An-Najah National University Faculty of Graduate Studies Characterization of Bioactive Phytochemicals from Micromeria Fruticosa Plant as an Approach to Develop Natural-based Drug Leads By Anwar Afeef Alhajeh Supervisor Prof. Mohammed Al-Nuri Co- Supervisor Dr. Ibrahim Abu-Reidah This Thesis is submitted in Partial Fulfillment of the Requirements for the Degree of Master of Chemistry, Faculty of Graduate Studies, An-Najah National University, Nablus, Palestine. 2019 iii Dedication The author would like to express sincere appreciation to my family and friends special thanks to my advisor and co advisor and to everyone of AN-Najah National University Chemistry Department. Bunches of thanks to Professor Ghaleb Odwan and Dr. Ghadeer Omar of Biology Department for assisting me in doing the experimental part of biological activity of the plant extracts. iv Acknowledgments Praise to almighty Allah who guided my steps in all my work till the very end. I would like to express my gratitude to my supervisors, Prof. Mohammed Al-Nuri and Dr. Ibrahim Abu-Reidah, who always supported me with their knowledge and experiences. I would also like to thank Mr. Nafith Dweikat, for facilitating my work in the labs and helping me during research. Specially thanks for my parents, sisters (Manar and Inas), brothers (Obada, Ahmad and Abed-alrahman) and my aunt Ms. Afaf Al-hajeh who supported me during my study. Thanks, are also due to my friends, particularly Ms. Nabaa Abu- Hafez and Ms. Zeinab Khaled, who always has been my best friend. Finally, thanks to chemistry department at my University. vi List of Contents Page Subject No. iii Dedication iv Acknowledgements v Declaration vi List of Contents viii List of Figures xi List of Tables xii List of Abbreviations xiii Abstracts Chapter One: Introduction 1 Traditional medicine 1.1 3 Main chemical constituents in medicinal plant 1.2 4 Methods of isolation and quantitative determination 1.3 4 Extraction 1.3.1 5 Chromatographic types and techniques 1.3.2 6 Biological Activities 1.4 6 Biological Activity of Some Modified Compound 1.4.1 6 Anti-microbial (antibacterial) 1.4.2 6 Anti-microbial (antifungal activities) 1.4.3 7 Anti-oxidants (free radical scavengers) 1.4.4 7 Aims of study 1.5 Chapter Two: Experimental 8 Collection of plant 2.1 8 Extraction 2.2 Chapter Three: GC-MS/MS Analysis 10 Sample preparation for GC-Analysis 3.1 10 GC-MS/MS techniques 3.2 10 GC-MS/MS Apparatus and Conditions 3.3 11 Results and Discussion for GC-ANALYSIS 3.4 Chapter Four: ICP-MS (Inductively coupled plasma mass spectrometry) analysis 52 Sample preparation for ICP-MS Analysis 1.4 52 ICP-MS techniques 1.9 52 Results and Discussion for ICP-MSANALYSIS 4.3 Chapter Five: Biological activities 57 Antimicrobial activity tests 5.1 vii Page Subject No. 57 Preparation of samples for testing 5.1.1 57 Media and Solutions 5.1.2 57 Nutrient Broth 5.1.2.1 57 Mueller-Hinton Broth 5.1.2.2 58 Mueller Hinton Agar (MHA) 5.1.2.3 58 Sabouraud Dextrose Agar 5.1.2.4 59 Normal Saline (0.9% NaCl) 5.1.2.5 59 Preparation of McFarland Turbidity Standard No.0.5 5.1.2.6 60 Test Microorganism 5.1.3 60 Determination of Minimum Inhibitory Concentration (MIC) 5.1.4 60 Determination of Minimum Inhibitory Concentration (MIC) against bacteria 5.1.4.1 61 Determination of Minimum Inhibitory Concentration (MIC) against yeast 5.1.4.2 62 Results and Discussion for Biological Activities 5.2 Chapter Six: Anti-oxidant activity (free radical scavenging activity) 68 General procedure of anti-oxidant test for Micromeria Fruticosa Plant Extract 6.1 69 Results and Discussion of Antioxidant 6.2 Chapter Seven: Conclusion 74 Conclusion 76 References الولخص ب viii List of Figures Page Title Figure No. 4 Common Hydroxybenzoic Acids Figure (1.1) 4 Structures of Hydroxycinnamic Acids Figure (4.9) 11 GC-MS Apparatus used for Analysis Figure (3.1) 18 Trace GC-MS Chromatogram of the ethanol flower extract of Micromeria Fruticosa plant Figure (3.2) 19 Trace GC-MS Chromatogram of the hexane flower extract of Micromeria Fruticosa plant Figure (3.3) 20 Trace GC-MS Chromatogram spectral of the ethanol flower extract of Micromeria Fruticosa plant at the R.T. (19.204) minute Figure (4.1) 21 Trace GC-MS Chromatogram of the menthol compound from ethanol flower extract of Micromeria Fruticosa plant Figure (3.5) 22 Trace GC-MS Chromatogram spectral of the hexane flower extract of Micromeria Fruticosa plant at the R.T. (58.950) minute. Figure (4.3) 23 Trace GC-MS Chromatogram of the 9- octadecenamide, (z)- compound from hexane flower extract of Micromeria Fruticosa plant Figure (3.7) 24 Trace GC-MS Chromatogram spectral of the hexane flower extract of Micromeria Fruticosa plant at the R.T. (18.144) minute Figure (4.0) 25 Trrace GC-MS Chromatogram of the CYCLOHEXANONE, 5-METHYL-2-(1-METHYLETHYL)-, TRANS compound from hexane flower extract of Micromeria Fruticosa plant Figure (3.2) 28 Trace GC-MS Chromatogram of the ethanol leaves extract of Micromeria Fruticosa plant Figure (3.48) 29 Trace GC-MS Chromatogram of the hexane leaves extract of Micromeria Fruticosa plant Figure (3.44) 30 Trace GC-MS Chromatogram spectral of the ethanol leaves extract of Micromeria Fruticosa plant at the R.T. (18.144) minute and the fragment for CYCLOHEXANONE, 5-METHYL-2-(1- METHYLETHYL)-, TRANS Figure (3.49) 31 Trace GC-MS Chromatogram spectral of the ethanol leaves extract of Micromeria Fruticosa plant at the R.T. (20.875) minute and the fragment for Pulegone Figure (3.44) ix Page Title Figure No. 32 Trace GC-MS Chromatogram spectral of the ethanol leaves extract of Micromeria Fruticosa plant at the R.T.(36.701) minute and the fragment for Cyclobutane, 1, 3-Diphenyl-, Trans. Figure (3.41) 39 Trace GC-MS Chromatogram of the ethanol stems extract of Micromeria Fruticosa plant Figure (3.45) 40 Trace GC-MS Chromatogram of the hexane stems extract of Micromeria Fruticosa plant Figure (3.43) 44 Trace GC-MS Chromatogram of the ethanol root extract of Micromeria Fruticosa plant Figure (3.47) 45 Trace GC-MS Chromatogram of the hexane root extract of Micromeria Fruticosa plant Figure (3.40) 46 Trace GC-MS Chromatogram spectral of the hexane root extract of Micromeria Fruticosa plant at the R.T. (54.433) minute and the fragment for 1, 2- BENZENEDICARBOXYLIC ACID, DIISOOCTYL ESTER compound Figure (3.42) 47 Trace GC-MS Chromatogram spectral of the hexane root extract of Micromeria Fruticosa plant at the R.T. (19.204) minute and the fragment for Menthol compound Figure (3.98) 48 Trace GC-MS Chromatogram spectral of the hexane root extract of Micromeria Fruticosa plant at the R.T. (42.159) minute and the fragment for N-hexadecanoic acid compound Figure (3.21) 49 Trace GC-MS Chromatogram spectral of the hexane root extract of Micromeria Fruticosa plant at the R.T. (57.555) minute and the fragment for HENTRIACONTANE compound Figure (3.22) 50 Trace GC-MS Chromatogram spectral of the ethanol root extract of Micromeria Fruticosa plant at the R.T. (54.428) minute and the fragment for DIDODECYL PHTHALATE compound Figure (3.23) 51 Trace GC-MS Chromatogram spectral of the ethanol root extract of Micromeria Fruticosa plant at the R.T. (42.929) minute and the fragment for ETHYL 14-METHYL-HEXADECANOATE compound Figure (3.24) 64 Minimum Inhibitory Concentration (µg/ml) of different leave extract types against different pathogens Figure (5.1) x Page Title Figure No. 65 Minimum Inhibitory Concentration (µg/ml) of different flower extracts types against different pathogens Figure (5.2) 66 Minimum Inhibitory Concentration (µg/ml) of different stem extracts types against different pathogens Figure 5.3 67 Minimum Inhibitory Concentration (µg/ml) of different root extracts types against different pathogens Figure 5.4 71 Anti-oxidant % Inhibition Concentration (µg/ml) of