Constantine 1
Mentouri Brothers University
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
Members of jury:
University Constantine President
Brothers
Akkal Salah Mentouri
Prof.
Supervisor
Oum El Bouaghi University
-Larbi Ben Mhidi
Zellagui Amar
Prof.
Prof. Rhouati Salah Mentouri Brothers University Constantine Co-Supervisor
Prof. Gherraf Noureddine Larbi Ben Mhidi - Oum El Bouaghi University Examiner
Ferhat Abbas setif university Examiner
Seddik Khennouf
Prof.
2017
Secondary metabolic components and biological
effectiveness study on two
species
Thymelaea
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.
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.
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.
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).
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. Coumarins………..………..……….. 09 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
2.3.3.5. 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 andHPLC-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
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 صخلملا
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.
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.
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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.
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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].
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Scheme 1: Major subgroups of flavonoids with their general structures, sources and general
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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
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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 sub-class 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.
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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
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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.
8 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
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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].
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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 coumarin
Chemical structure Example Pharmacological
activity 1 Simple coumarins Esculetin Antiadipogenic Antioxidant Neuroprotective Ammoresinol Antibacterial Ostruthin Antibacterial Antifungal Antibacterial Antifungal Osthole Anticancer Anticonvulsant Antioxidant Novobiocin Antibacterial Coumermycin Antibacterial Chartreusin Antibacterial Fraxin Anticancer Antiadipogenic Antioxidant Umbelliferone Antitubercular Fraxidin Antiadipogenic Antihyperglycemic Phellodenol A Antitubercular 2 Furano coumarins Imperatorin Anti-inflammatory Antibacterial Antifungal Antiviral Anticancer Anticonvulsant Psoralen Antifungal Anti-TB Bergapten Anti-TB
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3 Dihydrofurano
coumarins
Anthogenol Antibacterial
Felamidin Antibacterial
4 Pyrano coumarins are of two types
4a Linear type Grandivittin Antibacterial Agasyllin Antibacterial Aegelinol benzoate Antibacterial Xanthyletin Anti-TB 4b Angular type Inophyllum A, B, C, E, P, G1, and G2 Antiviral Calanolide A, B, and F Antiviral (+)-Dihydrocalanolide A and B Antiviral Pseudocordatolide C Antiviral 5 Phenyl coumarins Disparinol A Antiviral Isodispar B Antiviral 6 Bicoumarins Dicoumarol Anticoagulant
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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 oxygen” and “lignans without C9 (9´)-oxygen”. Figure 11.
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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
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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].
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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, 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].
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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)
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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
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Lasalocid Cannabidiolic acid
Cajaninstilbene acid (R1=H, R2=prenyl )
Isocajaninstilbene acid (R1=prenyl, R2=H)
Dicaffeoylquinic acid
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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
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important role as a source of effective anti- cancer 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].
21 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].
22
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].
23
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].
24
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 : Thymelaeaceae2.2 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.
25
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].
26
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
27 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
28
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.
29 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.
30
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.
31
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.
32
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)
33
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).
34
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: Diacetyl-viburnolide A, 4’,6’-Diacetyl-12- coumaroyl-viburnolide A and Tetraacetylviburnolide A, which extracted from the family of Thymeleaeceae [131]. Figure 27.
35
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, Hydroxybenzoic acid,
p-Hydroxyphenylacetic acid, Hydroxycinnamic acid, Vanillic acid, Protocatechuic acid, p-Coumaric acid, Ferulic acid, Caffeic acid and Gallic acid [132]. Figure 28.
36
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
37
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 Lacaita
23. Thymelaea sanamunda All.
9. Thymelaea gussonei 24. Thymelaea sempervirens
10. Thymelaea hirsuta (L.). 25. Thymelaea subrepens
11. Thymelaea lanuginosa (Lam.) Ceballos & C.Vicioso
26. Thymelaea tartonraira (L.) All.
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.
38 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]. thymelol ((C3H2O)n) [157].
stigmasterol, β-sitosterol, alcool aliphatic , l alcool aliphatique C12H22O, lactone C19H18O6 [158].
daphnoretine, β -sitosterol-β - D-glucoside [159]. daphnorine, daphnoretine, daphnine,
daphnetine, daphnetine-glucoside,
ombelliferone, scopoletine and esculetine (coumarines) [160].
2-vicenine (C-flavone) [161].
tiliroside (3-p – coumaroyl glucosyl kaempferol) (flavanol) [162].
lupeol, β-sitosterol, phytol, β-amyrine,
betuline, erythrodiol, cholesterol and lanosterol [163].
5,12-dihydroxy-6,7-epoxy-resiniferonol [164]. gnidicine, gniditrine, genkwadaphnine, 12-O - heptadecenoyl-5-hydroxy-6,7-epoxy-
Leaves
Twigs
Thymelaea hirsuta (L.)
39
resiniferonol-9, 13,14-orthobenzoate (diterpenes daphnane ([165]. Tanins. [166].
proteines [167].
daphnoretine (ether de dicoumaryl). [168].
Seeds
Roots
1-carboxylic acid bornane, limonene,
isobutyranilide, D-menthone, Pulegone, (6E)-2,5-dimethyl-1,6 octadiene, Perillal, 2-Undecanone, (Z,E)-α-Farnesene, 1-(2-Bromovinyl)-adamantane, Artemesiatriene [169].
pentacosane, triacontanol, sitosterol, stigmasterol, β-amyrine, ombelliferone et scopoletin. [170].
Aerial part T.microphylla Coss. et
Dur
Thymelaea passerina (L.) Coss. & Germ.
orientine, isoorientine, vitexine, 2-vicenine, kaempferol, daphnoretine, genkwanine, 5-o- D-genkwanine, primeverosyl (flavone- coumarine). [171].
Lipides, sucres and amidon. [172].
Whole plant Thymelaea tartonraira
(L.) All.
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
40
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.
Rogers
79. Gnidia meyeri Meisn.
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
Engl.
83. Gnidia montana H.Pearson
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.)
Baill.
86. Gnidia nana (L.f.) Wikstr.
41
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
42
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.
43 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].
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.
44 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, 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]. 7,7’-dihydroxy-3,8’-biscoumarin, 8-(6’’-Umbelliferyll)-apigenin, 4’,6’-Diacetyl-viburnolide A, 4’,6’-Diacetyl-12-coumaroyl-viburnolide A, Tetraacetyl4’,6’-Diacetyl-12-coumaroyl-viburnolide A [150]. Gnididin, Gnidicoumarin [118]. Aerial part Leaves and twigs - Gnidia involucrata Gnidia socotrana Gnidia lamprantha
Umbelliferone, syringin, 2-O-beta-D-glucosyloxy-4-methoxybenzenepropanoic acid [190,191] Gnididione [192] Stem Stem Gnidia polycephala Gnidia latifolia 12-Hydroxydaphnetoxin, Mezerein, Genkwadaphnin [193-195] Maltol, 3-Hydroxy-2-methyl-4H-pyran-4-one, Gnidilatin, Pimelea factor P2, Excoecariatoxin, Gnidilatidin,kraussianin [78,196]
-
-
Gnidia burchellii
Gnidia kraussiana
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].
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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].
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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
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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
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