(353)= 173(100); 179(79); 191(73); 154(1) 8 14.82 296, 324 515 515= 353 515=353(100); 203(7); 299 (6); 335 3,5-Di-O- (4); 179(2); 191(1). caffeoylquinic acid Ms3(353)=191(100); 179(55); 173(48). 9 15 296, 324 515 515= 353 515=353(100); 203(14); 299(12); 4,5-Di-O- 317(9); 335(6); 255(4); 179(2). caffeoylquinic acid Ms3(353)=173(100); 179(56); 191(25);135 (5). 10 19.96 236, 284, 345 330;315;301 - Myricetin –di 367 methyl ether 11 21 258, 351 359 359=344 359=344; 315; 223; 180. Quercetagetin- Ms3(344)=329; 301; 175. triamethyl ether Ms4(329)=314; 301; 286; 175.

12 23.9 255, 373 373= 359, 343, - Casticin 270sh, 328, 313 346

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4.3 Bioassays results and discussion: 4.3.1 Antiproliferative activity: The antiproliferative activities of all the samples from species T. microphylla; fractions (F6, F6- W42, F6-C5, F6-C9), pure compounds (2, 3, 5, 6, 7 and 10) and the standard (5-FU) were investigated on eight concentrations (5, 10, 20, 30, 40, 50, 75 and 100 µg/mL) against HeLa and C6 cells using BrdU cell proliferation ELISA assay Figure 32 (page 57). The xCELLigence was used to analyze the ability of extract B, the fractions (F5, F7, B-C8), and compound 1 to inhibit cell growth of HeLa cell line at the concentrations (10, 50, 100 and 250 μg/mL) Figure 32 (page 57).

The fraction F5 from the extract F exhibited dose- and time-dependent effects on HeLa cell growth by xCELLigence assay especially at 250 µg/mL concentration and we can observe the inhibitory effect even over time. Compound 1 (matairesinol) was isolated from fraction F5 have a weak antiproliferative effect against HeLa cells in comparison with the inhibition of fraction F5. The potency of inhibition for HeLa at 250 µg/mL concentration follows the order: F5 > Compound 1. Figure 174. Matairesinol is a central precursor in the biosynthesis of numerous lignans, including the important antiviral and anticancer [240]. Pervious study had been reported of lignin mixture isolated from the stem wood of Cedrus deodara consisted of (-)-wikstromal (75 - 79 %), (-)- matairesinol (9 - 13 %) and benzylbutyrolactol (7 - 11 %) showed significant dose-dependent effects against several cancer cell lines such as cervix, colon, liver, prostate and neuroblastoma at 10, 30 and 100 mg/mL, which displays the importance of lignin compounds [241].

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Compound 1

250µg/ml■ 100 µg/ml ■ 50 µg/ml ■ Medium■ Control■

Figure 174: Antiproliferative activity of F5 and compound 1 against HeLa cell lines.

On the other hand compound 2 (prestegane B) was isolated from fraction F5 showed weak activity compare with 5-FU cells against HeLa by using BrdU cell proliferation ELISA assay and high activity at 100 µg/mL and 75 µg/mL against C6 cell line and increase of activity with increasing concentration Figure 175. Among the pure compounds in the present study, the highest antiproliferative effect on C6 cell lines was observed for compound 2, it has shown selective activity on C6 cell lines in comparison with the performed study against five Human Cancer Cell Lines HL- 60 (human myeloid leukemia), SMMC-7721 (hepatocellular carcinoma), A549 (lung cancer), MCF- 7 (breast cancer), and SW480 (colon cancer), which exhibited inactive against this five Human Cancer Cell [242].

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Figure 175: Antiproliferative activity of compound 2 against C6 cell and HeLa cell lines.