different D.W extract types Figure 3.4 72 Anti-oxidant % Inhibition Concentration (µg/ml) of different Hexane extracts types Figure 3.9 73 Anti-oxidant % Inhibition Concentration (µg/ml) of different Ethanol extracts types Figure 3.4 xi List of Tables Page Subject Table No. 13 The similar compounds of hexane and ethanol flower extract from GC-MS/MS analysis Table (4.4) 14 The compound isolated from GC-MS/MS analysis of flower ethanol extract Table (3.2) 16 The compound isolated from GC-MS/MS analysis of flower hexane extract Table (3.3) 27 The similar compounds of hexane and ethanol leave extract from GC-MS/MS analysis Table (3.4) 33 The compound isolated from GC-MS/MS analysis of leave ethanol extract Table (3.5) 35 The compound isolated from GC-MS/MS analysis of leave hexane extract Table (3.6) 37 The compound isolated from GC-MS/MS analysis of stem hexane and ethanol extract Table (3.7) 41 The compound isolated from GC-MS/MS analysis of roots ethanol extract Table (3.8) 42 The compound isolated from GC-MS/MS analysis of roots hexane extract Table (3.9) 52 The concentration of various elements from leave aqueous extract using ICPMS spectrometer. Table (4.1) 54 The concentration of various elements from stem aqueous extract using ICPMS spectrometer. Table (4.2) 55 The concentration of various elements from flower aqueous extract using ICPMS spectrometer. Table (4.3) 56 The concentration of various elements from root aqueous extract using ICPMS spectrometer. Table (4.4) 63 Minimum Inhibitory concentration values (µg/ml) for different Micromeria frticosa extract types against different pathogens Table (5.1) 70 %Inhibition and IC50 for D.W, ethanolic and Hexane extracts Table (6.1) xii List of Abbreviations : retention time (Min) R.T. : retention index R.I. : molecular formula M.F. : molecular weight (g/mol) M.W. : Reference Ref. : distilled water D.W. xiii Characterization of Bioactive Phytochemicals from Micromeria Fruticosa Plant as an Approach to Develop Natural-based Drug Leads By Anwar Afeef Alhajeh Supervisor Prof. Mohammed Al-Nuri Co- Supervisor Dr. Ibrahim Abu-Reidah Abstract Four parts (flowers, leaves, stems, roots) from Micromeria Fruticosa plant used in traditional medicine in Palestine were tested for their biological activity against four types of bacteria and one type of fungi {S. sonnie (ATCC 25931), S. aureus (ATCC 25923), Escherichia coli (E. coli), MARSA, and Candida albicans (C. albicans) (ATCC 90028)} respectively. The plant was collected from Tulkarem region. The family name of plant is Micromeria Fruticosa (L) Druce ssp serpyllifolia (Lamiaceae) and is known as ashab a-shai in Arabic. The ethanolic, hexane and distilled water extracts of those parts of the plants were tested for their antioxidant activity through DPPH assay. The D.W. extract from leave has got the highest percentage inhibition (89%) at the concentration (100µg/ml). Some constituents were detected from ethanolic and hexane plants extracts using GC-MS/MS spectrophotometer and separated by flash chromatography the most similar compound found in all part in plant is 9- OCTADECENAMIDE, (Z)-, Menthol and CYCLOHEXANONE,5- xiv METHYL-2-(1-METHYLETHYL)-, TRANS with different high, and area percentage. Also, some elements were detected from distilled water plant extracts using ICPMS analysis, these elements are Fe, Zn, Sr with different percentage per extract part of plant. 1 Chapter one Introduction 1.1 Traditional medicine Thousands of years ago, herbs and plant products were used in folk medicine in treating a wide spectrum of ailments and diseases. Folk remedies are prepared as powders, poultices, ointments, baths, decoctions, infusions and teas. The interest in studying the biological effects of traditional medicinal plant or isolating their active components for treatment of illness has been increased all over the world and comprehensive screening programs have been established [1]. Currently, a great number of different medicinal plant products are available in markets including cosmetics and pharmaceuticals, which contain biologically active substances. Recently, some products of plant origins have shown biological activity without side effects. Such results have attracted the attention of many scientists and encouraged them to screen vast number of plants to test their biological activities [2]. Natural products have been a source of drugs and drug leads. In the past, it was extremely difficult, time consuming and invaluable efforts to build such library of purified natural products without applying improved technologies for separation, isolation and identification of such natural products. Natural products libraries have been established to preserve crude extracts, chromatographic fraction or semi -purified compounds. However, 2 the best result scan be obtained from fully identified pure natural products library since it provides scientists with the opportunity to handle the lead rapidly for further developed work [3]. Micromeria Fruticosa (L) Druc essp Serpyllifolia (Lamiaceae) is used widely in many Mediterranean regions for various inflammatory conditions and wound healers [4]. The name of the genus was derived from Greek words micro and meros (meaning small and part) because they were found as dwarf fragrant shrubs or perennial herbs which usually grow between rocks and in other dry open habitats. A number of Micromeria species are used in daily life for medicinal, insecticidal, herbicidal and culinary purposes [5]. Phenolics are plant metabolites with well-known protective action against various health diseases]. For instance, they possess numerous biological activities e.g. anti-inflammatory, anti-diabetic, antioxidant, cytotoxic and antitumor. Micromeria Fruticosa Druce (Wildflowers, White Micromeria) is used widely in many Mediterranean countries as herbal infusion for various inflammatory conditions and in wound healing. It is a member of genus Micromeria and is known as ashab a-shai in Arabic [6]. 3 Micromeria Fruticosa 1.2 Main chemical constituents in medicinal plant The plant extracts have shown that there are a large number of chemical compounds according to chemical analysis which have different functional groups are present in the plant [7]. Flavonoids and phenolic and their derivatives are found in most of plants. They are considered very important chemical groups for both human and plants [8]. They have many functions in plants. Flavonoids and phenolic acids also have antioxidative and anticarcinogenic effects [9]. Phenolic compounds present in plants as hydroxylated derivatives of benzoic and cinnamic acids [10]. They are important in the defense mechanisms of plants under different environmental stress conditions such as wounding, infection, and excessive light or UV irradiation [44]. There are two main groups of phenolic compounds that are listed below: 4 1. Hydroxybenzoic acids: Which they are derived from benzoic acid directly. Four acids are commonly present: syringic acid, hydroxyl benzoic acid, vanillic acid and protocatechuic acid as shown below in Figure (1.1) [49]. Figure (1.1): Common Hydroxybenzoic acids. 2. Hydroxycinnamic acids: The four common acids are distributed widely in plants are p-coumaric acid, sinapic acids, ferulic acid and caffeic acid shown in Figure (1.2) [49]. Figure (1.2): Structures of Hydroxycinnamic acids. 1.3 Methods of isolation and quantitative determination 1.3.1 Extraction Extraction is the next step in the study of medicinal plants after collection of the plant. Several methods of extraction plant content. There 5 are many factors that affect the extract like, pH of the medium of extract, stability of the effective constituent, and biological activity of the constituents of the plants. And Solvent type either organic or aqueous. Popular methods of extraction: a. Decoction: It is traditional extraction methods, in which the extract is prepared by putting the part of plant in cold water, then boil for 15 minutes, after that the extract is filtered or decanted. b. Soaking: The part of plant is soaked in organic or aqueous solvent for three days, and then the extract is decanted and dried [44]. 1.3.2 Chromatographic types and techniques A chromatographic technique was used for separation of colored pigments, and separation of mixtures that have high number of organic compounds. Chromatography avoids any reason for changing the structure of the contents in plant. So, it is being considered as a physical method of separation [41]. Several types of chromatographic techniques are: liquid chromatography (LC), thin layer chromatography (TLC), column chromatography, High-performance liquid chromatography (HPLC), and gas chromatography (GC) [45, 43]. 6 1.4 Biological Activities 1.4.1 Biological Activity of Some Modified Compound Biological activity refers to substances having effect on the living tissue or its ability to effect a change in a biological process. The importance of biological processes refers to the description of functional relationships between biological activities and the chemical substances that express them [47]. 1.4.1 Anti-microbial (antibacterial) Microbes are tiny organisms seen by a microscope. These microbes are found in air, soil, rocks, plants, bodies and water. Microbial organisms include bacteria, fungi, viruses and protozoa. Some microbes cause disease and are called pathogens [40]. Antimicrobial drugs are synthesized to inhibit the microbe without any side effects on the patients [42]. 1.4.3 Anti-microbial (antifungal activities) Medicine of antifungal agent makes selectivity to reduce fungal pathogens. Unlike diseases of bacteria, diseases of fungi are more difficult to treat. Often oral and topical treatments are long term and may be partially successful in controlling the fungus. Fungal infections are the most spread on skin of all mycoses [98]. 7 1.4.4 Anti-oxidants (free radical scavengers) Anti-oxidant are called "free radical scavengers" are substances which delay or sometimes prevent types of cell damage by blocking the activity and reactions of free radicals from causing the damage or by giving hydrogen atoms. Free radical is very reactive species which have an odd number of electrons. Some of damages may cause cancer. In biological systems reactive species like "reactive oxygen species" (ROS). "Reactive nitrogen species" (RNS) are example of reactive species plays a dual role as both deleterious and beneficial species [94, 99, 94]. Free radicals are two types. The first is synthesized naturally in the body. And other type introduced through external sources to our bodies. The sun and tobacco smoke can be considere source of free radical. Also, the body needs external sources of antioxidants sources like fruits and vegetables [91]. Free radical with high potential gives high reactivity which harms the cell. It is created when a molecule or atom either loses or gains an electron [95]. 1.5 The Aim of the study The main objectives of this study are the followings: 1- Identification and characterization of bioactive phytochemical compound from Micromeria Fruticosa plant. 2- Evaluate the biological activity (antifungal and antibacterial activities) and anti-oxidant for Micromeria Fruticosa plant extract of different part of it in Palestine. 8 Chapter Two Experimental Part All chemical were purchased from Sigma-Aldrich Chemical Company and used without further purifications The antibacterial activity of the extracts were determined against the following microorganisms; Escherichia coli, Shigella, MRSA, S. aureus. On the other hand, the antifungal activity test (Yeast) was done against Candida. 2.1 Collection of plant The leaves, stems, flowers and roots of the plant were obtained during April 2019 from the City of Tulkarm Mountains. The parts were placed in ventilated room temperature in a shaded area away from direct sunlight until is completely dry. 2.2 Extraction The dry leaves, stem, flower, and roots were grinded partially and soaked in hexane for 5 days at room temperature. The extract was obtained by suction filtration. The solvent were removed by evaporation (rotatory evaporation) to get the extract at 35 o C and then each part of the plant was soaked in ethanol, the filtered and 9 finally were soaked in water for 5 days and made the filtrate for each solvent. Water was removed by freeze drying for 3 days. After the removal of each solvent by evaporation, the extracts were subjected to biological and chemical analysis. 10 Chapter Three GC-MS/MS Analysis 3.1 Sample preparation for GC-ANALYSIS A 2 mg of Organic plant extracts (ethanol and hexane extracts) were dissolved in 2ml of acetonitrile. 3.2 GC-MS/MS techniques Gas chromatography-mass spectrometry (GC-MS) is an important technique for qualitative and quantitative analysis for plant extract. It is fast and sensitive, provides a high peak capacity and allows determination of thermally stable and volatile compounds [93]. GC-MS used for the separation of a mixture that contains a large number of organic compounds. Chromatography is a physical method that helps to avoid any reaction that may change the structure of the original compounds in the plant [97]. 3.3 GC-MS/MS Apparatus and Conditions The Clarus 500GC MS used in the analysis employed a fused silica column packed with Elite-1 (100% dimethyl poly siloxane, 30 m × 0.25 mm ID × 0.25µm df) and the components were separated using Helium as carrier gas at a constant flow of 1.1ml/min. The µL sample extract injected into the instrument was detected by the Turbo gold mass detector (Perkin 11 Elmer) with the aid of the Turbo mass 5.1 software. During the 62.5minute GC extraction process, the oven was maintained at a temperature of 50 o C with 5 minutes holding. The injector temperature was set at 250 o C (mass analyzer). The different parameters involved in the operation of the Clarus 500 MS, were also standardized (Inlet line temperature: 200 0 C; Source temperature: 200 0 C). Mass spectra were taken at 4 min; a scan interval of 0.2 s and fragments from 50 to 500 Da. Figure ( 3.1): GC-MS/MS system. 3.4 Results and Discussion for GC-ANALYSIS The analysis was carried out using GC-MS system. The compounds were identified from ethanol and hexane flower extract of Micromeria Fruticosa plant as shown in Figure 3.2, 3.3 respectively. The retention time (RT), percentage peak of the bioactive compounds and biological study are presented in Table 3.2 and Table 3.3. 12 There are similar compounds in the hexane and ethanol flower extract and with different percentage and these compounds are shown in Table 3.1 and Figure 3.5, 3.7, 3.9. The presence of these compounds in the flower makes this part of the plant to be so effective biologically and antioxidants activities. 13 Table (3.1): The similar compounds of hexane and ethanol flower extract from GC-MS/MS analysis Similar Compounds Name Molecular formula %high peak (flower ethanol extract) %high peak (flower hexane extract) CYCLOHEXANONE, 5-METHYL-2- (1-METHYLETHYL)-,TRANS C10H18O 35.86411% 5.75458% MENTHOL C10H20O 15.77273% 51.48503% 9-OCTADECENAMIDE, (Z)- C18H35NO 9.635816% 3.250345% 14 Table (3.2): The compound isolated from GC-MS/MS analysis of flower ethanol extract Extract Type R.T (min.) R.I Possible Compound Name High Area %High %Area M. F M.W (g/mol) Biological Study Ref F lo w er E th a n o l 1 8 .4 4 7 9 6 CYCLOHEXANO, 5-METHYL-2-(1- METHYLETHYL), TRANS 4 7 7 3 2 1 8 6 9 3 1 7 5 4 4 3 5 .8 6 4 1 1 3 0 .0 4 8 8 2 C 1 0 H 1 8 O . 