Fraction F6 which separated from CH2Cl2: MeOH (1:1) extract and F6-W42 which was subfraction of F6 have shown high activity at 100 µg/mL against C6 cell line, Figure 176 displays the antiproliferative activities of these constituents and compound 3 (daphnoretin) which isolated from F6-W42 against C6 cell and HeLa cell. It is clear increases the activity with increasing concentration for these samples. Compound 3 has shown moderate activity at 100 µg/mL, and the potency of inhibition against C6 follows the order: F6 > F6-W42 > 5-FU> Compound 3. The antiproliferative effect of F6, F6-W42 and compound 3 against HeLa cell at 100 µg/mL follows the order: 5-FU > F6-W42 > Compound 3 >F6. Compare with HeLa cell antiproliferative effect of F6, F6-W42 and compound 3, all of these samples have shown a selective antiproliferative activity against C6 cell line. Compound 3 (daphnoretin) had been reported to inhibit the growth of A549 (lung cancer cells)

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[243], leukaemia [244], as well as against four cancer cells Huh7 (human hepatocellular carcinoma cells), A2058 (melanoma gastric cancer cells), B16 (mice melanoma cancer cells) and AGS (human gastric cancer cells) [245]. From the overall results suggest that daphoretin may have potential as a novel anticancer agent.

Figure 176: Antiproliferative activity of compound 3, F6 and F6-W42 against C6 cell and HeLa cell lines.

Also, F6-C5 and F6-C9 are a subfraction of F6, compound 5 (microphynolide A) from F6-C5 and compound 6 (microphynolide B) from F6-C9 are spiro lactones type and the first time reported compounds with the rare spiro-bis-γ-lactone structure (4’,6’-Diacetyl-viburnolide A), (4’,6’- Diacetyl-12- coumaroyl-viburnolide A) and (Tetraacetylviburnolide A), in the family of Thymelaeaceae [95].

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The subfraction F6-C5 showed significant activity more than F6 and the control 5-FU at 100 µg/mL and 75 µg/ mL, the potency of inhibition order against C6 as follows: F6-C5> F6> 5-FU> compound 5. The antiproliferative activities of F6, F6-C5, compound 5 and 5-FU at 100 µg/mL exhibit inhibition against HeLa cell follows the order: 5-FU > Compound 5> F6-C5 > F6. Compare with HeLa cell antiproliferative effect, the fraction F6 and subfraction F6-C5 have shown a selective antiproliferative activity against C6 cell line. Figure 177.

Figure 177: Antiproliferative activity of compound 5, F6 and F6-C5 against C6 cell and HeLa cell lines.

The fraction F6 and F6-C9 were found to be more potent than 5-FU at 100 and 75µg/mL. The antiproliferative activities of compound 6, F6-C9 and F6 against HeLa cell showed weak activity compare with 5-FU at the highest concentration. At 100 µg/mL the potency of inhibition against

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HeLa follows the order: 5-FU > Compound 6> F6-C9>F6. Compare with HeLa cell antiproliferative effect, F6 and F6-C9 have shown a selective antiproliferative activity against C6 cell line. Figure 178. Compound 5 and 6 have shown inactive behavior against C6 cell and HeLa cell lines and this maybe refer to the Murray and Bradshaw (1966) explain, which discussed a possible role of these substances as carbohydrate storage compounds or as products of a detoxification mechanism that removes phenolic compounds [246].

Figure 178: Antiproliferative activity of compound 6, F6 and F6-C9 against C6 cell and HeLa cell lines.

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In comparison with the fraction F7 antiproliferative activity of HeLa cell lines Figure 179, and compound 7 (trans-tiliroside) Figure 180, which isolated from fraction F7; compound 7 has shown less activity than fraction F7 at 100 µg/mL concentration.

According to Figure 180, the antiproliferative activities of compound 7 has shown activity with increasing depending to concentration against HeLa cell lines and showed a moderate activity in comparison with 5-FU at high concentrations 100 and 75 µg/mL. However the antiproliferative activities of compound 7 (trans-tiliroside) against C6 cell lines showed no inhibit proliferation at low concentrations 5, 10 and 20 µg/mL and exhibit activity with increasing depending to concentrations at 30 µg/mL - 100 µg/mL. Tiliroside which isolated from Daphne genkwa Sieb.et Zucc. also, had been reported a moderate inhibit proliferation in six cancer cell lines; Huh 7 (human hepatocellular carcinoma cells), A 2058 (human skin cancer cells), MDA-MB-231 (breast cancer cell line), MCF-7 (human breast cancer cells), COLO 205 (human colon cancer cells), AGS (human gastric cancer cells) and HT-29 [245]. Which have demonstrated to inhibit proliferation in many types of cultured cancer cell lines.

Figure 179: Antiproliferative activity of F7 against HeLa cell line.

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Figure 180: Antiproliferative activity of compound 7 against C6 cell and HeLa cell lines.