1 5 4 .2 4 9 3 . antimicrobial activity, potential antibiofilm, antitumor activities [3 5 ] 1 9 .2 0 4 8 6 6 MENTHOL 2 0 9 9 2 2 1 4 8 9 6 1 4 0 4 1 5 .7 7 2 7 3 2 1 .2 2 4 5 3 C 1 0 H 2 0 O 1 5 6 .2 7 Antibacterial ,antifungal ,antipruritic, anticancer, cooling effects and toxicity, Antioxidant. [2 8 ] 2 0 .8 2 5 8 1 6 PULEGONE 4 2 1 8 9 1 4 7 3 3 4 0 1 2 0 3 1 .6 9 9 2 8 3 1 .7 9 2 5 9 C 1 0 H 1 6 O 1 5 2 .2 3 3 4 Antimicrobial , Antioxidant, anti-inflammatory, anti-ulcer, insecticidal properties. [2 9 ] 2 5 .1 2 2 6 1 4 4,6-DECADIENE 9 6 6 8 8 1 9 7 9 6 3 2 0 .7 2 6 4 7 6 0 .8 5 8 1 6 1 C 1 0 H 1 8 1 3 8 .2 5 Antioxidant, antiasthmatics, antibacterial, for sexual and urinary system disorders, and also used for enhancing fertility, bioactive. [3 5 , 3 6 ] 7 9 4 CARYOPHYLLENE 4 9 7 7 4 7 9 5 5 0 8 6 3 .7 3 9 8 7 8 4 .1 4 0 2 5 C 1 5 H 2 2 0 4 .3 anti-inflammatory, antibiotic, antioxidant, anticarcinogenic, local anaesthetic activities, anticancer activity. antinociceptive, neuro protective, anxiolytic, anti depressent, antialcoholism, anti pyritics, anti-microbial, anti-carcinogenic, anti dermatitic, allergenic, aldose- reductase inhibitor, anti-acne, anti-asthmatic, anti- ulcer, anti proliferants, cyto protective, gastro protective, sedative, anti-spasmodic, flavour. [3 0 , 4 0 ] 15 2 9 .0 3 8 7 2 5 1,6CYCLODECADI ENE, 1-METHYL-5- METHYLENE-8-(1- METHYL ETHYL)-,[S-(E-E)]- 1 6 8 4 5 8 3 3 2 0 4 2 8 1 .2 6 5 7 2 8 1 .4 3 9 3 9 C 1 5 H 2 4 2 0 4 .3 6 antioxidant activity [4 9 ] 3 6 .7 1 1 7 7 0 1,2-(1,2- cyclobutanediyl) bis-, trans 7 4 8 9 1 1 2 9 2 4 5 3 0 .5 6 2 7 0 2 0 .5 6 0 2 7 2 C 1 6 H 1 6 2 0 8 .2 9 8 antitumor activity [5 1 ] 5 4 .6 5 3 5 5 8 (2,3- DIPHENYLCYCLO PROPYL)METHYL PHENYL SULFOXIDE, TRANS- 9 7 5 9 4 1 6 9 7 7 1 9 0 .7 3 3 2 8 3 0 .7 3 5 9 5 3 C 2 2 H 2 0 O S 3 3 2 .4 6 1 anticandidal and antioxidant activity. [5 0 ] 5 9 .0 1 5 5 5 9 9- OCTADECENAMI, (Z)- 1 2 8 2 4 4 8 2 1 2 2 2 9 0 0 9 .6 3 5 8 1 6 9 .2 0 0 0 2 5 C 1 8 H 3 5 N O 2 8 1 .4 7 7 Anti-inflammatory activity, antibacterial activity and Antioxidant Activities. [3 1 ] R.T: retention time (Min), R.I: retention index, M.F: molecular formula, M.W: molecular weight (g/mol), Ref: reference. 16 Table (3.3): The compound isolated from GC-MS/MS analysis of flower hexane extract Extract Type R.T )min.) R.I Possible Compound Name High Area %High %Area M.F M.W )g/mol( Biological Study Ref F lo w er H ex a n e 1 8 .1 4 4 8 4 9 CYCLOHEXANONE, 5- METHYL-2- (1METHYLETHYL), TRANS 4 7 7 3 2 1 8 6 9 3 1 7 5 4 4 5 .7 5 4 5 8 5 .4 2 3 1 5 C 1 0 H 1 8 O 1 5 4 .2 4 9 antimicrobial activity, potential antibiofilm, antitumor activities. [3 2 ] 1 9 .1 4 4 8 7 2 MENTHOL 4 2 7 0 4 9 2 0 5 9 3 0 6 2 5 9 2 5 1 .4 8 5 0 3 4 6 .3 9 9 0 9 C 1 0 H 2 0 O 1 5 6 .2 7 Antibacterial, antifungal, antipruritic, anticancer, cooling effects & toxicity, Antioxidant. [2 8 ] 2 2 .6 3 1 7 7 7 (-)-1R-8-HYDROXY-P- MENTH-4-EN-3-ONE 7 1 6 8 6 1 5 1 1 4 5 0 1 2 8 0 8 .6 4 2 4 7 8 8 .9 5 8 1 6 9 C 1 0 H 1 6 O 2 1 6 8 .2 3 2 No activity was recorded 2 5 .4 9 7 7 0 2 CYCLOHEXENE,4- PENTYL-1- (4- PROPYLCYCLOHEXYL) - 8 0 0 3 8 4 1 7 2 5 9 0 9 6 0 .9 6 4 9 4 2 1 .3 5 0 2 9 C 2 0 H 3 6 2 7 6 .5 0 8 No activity was recorded [3 3 ] 2 9 .7 3 4 7 2 2 ethyl 14-methyl- hexadecanoate 1 1 7 6 3 0 6 2 4 9 4 4 7 8 6 1 .4 1 8 1 5 4 1 .9 5 1 5 9 C 1 8 H 3 6 O 2 2 8 4 Antioxidant, Hypocholesterolemic Nematicide, Antiandrogenic, Flavor, Hemolytic. [5 2 ] 17 3 2 .1 0 0 8 4 6 1-FORMYL-2,2- DIMETHYL-3-TRANS- (3-METHYL-BUT-2- ENYL) -6-METHYLIDENE- CYCLOHEXANE 1 3 5 6 5 8 9 2 2 5 4 8 5 7 2 1 .6 3 5 5 0 3 1 .7 6 4 1 1 9 C 1 5 H 2 4 O 2 2 0 .3 5 6 Antifungal, Insecticidal and larvicidal, Agent. [4 2 ] 4 2 .1 2 9 8 0 8 N-HEXADECANOIC ACID 3 1 6 7 7 3 5 4 6 4 0 1 5 2 4 3 .8 1 9 0 1 9 3 .6 3 0 2 8 8 C 1 6 H 3 2 O 2 2 5 6 .4 Antioxidant, anticancer, food additive, anti- inflammation. [3 7 ] 4 2 .8 7 9 8 9 0 ETHYL 13-METHYL- TETRADECANOATE 1 7 0 7 6 3 6 4 3 0 8 0 0 4 0 0 0 2 0 .5 8 7 2 5 2 4 .0 9 7 1 3 C 1 7 H 3 4 O 2 2 7 0 .4 5 7 antioxi-dant, β-glucuronidase inhibitory and anticancer activities [4 3 ] 4 7 .4 8 6 8 6 7 10-BROMODECANOIC ACID, ETHYL ESTER 1 5 6 8 2 8 5 3 1 1 0 2 5 6 6 1 .8 9 0 7 2 3 2 .4 3 3 3 5 3 C 1 2 H 2 3 B rO 2 2 7 9 .2 1 4 antidiabetic activities [4 4 ] 5 1 .7 2 2 8 4 1 METHYL 17-METHYL- OCTADECANOATE 4 5 7 8 3 6 7 6 8 1 9 0 0 0 .5 5 1 9 6 7 0 .6 0 1 0 0 4 C 2 0 H 4 0 O 2 3 1 2 .5 3 8 Saturated fatty acid, antiasthmatics, antibacterial agents, antioxidants, anticancer, also used for urinary or sexual disorders treatment. [5 3 ] 5 8 .9 5 0 7 8 1 9-OCTADECENAMIDE, (Z)- 2 6 9 6 0 4 1 4 3 3 5 3 4 7 6 3 .2 5 0 3 4 5 3 .3 9 1 8 2 C 1 8 H 3 5 N O 2 8 1 .4 7 7 Anti-inflammatory activity, Antibacterial activity and Antioxidant Activities. [3 1 ] *R.T: retention time (Min), R.I: retention index, M.F: molecular formula, M.W: molecular weight (g/mol), Ref: reference. 18 Fig. (3.1): Trace GC-MS Chromatogram of the ethanol flower extract of Micromeria Fruticosa plant. 19 Fig. (3.3): Trace GC-MS Chromatogram of the hexane flower extract of Micromeria Fruticosa plant. 20 Fig. (3.4): Trace GC-MS Chromatogram spectrum of the ethanol flower extract of Micromeria Fruticosa plant at the R.T. (19.204) minute. 21 Fig. (3.5): Trace GC-MS Chromatogram of the menthol compound from ethanol flower extract of Micromeria Fruticosa plant. 22 Fig. (3.6): Trace GC-MS Chromatogram spectrum of the hexane flower extract of Micromeria Fruticosa plant at the R.T. (58.950) minute. 23 Fig. (3.7): Trace GC-MS Chromatogram of the 9-octadecenamide, (z) - compound from hexane flower extract of Micromeria Fruticosa plant. 24 Fig. (3.8): Trace GC-MS Chromatogram spectrum of the hexane flower extract of Micromeria Fruticosa plant at the R.T. (18.144) minute. 25 Fig. (3.9): Trace GC-MS Chromatogram of the CYCLOHEXANONE, 5-METHYL-2-(1-METHYLETHYL)-, TRANS compound from hexane flower extract of Micromeria Fruticosa plant. 26 The GC-MS chromatogram of hexane extract of leaves in Figure 3.11, ten compounds were identified. Some of the compounds identified were Menthol (21.2568%) and 9-OCTADECENAMIDE, (Z)- (34.34091%). The details of these bioactive compounds were given in Table 3.6. The compounds were identified in ethanolic leave extract as shown in Table 3.5 and the GC-MS of this extract are shown in Figure 3.10. There are similar compounds in the hexane and ethanol leave extract but with different percentage and these compounds are as shown in Table 3.4 and Figure 3.12, 3.13, 3.14. The presence of these compounds in the leave makes this part of the plant is highly effective when the biological and antioxidants activation have been examined. 27 Table )3.4(: The similar compounds of hexane and ethanol leave extract from GC-MS/MS analysis. Similar Compounds Name Molecular formula %high peak In leave ethanol extract %high peak in leave hexane extract CYCLOHEXANONE,5-METHYL -2-(1-METHYLETHYL)-, TRANS C10H18O 13.33935% 7.407839% PULEGONE- C10H16O 63.71028 9.038732 CYCLOBUTANE,1,3-DIPHENYL-, TRANS C16H16 1.112354 3.677446 28 Fig. (3.10): Trace GC-MS Chromatogram of the ethanol leaves extract of Micromeria Fruticosa plant. 29 Fig. (3.11): Trace GC-MS Chromatogram of the hexane leaves extract of Micromeria Fruticosa plant. 30 Fig. (3.11): Trace GC-MS Chromatogram spectrum of the ethanol leaves extract of Micromeria Fruticosa plant at the R.T. (18.144) minute and the fragment for CYCLOHEXANONE, 5-METHYL-2-(1-METHYLETHYL)-, TRANS. 31 Fig. (3.13): Trace GC-MS Chromatogram spectrum of the ethanol leaves extract of Micromeria Fruticosa plant at the R.T. (20.875) minute and the fragment for Pulegone. 32 Fig. (3.14): Trace GC-MS Chromatogram spectrum of the ethanol leaves extract of Micromeria Fruticosa plant at the R.T. (36.701) minute and the fragment for Cyclobutane, 1, 3-Diphenyl-, Trans. 33 Table )3.5(: The compound isolated from GC-MS/MS analysis of leave ethanol extract Extract Type R.T )min.) R.I Possible Compound Name High Area %High %Area M.F M.W (g/mol) Biological Study Ref. L ea v e E th a n o l 1 8 .4 4 9 6 6 CYCLOHEXANON E, 5-METHYL-2- (1- METHYLETHYL), TRANS 1 9 8 4 4 9 6 4 2 6 1 4 7 9 6 1 3 .3 3 9 3 5 1 4 .5 9 0 8 7 C 1 0 H 1 8 O 1 5 4 .2 4 9 antimicrobial activity, potential antibiofilm, antitumor activities [3 2 ] 2 0 .8 7 5 8 7 0 PULEGONE 9 4 7 8 1 8 3 1 8 7 2 5 2 7 0 4 6 3 .7 1 0 2 8 6 4 .1 1 3 4 3 C 1 0 H 1 6 O 1 5 2 .2 3 3 4 Antimicrobial , Antioxidant, anti-inflammatory, anti-ulcer, insecticidal properties. [2 9 ] 2 7 .0 8 8 9 0 5 CARYOPHLLENE 1 8 1 3 8 0 0 3 5 0 2 0 7 7 6 1 2 .1 9 1 9 7 1 1 .9 9 0 7 6 C 1 5 H 2 4 2 0 4 .3 6 anti-inflammatory, antibiotic, antioxidant, anticarcinogenic, local anaesthetic activities, anticancer activity. antinociceptive, neuro protective, anxiolytic, anti depressent, antialcoholism, anti pyritics, anti-microbial, anti- carcinogenic, anti dermatitic, allergenic, aldose- reductase inhibitor, anti-acne, anti- asthmatic, anti-ulcer, anti proliferants, cyto protective, gastro protective, sedative, anti- spasmodic, flavour. [3 0 , 4 0 ] 34 2 9 .0 3 3 8 6 5 1H- CYCLOPENTA[1,3 ]CYCLOPROPA[1, 2] BENZENE,OCTAH YDRO-7- METHYL-3- METHYLENE-4- (1- METHYLETHYL)- 6 1 4 5 4 1 1 1 5 5 9 9 9 1 4 .1 3 0 8 1 1 3 .9 5 8 0 2 4 C 1 5 H 2 4 2 0 4 .3 5 1 1 antibacterial activity. [3 8 ] 3 6 .7 0 1 8 6 3 CYCLOBUTANE, 1,3-DIPHENYL-, TRANS 1 6 5 4 8 5 3 0 0 3 7 5 1 1 .1 1 2 3 5 4 1 .0 2 8 4 5 4 C 1 6 H 1 6 2 0 8 .3 0 4 estrogen screening assay and estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. [3 9 ] 4 6 .2 6 0 8 8 9 METHYL 8, 11, 14- HEPTADECATRIE NOATE 8 2 0 5 0 2 1 2 6 1 2 7 0 5 5 .5 1 5 2 3 6 4 .3 1 8 4 6 2 C 1 8 H 3 0 O 2 2 7 8 .4 3 6 No activity was recorded *R.T: retention time (Min), R.I: retention index, M.F: molecular formula, M.W: molecular weight (g/mol), Ref: reference. 35 Table )3.6(: The compound isolated from GC-MS/MS analysis of leave hexane extract Extract Type R.T )min.) R.I Possible Compound Name High Area %High %Area M.F M.W )g/mol( Biological Study Ref. L ea v e H ex a n e 7 .1 4 0 8 9 8 STYRENE 1 1 1 2 9 2 2 6 3 4 6 7 2 3 .8 4 8 8 7 5 .5 5 0 9 7 C 8 H 8 1 0 4 .1 5 No activity was recorded 1 7 .6 1 9 7 1 7 CYCLOHEXENE, 1-METHYL-4- (1- METHYLETHYLI DENE)- 6 0 2 2 4 1 2 1 4 1 3 7 2 .0 8 2 7 6 2 2 .5 5 8 0 5 9 C 1 0 H 1 6 1 3 6 .2 3 4 Antioxidant, antiasthmatics, antibacterial agents, for wound treatment, food, flavouring agent, also used for urinary and sexual disorder, bioactive. [3 3 , 3 4 ] 1 8 .1 4 4 9 1 9 CYCLOHEXANO NE, 5-METHYL-2- (1- METHYLETHYL) -, TRANS 1 1 4 1 9 1 4 3 1 9 8 5 1 7 .4 0 7 8 3 9 9 .1 0 1 4 7 2 C 1 0 H 1 8 O . 1 5 4 .2 4 9 antimicrobial activity, potential antibiofilm, antitumor activities [3 2 ] 1 9 .1 9 9 8 5 3 MENTHOL 6 1 4 6 5 0 8 4 5 6 7 0 1 2 1 .2 5 6 8 1 7 .8 1 7 3 8 C 1 0 H 2 0 O 1 5 6 .2 7 Antibacterial ,antifungal ,antipruritic, anticancer, cooling effects and toxicity, Antioxidant. [2 8 ] 2 0 .8 8 0 8 2 3 PULEGONE 2 6 1 3 5 9 5 0 6 0 8 9 1 9 .0 3 8 7 3 2 1 0 .6 6 2 7 6 C 1 0 H 1 6 O 1 5 2 .2 3 3 4 Antimicrobial, Antioxidant, anti- inflammatory, anti-ulcer, insecticidal properties. [2 9 ] 36 2 2 .6 7 6 7 0 1 (-)-1R-8- HYDROXY-P- MENTH-4-EN-3- ONE 1 1 4 2 2 9 1 9 1 1 7 6 9 3 .9 5 0 4 4 9 4 .0 2 7 8 9 6 C 1 0 H 1 6 O 2 1 6 8 .2 3 2 No activity was recorded 3 6 .6 9 1 8 5 8 CYCLOBUTANE, 1,3-DIPHENYL-, TRANS 1 0 6 3 3 5 1 9 7 8 4 9 7 3 .6 7 7 4 4 6 4 .1 6 8 4 8 4 C 1 6 H 1 6 2 0 8 .3 0 4 estrogen screening assay and estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. [3 9 ] 4 2 .1 0 8 5 8 5 N- HEXADECANOIC ACID 1 8 0 4 7 3 3 0 0 3 9 0 0 6 .2 4 1 4 0 4 6 .3 2 8 9 C 1 6 H 3 2 O 2 2 5 6 .4 Antioxidant, anticancer, food additive, anti-inflammation. [3 7 ] 4 6 .2 1 5 8 4 7 METHYL 2- HYDROXY- OCTADECA- 9, 12 ,15- TRIENOATE 2 3 5 7 9 9 3 6 0 4 2 5 3 8 .1 5 4 7 7 5 7 .5 9 3 7 8 1 C 1 9 H 3 2 O 3 3 0 8 .4 6 2 No activity was recorded 5 8 .9 4 0 7 7 1 9- OCTADECENAMI DE, (Z)- 9 9 2 9 8 3 1 5 2 7 8 5 5 0 3 4 .3 4 0 9 1 3 2 .1 9 0 2 9 C 1 8 H 3 5 N O 2 8 1 .4 7 7 Anti-inflammatory activity, antibacterial activity and Antioxidant Activities. [3 1 ] *R.T: retention time (Min), R.I: retention index, M.F: molecular formula, M.W: molecular weight (g/mol), Ref: reference. 37 Table (3.7): The compound isolated from GC-MS/MS analysis of stem hexane and ethanol extract Extract Type R.T )min.) R.I Possible Compound Name High Area %High %Area M.F M.W )g/mol( Biological Study Ref. S te m H ex a n e 1 9 .1 9 9 8 8 0 MENTHOL 4 6 4 1 0 9 2 7 9 5 6 8 3 3 6 6 3 .2 4 8 9 6 6 .6 1 1 6 C 1 0 H 2 0 O 1 5 6 .2 7 Antibacterial ,antifungal ,antipruritic, anticancer, cooling effects and toxicity, Antioxidant. [28] 5 4 .7 5 3 8 8 7 HENTRIAC ONTANE 4 7 1 3 2 9 5 2 5 6 4 1 0 6 .4 2 3 2 8 5 4 .4 0 0 4 6 8 C 3 1 H 6 4 4 3 6 .8 5 Antifugal against fungal spores germination, Antioxidant, antitumor activity and antibacterial. [41] 5 8 .9 3 5 7 6 6 9- OCTADECE NAMIDE, (Z)- 2 2 2 5 3 9 8 3 4 6 2 6 4 2 0 3 0 .3 2 7 7 9 2 8 .9 8 7 9 3 C 1 8 H 3 5 N O 2 8 1 .4 7 7 Anti-inflammatory activity, antibacterial activity and Antioxidant Activities. [31] S te m E th a n o l 1 9 .2 1 4 8 6 4 MENTHOL 1 3 7 2 4 9 6 1 7 9 2 1 3 8 8 1 0 0 1 0 0 C 1 0 H 2 0 O 1 5 6 .2 7 Antibacterial ,antifungal ,antipruritic, anticancer, cooling effects and toxicity, Antioxidant. [28] *R.T: retention time (Min), R.I: retention index, M.F: molecular formula, M.W: molecular weight (g/mol), Ref: reference. 38 The GC-MS chromatogram of ethanol and hexane stem extract as shown in Figure 3.15, 3.13. Three compounds were identified as shown in Table 3.7. The compound that had the highest percentage peak is Menthol (63.2489%, 100%) in stem hexane and ethanol extract respectively, and this compound has strong antioxidant, antibacterial activity when we were estimated. The ethanolic extract had antifungal activity in [C. albicans (ATCC 90028)] as shown in chapter five. 39 Fig. (3.15): Trace GC-MS Chromatogram of the ethanol stems extract of Micromeria Fruticosa plant. 40 Fig.3.16. Trace GC-MS Chromatogram of the hexane stems extract of Micromeria Fruticosa plant. 41 Table )3.8(: The compound isolated from GC-MS/MS analysis of roots ethanol extract Extract Type R.T )min.) R.I Possible Compound Name High Area %High %Area M.F M.W )g/mol( Biological study Ref. R o o ts E th a n o l 3 7 .9 0 2 8 2 4 CIS-1- CHLORO-9- OCTADECE NE 3 4 0 1 6 4 6 4 0 4 5 4 4 3 .5 6 2 7 8 .3 0 0 1 2 2 C 1 8 H 3 5 C l 2 8 6 .9 2 3 Antibacterial activity. [45] 4 2 .9 2 9 8 8 4 ETHYL 14- METHYL- HEXADECA NOATE. 1 1 3 9 3 1 9 1 9 6 9 8 1 9 1 1 1 .9 3 3 0 3 2 6 .8 2 4 3 3 C 1 8 H 3 6 O 2 2 8 4 .4 7 7 Antifungal, antitumor activity, Antibacterial. [46] *R.T: retention time (Min), R.I: retention index, M.F: molecular formula, M.W: molecular weight (g/mol), Ref: reference. 42 Table )3.9(: The compound isolated from GC-MS/MS analysis of roots hexane extract Extract Type R.T (min.) R.I Possible Compound Name High Area %High %Area M.F M.W (g/mol) Biological study Ref Roots Hexane 1 9 .2 0 4 8 0 1 MENTHOL 3 0 7 9 0 5 5 1 7 5 2 0 2 4 .1 3 9 9 9 3 .6 9 9 8 7 C 1 0 H 2 0 O 1 5 6 .2 7 Antibacterial, antifungal ,antipruritic, anticancer, cooling effects and toxicity, Antioxidant. [2 8 ] 4 1 .1 5 9 8 1 4 N- HEXADECANOIC ACID 6 8 1 1 5 3 1 1 8 1 3 7 4 7 9 .3 7 2 9 9 4 8 .2 4 0 4 7 C 1 6 H 3 2 O 2 1 5 6 .4 Antioxidant, anticancer, food additive, anti-inflammation. [3 7 ] 4 2 .9 3 4 9 0 2 ETHYL 13- METHYL- TETRADECANOAT E 3 1 8 0 2 4 6 6 8 1 2 9 4 0 8 4 3 .6 9 1 1 6 4 7 .5 2 2 4 C 1 7 H 3 4 O 2 2 7 0 .4 5 7 antioxi-dant, β-glucuronidase inhibitory & anticancer activities [4 3 ] 5 4 .4 3 3 8 1 3 1,2- BENZENEDICARB OXYLIC ACID, DIISOOCTYL ESTER. 