Compound 10 (microphybenzimidazole) has been isolated from fraction B-C8 which it’s separated from the extract B (70% MeOH- H2O), both of them fraction B-C8 and extract B exhibited dose- and time-dependent effects on HeLa cell growth by xCELLigence assay. In comparison the antiproliferative activities of fraction B-C8 and extract B with Compound 10 (microphybenzimidazole) on HeLa cell, we can observe that the pure compound has a less activity than the fraction and the extract which isolated from, fraction B-C8 and extract B determined activity with increasing depending to concentration against HeLa cell lines Figure 181. According to Figure 182, the antiproliferative activities of Compound 10 (microphybenzimidazole) observed a weak activity with increasing depending to concentration against C6 cell. The benzimidazole nucleus is an important heterocyclic ring, because of its synthetic utility and broad range of pharmacological activities. The benzimidazole ring, containing two nitrogen atoms, plays an important role in the constitution of many of the natural products as well as synthetic compounds. Benzimidazole derivatives possess various pharmacological activities including antibacterial [247], antifungal [248], antiviral [249], antiparasitic [250], antiprotozoal [251], antihelmintic [252], anti- inflammatory [253] anti-ulcer [254], anti-hypertensive [255], anticonvulsant [256], anticancer activity

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CHAPTER 4 Results and Discussion against Ehrlich ascites carcinoma cell (EACC) [257], and some new benzimidazole-4,7-diones were reported that the compounds perform excellent cytotoxic activity against colon (HT29), breast (T47D) and lung (A549) cancer cell lines and shown the lowest activity [258]. Although the compound 10 is a benzimidazole derivative and demonstrated a weak growth inhibitory activity against HeLa cell and C6 cell lines and has almost the same inhibitory activity with compare the results with the similar compound (Arvense 1) against HeLa cell and C6 cell lines [212]. The linked of sugar in compound (Arvense 1) and sugar with addition of an ethyl group on 3′ oxygen atom in Compound 10 (microphybenzimidazole), perhaps reduced their effectiveness.

250µg/ml■ 100 µg/ml ■ 50 µg/ml ■ Medium■ Control■

B-C8

Figure 181: Antiproliferative activity of B extract and B-C8 fraction against HeLa cell line.

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Figure 182: Antiproliferative activity of compound 10 against C6 cell and HeLa cell lines.

In conclusion, it is clear that the results showed the highest effect of pure compounds on HeLa cell line is compound 7 and for C6 cell line was observed compound 2 and compound 4. However; the extracts have the highest activity than isolated compounds. Therefore, we refer these extracts activities perhaps to synergic effects. The toxicity of plants in the Thymelaeaceae is of considerable economic importance beyond its effects on the livestock industry. Clinical trials are being conducted in China on preparations of Daphne, Gnidia, Wikstroemia and Pimelea species that have been reported to have anticancer activity [259, 260, 261, 262, 263, 264 and 265]. The anticancer activities of these products have been

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CHAPTER 4 Results and Discussion shown to be due primarily to the presence of daphnane esters. Plants of this family have been included in several large-scale screening studies [266, 267, 268, 269, and 270] investigating various biological activities.

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CONCLUSION

In the course of the present work, aiming to knowledge of the Thymelaeaceae family in through the study of their secondary metabolites, 3 crude extracts from 2 species Thymelaea microphylla Coss. et Dur. and Gnidia somalensis Gilg. var.sphaerocephala (Bak.) Gastald. were evaluated by using separation, analyses and biological methods. The use of T. microphylla in flok medicine for treatment of wounds, erysipelas, abscesses, and the results of previous studies in antibacterial and antioxidant activity, in addition to the absence of phytochemical studies and cancer activity on this plant; all of that have led us to study the CH2Cl2: MeOH (1:1) extract and 70% (v:v) MeOH-H2O extract of aerial parts of T. microphylla. The isolation by CC and TLC, and the structural identification by NMR and mass spectrometry allowed to obtain 10 compounds:  Two lignan lactones.  Bicoumrin.  Four flavonids.  Two spiro lactones.  One new compound microphybenzimidazole (alkaloid type).

Prestegane B. Matairesinol.