4 8 6 0 5 4 8 9 6 3 9 4 6 6 .6 7 7 5 5 4 6 .2 5 2 6 4 C 2 4 H 3 8 O 4 3 9 0 .5 5 6 Antioxidant, Antimicrobial, Antifoulin. [4 7 , 4 8 ] 5 8 .9 6 5 7 5 0 9- OCTADECENAMID E, (Z)- 1 2 1 7 1 2 6 2 0 9 4 1 2 7 8 1 6 .7 2 1 2 4 1 4 .6 0 7 2 C 1 8 H 3 5 N O 2 8 1 .4 7 7 Anti-inflammatory activity, antibacterial activity and Antioxidant Activities. [3 1 ] 6 1 .0 3 6 9 2 6 HENTRIACONTAN E 1 4 0 5 3 3 9 2 8 3 3 8 9 4 0 1 9 .3 0 6 9 6 1 9 .7 6 7 3 C 3 1 H 6 4 4 3 6 .8 5 Antifugal against fungal spores germination, Antioxidant, antitumor activity and antibacterial. [4 1 ] *R.T: retention time (Min), R.I: retention index, M.F: molecular formula, M.W: molecular weight (g/mol), Ref: reference. 43 The GC-MS chromatogram of root ethanol extract, three compounds were identified and are shown in Table 3.8 and Figure 3.17. These compound DIDODECYL PHTHALATE (84.5%) and ETHYL 14-METHYL-HEXADECANOATE (11.9%) are shown in Figure 3.23, 3.24 respectively. However root hexane extract, six compounds were identified as shown in Table 3.9 and Figure 3.18. That compound include ETHYL 13- METHYL-TETRADECANOATE (43.69%), HENTRIACONTANE (19.3%) as shown in Figure 3.22, 9-OCTADECENAMIDE, (Z)- (16.7%), N-HEXADECANOIC ACID (9.3%) as shown in Figure 3.21. 1,2-BENZENEDICARBOXYLIC ACID, DIISOOCTYL ESTER (6.6%) as shown in Figure 3.19, and MENTHOL (4.23%) as shown in Figure 3.20. 44 Fig. (3.17): Trace GC-MS Chromatogram of the ethanol root extract of Micromeria Fruticosa plant. 45 Fig. (3.18): Trace GC-MS Chromatogram of the hexane root extract of Micromeria Fruticosa plant. 46 Fig. (3.19): Trace GC-MS Chromatogram spectrum of the hexane root extract of Micromeria Fruticosa plant at the R.T. (54.433) minute and the fragment for 1, 2-BENZENEDICARBOXYLIC ACID, DIISOOCTYL ESTER compound. 47 Fig. (3.20): Trace GC-MS Chromatogram spectrum of the hexane root extract of Micromeria Fruticosa plant at the R.T. (19.204) minute and the fragment for Menthol compound. 48 Fig. (3.21) Trace GC-MS Chromatogram spectrum of the hexane root extract of Micromeria Fruticosa plant at the R.T. (42.159) minute and the fragment for N-hexadecanoic acid compound. 49 Fig. (3.22): Trace GC-MS Chromatogram spectrum of the hexane root extract of Micromeria Fruticosa plant at the R.T. (57.555) minute and the fragment for HENTRIACONTANE compound. 50 Fig. (3.23):Trace GC-MS Chromatogram spectrum of the ethanol root extract of Micromeria Fruticosa plant at the R.T. (54.428) minute and the fragment for DIDODECYL PHTHALATE compound. 51 Fig. (3.24): Trace GC-MS Chromatogram spectrum of the ethanol root extract of Micromeria fruticosa plant at the R.T. (42.929) minute and the fragment for ETHYL 14-METHYL-HEXADECAOATE compound. 52 Chapter Four ICPMS (Inductively coupled plasma mass spectrometry) Analysis 4.1 Sample preparation for ICPMS-Analysis A 50 mg of aqueous plant extract was dissolved in 50 ml de-ionized distilled water. 4.2 ICPMS techniques ICP-MS is used for quantification and qualification measurements of heavy metal in samples. Samples are decomposed to neutral elements in high-temperature argon plasma and analyzed based on their masse to charge ratios using a mass spectrometer system. Elements can be analyzed at the parts-per-million (PPM) to parts- per-trillion (PPT) concentration levels. ICP-MS is also capable of monitoring isotopic specification for the ions of choice. 4.3 Results and Discussion for ICPMS ANALYSIS The elements shown in Table 4.1. were present at high concentration in leaves aqueous extract. These elements were Fe, Zn, Mn, and Sr (346, 85.8, 81.7, and 67.7 ppb) respectively. However, in stem aqueous extract, only Fe and Zn (165 and 77 ppb) had a high concentration in this part of the plant as shown in Table 4.2. 53 Table (4.1): The concentration of various elements from leave aqueous extract using ICPMS spectrometer. Extract type Analyte Conc. (ppb) Dilution factor Leave D.W Ag 0.286 286 Al 33.514 33514 Ba-1 21.494 21494 Cd 0.067 67 Cr 7.783 7783 Co 0.344 344 Cs 0.020 20 Cu 8.969 8969 Fe 346.751 346751 Ga 0.174 174 Li 1.305 1305 Mn 81.700 81700 Mo 1.245 1245 Ni 10.784 10784 Pb 1.066 1066 Rb 13.949 13949 Sr 67.704 67704 V 0.181 181 Zn 85.881 85881 54 Table (4.2): The concentration of various elements from stem aqueous extract using ICPMS spectrometer Extract type Analyte Conc. (ppb) Dilution factor Stem D.W Ag 0.043 43 Al 17.991 17991 Ba-1 14.663 14663 Cd 0.257 257 Cr 4.977 4977 Co 0.194 194 Cs 0.041 41 Cu 6.882 6882 Fe 165.348 165348 Ga 0.115 115 Li 1.006 1006 Mn 23.311 23311 Mo 0.607 607 Ni 3.492 3492 Pb 1.494 1494 Rb 14.384 14384 Sr 23.018 23018 V 0.095 95 Zn 77.144 77144 In Table 4.3 high concentration of elements from flower aqueous extract were Fe, Zn and Sr (238, 58, 38.9 ppb) respectively. As shown in Table 4.4, Fe and Al (228 and 65 ppb) had the highest concentration in root aqueous extract of the plant. 55 Table (4.3): The concentration of various elements from flower aqueous extract using ICPMS spectrometer Extract type Analyte Conc. (ppb) Dilution factor Flower D.W Ag 0.145 145 Al 27.264 27264 Ba-1 15.640 15640 Bi 0.021 21 Cd 0.086 86 Cr 5.891 5891 Co 0.441 441 Cs 0.062 62 Cu 18.716 18716 Fe 238.593 238593 Ga 0.123 123 Li 0.859 859 Mn 43.494 43494 Mo 0.305 305 Ni 9.320 9320 Pb 0.908 908 Rb Sr 38.965 38965 V 0.091 91 Zn 58.116 58116 *D.W: distilled water, Conc.: Concentration (PPb). 56 Table (4.4): The concentration of various elements from root aqueous extract using ICPMS spectrometer Extract type Analyte Conc. (ppb) Dilution factor Root D.W Ag 0.204 204 Al 65.201 65201 Ba-1 19.218 19218 Bi 0.021 21 Cd 0.136 136 Cr 6.972 6972 Co 1.643 1643 Cs 0.024 24 Cu 11.910 11910 Fe 228.406 228406 Ga 0.174 174 Li 0.496 496 Mn 48.519 48519 Mo 1.102 1102 Ni 6.977 6977 Pb 2.270 2270 Rb 12.532 12532 Sr 25.692 25692 Continue to Table 4.4 V 1.170 1170 Zn 25.926 25926 *D.W: distilled water, Conc.: Concentration (PPb). 57 Chapter Five Biological activities 5.1 Antimicrobial and antifungal activity tests 5.1.1 Preparation of samples for testing Organic plant extracts (ethanol and hexane extracts) were dissolved in sterile 10% Dimethyl sulfoxide (DMSO) to obtain a concentration of 100 mg/ml, while aqueous extracts were dissolved in sterile distilled water to obtain a concentration of 100mg/ml. The dissolved extracts stored at 4°C for further assays. 5.1.2 Media and Solutions 5.1.2.1. Nutrient Broth Nutrient broth (ACUMEDIA) was prepared according to manufacturer's instructions labeled on the bottle. 2.0 g of nutrient broth medium was dissolved in 250 ml de-ionized water. The broth was then distributed into tubes to have 5-10 ml each and plugged with cotton. The tubes were autoclaved at 121°C for 15 minutes, and allowed to cool to room temperature, then stored in refrigerate. 5.1.2.2. Mueller-Hinton Broth Mueller-Hinton broth (Hi Media Laboratories) was prepared according to manufacturer's instructions labeled on the bottle. 5.95 g of 58 Mueller –Hinton Broth medium was dissolved in 250 ml de-ionized water. The broth was then distributed into tubes to have 5-10 ml each and plugged with cotton. The tubes were autoclaved at 121°C for 15 minutes, allowed to cool and then kept in refrigerator at 4-6°C. 5.1.2.3. Mueller Hinton Agar (MHA) Mueller Hinton agar (BD) was prepared according to manufacturer's instructions labeled on the bottle. 19 g of Mueller –Hinton Agar medium was dissolved in 0.5 L de-ionized water. The solution allowed to boil for 1 minute, and then autoclaved at 121°C for 15 minutes. After that it was allowed to cool to about 55°C, and the media was poured into sterile Petri dishes to have (25-30) ml each, then the plates were left overnight at room temperature. The following morning the Petri dishes were turned upside down and kept in refrigerator at 4-6°C. 5.1.2.4. Sabouraud Dextrose Agar Sabouraud dextrose agar (Hi Media Labratories) was prepared according to manufacturer's instructions labeled on the bottle. 16.25 g of Sabouraud Dextrose Agar medium was dissolved in 0.25 L de-ionized water. The solution allowed to boil for 1 minute, and then autoclaved at 121°C for 15 minutes. After that it was allowed to cool to about 55°C, and the medium was poured into sterile Petri dishes to have (25-30) ml each, then, the plates were left overnight at room temperature. The following 59 morning the Petri dishes were turned upside down and kept in refrigerator at 4-6°C. 5.1.2.5. Normal Saline (0.9% NaCl) Normal saline solution (0.9% NaCl, MWt 58.44) was prepared by dissolving 2.25 g NaCl in 250 ml de-ionized water. The saline solution was then distributed into tubes to have 5-10 ml each and plugged with cotton. The tubes were autoclaved at 121°C for 15 min, allowed to cool and then kept in refrigerator at 4-6°C. 5.1.2.6. Preparation of McFarland turbidity standard No. 0.5 McFarland 0.5 turbidity standard was prepared by mixing 50 μl of a 1.175% (w/v) barium chloride dihydrate (BaCl2•2H2O) solution and 9.95 ml of 1% (v/v) sulfuric acid. The tube which had the 0.5 McFarland standard was then sealed with parafilm to prevent evaporation and stored in the dark at room temperature. The 0.5 McFarland standard was vigorously mixed on a vortex mixer before use. As with the barium sulfate standards, a 0.5 McFarland standard is comparable to a bacterial suspension of 1-5 x 10 8 colony-forming units (CFU)/ml (Andrews, 2006) [54] or 0.5 McFarland standard is comparable to Candida albicans suspension of 1-5 x 10 6 yeast cells/mL (Branda and Kratz, 2006) [55]. Three to four colonies of each bacteria were transferred into tubes had 5.0-10 mL of sterile normal saline, the turbidity of the bacterial suspensions was adjusted to have similar turbidity of 0.5 McFarland 60 standard with bacterial suspension of about 1.5 x10 8 cfu/mL. Few colonies from C. albicans sub-cultured on Sabouraud Dextrose Agar were transferred into tube had 5.0-10 mL of sterile normal saline. The turbidity of the yeast C. albicans suspension of about 1-5 x 10 6 yeast cells/mL. 5.1.3. Test Microorganisms Microorganisms used in this study represent pathogenic species commonly associated with infections. The microorganisms were stored in the Microbiology research laboratory at An-Najah National University, Nablus-Palestine. These microorganisms consisted of 2 Gram-positive strains Staphylococcus aureus (S. aureus) (ATCC 25923), clinical isolate of methicillin resistant staphylococcus aureus (MRSA), two Gram-negative strains, Shigellasonnie (S. sonnie) (ATCC 25931) and multidrug clinical Escherichia coli (E. coli) isolate and one yeast strain Candida albicans (C. albicans) (ATCC 90028). All the bacterial strains were subcultured on Mueller-Hinton agar while C. albicans was sub-cultured on Sabouraud Dextrose Agar. 5.1.4 Determination of Minimum Inhibitory concentration (MIC) 5.1.4.1Determination of Minimum Inhibitory concentration (MIC) against bacteria Minimum Inhibitory concentration (MIC) of organic plant extracts (ethanol and hexane) and aqueous plant extracts was determined by the broth microdilution method in sterile 96- wells microtiter plates according 61 to standard method described previously by the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2017). The organic plant extracts and aqueous plant extracts were dissolved in sterile 10% DMSO and sterile distilled water, respectively, to a final concentration 100mg/ml. Both extracts organic and water and 10% DMSO (negative control) were two- fold-serially diluted in nutrient broth in the wells of the plates in a final volume of 100μL. After that, a bacterial inoculum size of 10 5 CFU/ml was added to each well. Other negative control wells containing either 100μL nutrient broth only, or organic plant extracts (or aqueous plant extracts) and nutrient broths without bacteria were included in these experiments. Each plant extract was run in duplicate. The microtiter plates were then covered and incubated at 37°C for 24 hours. The MIC was considered as the lowest concentration of the plant extract which inhibited the bacterial growth. 5.1.4.2 Determination of Minimum Inhibitory concentration (MIC) against yeast Minimum Inhibitory concentration (MIC) of organic plant extracts (ethanol and hexane extracts) and aqueous plant extracts was determined by the broth microdilution method in sterile 96- wells microtiter plates according to standard method described previously (CLSI, 2017) [56]. The organic plant extracts and aqueous plant extracts were dissolved in sterile 10% DMSO and sterile distilled water, respectively, to a final concentration 100mg/ml. Both extracts organic and water and 10% DMSO (negative control) were two-fold-serially diluted in Mueller-Hinton broth in 62 the wells of the plates in a final volume of 100μL. After that, a C. albicans inoculum size of0.5 × 10 5 to 2.5 × 10 5 CFU/ml was added to each well. Other negative control wells containing either 100μL Mueller-Hinton broth only, or organic plant extracts (or aqueous plant extracts) and Mueller- Hinton broth without bacteria were included in these experiments. Each plant extract was run in duplicate. The microtiter plates were then covered and incubated at 37°C for 48 h. The MIC was considered as the lowest concentration of the plant extract which inhibited the yeast growth. 5.2 Results and Discussion for biological activities The antibacterial activities of the extracts obtained from the Micromeria Fruticosa under study by the broth microdilution method against different pathogens are shown in Table 5.1 and Figures 5.2, 5.3, 5.4, and 5.5. 63 Table (5.1): Minimum Inhibitory concentration values (µg/ml) for different Micromeria Fruticosa extract types against different pathogens Microorganism Type of extract Leave extract Flower extract Stem extract Root extract W E H W E H W E H W E H MIC (µg/ml) E. coli 12.5 0.78 100 49.5 49.5 58 8.70 95 488 49.5 488 488 S.sonnie (ATCC 25931) 3.195 6.25 50 4.53 3.95 58 4.53 488 488 4.495 95 488 MRSA 6.25 12.5 50 4.495 3.95 95 4.53 488 488 3.95 95 488 S. aureus (ATCC 25923) 3.125 6.25 50 49.5 4.495 58 4.53 95 488 3.95 3.95 488 C. albicans (ATCC 90028) 100 3.125 50 488 58 95 488 95 488 488 95 488 * W: aqueous extract, E: ethanol extract, H: hexane extract 64 Results of the current study showed that aqueous leaf extract had the highest antimicrobial activity against S. sonnie (ATCC 25931) and S. aureus (ATCC 25923), while the ethanolic leaf extract had the highest antimicrobial and antifungal activity against E. coli and C. albicans (ATCC 90028). Minimum Inhibitory concentration of different leaf extract types against different pathogens are presented in Figure 5.1. Figure (5.1): Minimum Inhibitory concentration (µg/ml) of different leave extract types against different pathogens. 0 20 40 60 80 100 120 E.coli S.sonnie (ATCC 25931) MRSA S.aureus (ATCC 25923) C.albicans (ATCC 90028) M IC ( µ g/ m l ) Studied microorganisms Leaf D.W Leaf Ethanol Leaf Hexane 65 Results of this study showed that aqueous flower extract had the highest antimicrobial activity against S. sonnie (ATCC 25931) and MRSA, while ethanolic flower extract had the highest antimicrobial activity against S. aureus (ATCC 25923). Hexane flower extract had the highest ativity against C. albicans (ATCC 90028) compared with aqueous and ethanolic flower extracts. Minimum Inhibitory concentration of different flower extract types against different pathogens are presented in Figure 5.2. Figure (5.2): Minimum Inhibitory concentration (µg/ml) of different flower extract types against different pathogens. 