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Daphnoretin Isochamaejasmin

Microphynolide A Microphynolide B

trans-tiliroside

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cis-tiliroside

Yuankanin

microphybenzimidazole

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The results of the present study of antiproliferative activitiy against HeLa cell lines and C6 cell lines of T. microphylla fractions and pure compounds showed the significant anti-cancer activity which may lead in future to isolate a new anticancer compounds.

The applications of advanced coupled analytical method of the LC-MSn are a complement Indispensable for the study of Thymelaeaceae family. Indeed, the unknown species G. somalensis studied with modern tools, allowing to know a lot of information in a time very short. In addition, very small quantities of plant material are required. The identification was carried out based on the retention behavior, as well as UV absorption and mass spectral characteristics.

The use of the LC / MSn allowed us to identify 12 compounds:  Six polysubstituted sinnamic acids.  Six flavonoids.

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Phenolic compounds have a high biological activity. The analysis of G. somalensis extract by LC- MSn allowed us to identify the nature of separated compounds, from this informations we can proceed to obtain these compounds by isolating, purifying and testing them to evaluate the biological activity to get a new active compounds which may have a high anticancer activity from this species of Thymelaeaceae family. The most important point is separation and purification, and it should be deeply studied to obtain others compounds.

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Abstract

The main objective of our work is phytochemical studies and evaluation of secondary metabolite products extracted from Thymelaeaceae family which is rich in biological active phenolic compounds. So, we choose Thymelaea microphylla, which grows in Algeria. In addition to another plant from the same family; Gnidia somalensis growing in Yemen. Our study also, devoted to the evaluation and effectiveness of the plant extracts and pure compounds against cancer cells. We used the ELISA and xCELLigence assay technics for determining the inhibition of fractions and pure compounds in vitro, against HeLa cells (human uterus carcinoma) and C6 cells (rat brain tumor). Which have shown positive results, especially for fractions that have a high activity compared with the reference compound. In addition, we have got a significant results by testing the pure compounds (Prestegane B, daphnoretin and trans-tiliroside) which gave a positive results. On the other hand, we were able to isolated 10 compounds from T. microphylla: • Two lignan lactones. • One bicoumarin. • Four flavonoids. • Two spiro lactones. • One new alkaloid (microphybenzimidazole).

Also, we have separated 12 phenolic compounds from the plant Gnidia somalensis: 6 polysubstituted cinnamic acid derivatives and 6 flavonoids.

We used different chromatographic methods for separation and purification (Column chromatography CC, thin layer chromatography TLC), and spectroscopic tools for structure identification (HPLC LC-MS, HPLC- TOF), ID (Tf -NMR, 13C- NMR and DEPT) and 2D (HMQC, HMBC and COSY).

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L'objectif principal de cette recherche est l'extraction, séparation et l’identification des métabolites secondaires à partir de deux plantes de la famille des Thymelaeacea qui sont riche en composés phénoliques bioactifs une plante endémique algérienne Thymelaea microphylla. Ainsi que d'autre plante de la même famille qui pousse au Yémen: Gnidia somalensis.

Cette étude est suivie par l’évaluation de l'active anticancéreuse. On a utilisés deux techniques ELISA et xCELLigence pour déterminer l’effet inhibiteur des fractions et des composés purs vis-à-vis des cellules cancéreuses HeLa (cancer de l'utérus humain) et l'activité des cellules C6 (rat de tumeur cérébrale), qui ont montré des résultats positifs, en particulier pour les fractions qui ont une activité élevée en comparaison avec le contrôle. En plus des résultats significatifs ont été obtenus avec les composés purs (Prestegane B, daphnoretine et trans-tiliroside).

Nous avons isolé 10 composés phénoliques de la plante T. microphylla: • Deux lignane lactones. • Une bicoumarine. • Quatre flavonoides. • Deux spiro lactones. • Un nouvel alcaloide (microphybenzimidazole). Et 12 composés phénoliques de l'espèce Gnidia somalensis: 6 dérivés d’acide cinnamique polysubstitués et 6 flavonoides.

Le processus de séparation s’est basé sur les techniques chromatographique telle que la chromatographie sur colonne (CC) et sur couche mince (CCM) ainsi des techniques physiques (HPLC, LC-MS, HPLC- TOF), alors que l’identification structurale a été réalisé par RMN 1D (1H- RMN, 13C-RMN et DEPT) et 2D (HMQC, HMBC et COSY).

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