0 20 40 60 80 100 120 E.coli S.sonnie (ATCC 25931) MRSA S.aureus (ATCC 25923) C.albicans (ATCC 90028) M IC ( µ g/ m l) Studied microorganisms Flower D.W Flower Ethanol Flower Hexane 66 Results of the current study showed that aqueous stem extract had the highest antimicrobial activity against both Gram-positive (S. aureus (ATCC 25923) and MRSA) and Gram-negative bacteria (S. sonnie (ATCC 25931) and E. coli). Hexane stem extract had the highest activity against C. albicans (ATCC 90028) compared with aqueous and ethanolic stem extracts. Minimum Inhibitory concentration of different stem extract types against different pathogens are presented in Figure 5.3. Figure (5.3): Minimum Inhibitory concentration (µg/ml) of different stem extract types against different pathogens. 0 20 40 60 80 100 120 E.coli S.sonnie (ATCC 25931) MRSA S.aureus (ATCC 25923) C.albicans (ATCC 90028) M IC ( µ g/ m l) Studied microorganisms Stem D.W Stem Ethanol Stem Hexane 67 Results of the current study showed that aqueous root extract had the highest antibacterial activity against both Gram-positive (S. aureus (ATCC 25923) and MRSA) and Gram-negative bacteria S. sonnie (ATCC 25931). Ethanol root extract had the highest activity against C. albicans (ATCC 90028) compared with aqueous and haxane root extracts. Minimum Inhibitory concentration of different root extract types against different pathogens are presented in Figure 5.4. Figure (5.4): Minimum Inhibitory concentration (µg/ml) of different root extract types against different pathogens. 0 20 40 60 80 100 120 E.coli S.sonnie (ATCC 25931) MRSA S.aureus (ATCC 25923) C.albicans (ATCC 90028) M IC ( µ g/ m l) Studied microorganisms Root D.W Root Ethanol Root Hexane 68 Chapter six Anti-oxidant activity (free radical scavenging activity) 6.1. General procedure of anti-oxidant test for Micromeria Fruticosa plant extract. The hydrogen atom or electron donation abilities of the corresponding compounds were measured from the bleaching of the purple- colored methanolic solution of DPPH (1, 1- Diphenyl -2-picryl-hydrazyl). This spectrophotometric assay uses the stable radical DPPH as a reagent. A stock solution of a concentration of 1mg/ml in methanol was initially prepared for plant extract. Stock solutions were used to prepare working solutions with the following concentrations (2, 5, 10, 30, 50, 80, 100μg/ml) by using serial dilution in methanol. A solution of DPPH was freshly prepared at a concentration of 0.002% w/v. Then, it was mixed with methanol along with each of the working concentration in ratio of 1:1:1. The spectrophotometer was zeroed using methanol as a blank solution. The first solution of the series concentration was DPPH with methanol only. The solutions were incubated at room temperature in a dark cabinet for about 30 minutes. Then, their optical densities were determined by using the spectrophotometer at a wavelength of 517nm. 69 The percentage of antioxidant activity of plant extract were calculated the following formula: DPPH inhibition activity (I %) = (Ablank-Asample)/Ablank*100%. 6.2. Results and discussion Values of %Inhibition for the Micromeria Frtcosa plant extract shown in Table 6.1. In D.W and Hexane extract the leaf had high %Inhibition at low concentration while root extract had minimum %Inhibition. But in ethanol extract the root had high %Inhibition at low concentration. As shown in Figures 6.4, 6.9 and 6.4. 70 Table 6.1: %Inhibition and IC50 for D.W, Ethanolic and Hexane extracts. Conc. %Inhibition for Different extracts Extract type D.W from leaf D.W from root D.W from stem D.W from flower Hexane from leaf Hexane from root Hexane from stem Hexane from flower Ethanol from leaf Ethanol from root Ethanol from stem Ethanol from flower 2 18.35 1.56 13.39 10.98 44.08 25.72 32.24 28.26 10.86 44.56 0 44.2 5 26.68 1.56 18.96 13.28 49.27 27.29 32.24 29.22 11.59 44.56 0 44.32 10 30.67 6.51 23.06 21.613 49.27 28.13 35.14 29.46 11.59 45.65 5.79 44.32 30 50.23 10.5 35.15 40.81 49.27 28.74 35.14 31.88 11.59 51.8 11.955 49.63 50 64.84 15.45 52.65 54.22 51.811 31.15 39.85 31.88 11.83 51.811 23.18 54.71 80 82.36 24.4 65.57 76.81 53.742 32.96 39.85 34.05 13.16 52 46.13 60.38 100 89.73 34.05 71.13 80.07 63.768 32.96 39.85 35.86 13.88 52.17 60.09 67.38 .* D.W: aqueous extract. 71 Figure (6.1): Anti-oxidant % Inhibition concentration (µg/ml) of different D.W extract types. 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 % In h ib it io n Concentration (µg/ml) D.W from leaf D.W from root D.W from stem D.W from flower 72 Figure (6.1): Anti-oxidant % Inhibition concentration (µg/ml) of different Hexane extract types. 0 10 20 30 40 50 60 70 0 20 40 60 80 100 120 % In h ib it io n Concentration (µg/ml) Hexane from leaf Hexane from root Hexane from stem Hexane from flower 73 Figure (6.3): Anti-oxidant % Inhibition concentratio (µg/ml) of different Ethanol extract types. -10 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 % In h ib it io n Concentration (µg/ml) Ethanol from leaf Ethanol from root Ethanol from stem Ethanol from flower 74 Chapter Seven Conclusion Antifungal test: Ethanolic leave extract showed complete inhibition against C.albicans (ATCC 90028) at 3.125 µg/ml. Antibacterial test: Ethanolic leave extract and aqueous stem extract showed complete inhibition against E.coli at 0.78 µg/ml. Aqueous flower extract and aqueous stem extract showed complete inhibition against S.sonnie (ATCC 25931) at 1.56 µg/ml. Aqueous stem extract showed complete inhibition against S.aureus (ATCC 25923) and MRSA at 1.56 µg/ml. Antioxidant test: In aqueous and hexane extracts the leaf showed good inhibition at low concentration 2µg/ml. Ethanolic root extract is useful showed high inhibition 44.56% at 2µg/ml. GC-MS/MS analysis: Some constituents were detected from ethanolic and hexane plants extracts using GC-MS/MS spectrophotometer and separated by flash 75 chromatography the most similar compound found in all part in plant is 9- OCTADECENAMIDE, (Z)-, Menthol and CYCLOHEXANONE,5- METHYL-2-(1-METHYLETHYL)-, TRANS with different high, and area percentage. 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Wayne, PA, USA: Clinical Laboratory Standards Institute; 2017. https://www.ncbi.nlm.nih.gov/pubmed/?term=Branda%20JA%5BAuthor%5D&cauthor=true&cauthor_uid=16891201 https://www.ncbi.nlm.nih.gov/pubmed/16891201 أ‌ جامعة النجاح الوطنية كمية الدراسات العميا توصيف المواد الكيميائية النباتية النشطة بيولوجيا من نبات الزعتمان (Micromeria fruticosa) كنهج لتطوير ادوية من مصدر طبيعي إعداد أنوار عفيف عبد الرحمن الحجة إشراف د محمد النوري أ. د. أبراهيم ابو ريدة في الكيمياء بكمية قدمت هذه األطروحة استكماال لمتطمبات درجة الماجستير فمسطين. -الوطنية، نابمس الدراسات العميا في جامعة النجاح 9102 ب‌ توصيف المواد الكيميائية النباتية النشطة بيولوجيا من نبات الزعتمان (Micromeria fruticosa) كنهج لتطوير ادوية من مصدر طبيعي إعداد أنوار عفيف عبد الرحمن الحجة إشراف النوريد محمد أ. د. أبراهيم ابو ريدة الممخص نبتة يهدف هذا البحث الى فحص مستخمصات اربعة اجزاء )اوراق, زهرة, ساق, جذور( من في فمسطين لعالج امراض مختمفة و اجراء بعض التي تستخدم في الطب الشعبيالزعتمان البكتيريا ونوع واحد من الفحوصات الحيوية عميها مثل أثر المستخمصات عمى أربعة أنواع من الفطريات بحث أكدت النتائج المخبرية عمى أن بعض المستخمصات لها أثر حيوي في بعض الفحوصات التي تمت. تم جمع هذه النبتة من جبال مدينة طولكرم وتعرف هذه النيتة ايضا بأسم عشبة الشاي في بعة من هذه النيتة بااليثانول المستخمصات الناتجة من نقع هذه االجزاء االر هذه المناطق والهكسان والماء المقطر تم فحصها ايضا لمدى أثرها في منع بحيث كانت فعالية المستخمص الذي نقع في الماء المقطر في منع عممية التأكسد (DPPH assay) 89تصل الى نسبة . او وقف عممية التأكسد بأستخدام%. لتعرف عمى المركبات الفعالة المكونة لكل جزء تم أما عممية فصل المستخمصات العضوية وا .أستخدام جهاز الكروموتغرافي (GC-MS/MS)بحيث تم إيجاد مركبات فعالة متشابهة في مكونات النبتة األربعة. وتم ايضا استخدام (ICPMS) لمتعرف عمى المعادن واالمالح الموجودة في المستخمصات المائية .جهاز