Supervisor: Prof

Al-Neelain University

Graduate College

The Effect of Drying on Chemical Composition and Antioxidant activity ofCymbopogon citratus and Cymbopogon schoenanthus

(L.) spring (proximus) Growing in Sudan

A Thesis Submitted for Fulfillment of the Requirements of Master Degree in Chemistry By

Balgies Abd Alrouf Abd Alrahman

Sudan University of Science and Technology

(B.sc) of Science Section of Chemistry

Dec. / 2006 Supervisor: Prof. Dr.Saad Mohamed Hussein Ayoub Khartoum

October . / 2017

ﭧ ﭨ ﭽ ﯱ ﯲ ﯳ ﯴ ﯵ ﯶ ﯷ ﯸ ﯹ ﯺ ﯻ ﯼ ﯽ ﯾ

ﯿ ﰀ ﰁ ﰂ ﰃ ﭼ الكهف: ٩٠١

DEDICATION

I would like to dedicate my thesis to my Father, Mother, Brother and my friends

ACKNOWLEDGMENTS

All praise be to Allah, who gave me the ability to complete this I would like to express my deepest gratitude to my work. Supervisor

Prof. Saad Mohamed Hussein Ayoub

My thanks are also to

Dr. Itmad Awad Elhassan and special thanks to

Dr.Mubark El-Siddig Elamin

Special thanks to

Dr.Zeinab Eldosh and my friends

III

Abstract

The essential oils prepared by water distillation of fresh and dried leaves of Cymbopogonproximus and C.citratus family (Poaceae) were investigated by GC-MS analysis to determine the effect of drying on their chemical composition and antioxidant activity. The oil content based on weight of sample was different: 1% and 2.87% in fresh and dried leaves of C.proximus; 0.5% and 1.5% in fresh and dried leaves of C.citratus.Physico- chemical properties of the oils prepared from fresh and dried samples were not compatible with published data due to different geographical locations and seasonal variations.

The antioxidant activity of the oils prepared from fresh and dried leaves of C.proximus was below 50% (08±03 and 28±01%), while that of C.citratus has increased significantly from 43±0.03% for fresh leaves to 66±0.0% for the dried leaves. This was confirmed by GC-MS analysis of the four oil samples from the two plants.

In C.proximus oil the proportion of mono and sesquiterpenes was not effected by drying (2:1) with regard to pipertetone, 2-carene and D- limonene. The case was different in C.citratus oil where new compounds emerged and their content was high: (+)-3-(2-Hydroxy-2-methyl propyl)-2,2- Dimethylcycloprpanone-trans-1-Carboxylic acid (10.84%) and 2,2- Dimethyl-1-oxa-Spiro(2,4)heptanes (15.08%), with regard to β-Citral, α- Citral and β-myrcene.

ملخص البحث

فً هذا البحث حن اسخخالص الشيج هي ًباحً الوحزيب وحشيشت الليوىى عي طزيق الخقطيز البخاري وهي ثن اجزي الخحليل االولً لوعزفت هكىًاث الشيج باسخخذام حقٌيت كزوهاحىغزافيا الغاس الولحق بالوطياف الكخلً لوعزفت حاثيز الخجفيف فً كال الٌباحيي وححذيذ فعاليت الشيج هي ًاحيت

هضاداث االكسذة وًسبت الشيج فً الٌباحيي وحخزاوح قيوخها بيي )-1 %8.8(

Table of Contents

I االيت

Dedication II

Acknowledgments III

English Abstract IV

Arabic Abstract V

Table of contents VI

List of Tables X

List of Figures XII

Chapter one

1 Introduction and literature review 1

1.1 General introduction 1

1.2 Literature review 5

1.2.1 Essential oils 5

1.2.2 Methods of preparation of essential oils 5

1.2.2.1 Distillation 5

1.2.2.2 Hydro- distillation 5

1.2.2.3 Hydro- diffusion 5

1.2.2.4 Solvent extraction 6

1.2.2.5 Super critical carbon dioxide 6

1.2.3 Chemical constituents of essential oil 6

1.2.4 Uses of essential oils 8

1.2.5 Cymbopogon proximus plant 8

1.2.6 Taxonomy 9

1.2.7 Botanical characteristics 10

1.2.8 Geographical distribution 10

1.3 Chemistry of the C.proximus 10

1.3.1 Chemical composition of C.proximus 10

1.4 Medicinal uses of C.proximus 14

1.4.1 Antimicrobial activity 14

1.4.2 Antioxidant activity 15

1.5 Cymbopogon citratus plant 15

1.6 Taxonomy 16

1.7 Botanical characteristics 16

1.8 Chemistry of C.citratus 17

1.8.1 Chemical composition of C.citratus 17

1.9 Pharmacological 23

1.9.1 Anti-amebic effect 23

1.9.2 Antibacterial activity 23

1.9.3 Ant diarrheal activity 23

1.9.4 Ant filarial activity 23

1.9.5 Antifungal activity 23

1.9.6 Anti- inflammatory activity 24

1.9.7 Anti malarial activity 24

1.9.8 Antimutagenicity 24

VII

1.9.9 Antimyco bacterial activity 24

1.9.10 Antinociceptive effect 24

1.9.11 Antiprotozoan activity 25

1.9.12 Ascaricidal activity 25

1.9.13 Antioxidant capacity 25

1.9.14 Hypocholesterolemic effect 25

1.9.15 Hypoglycemic and Hypolipidemic effects 25

1.9.16 Larvicidal Activity 25

1.9.17 Neurobehavioral effect 25

1.10 Traditional uses of Cymbopogon citratus 26

Chapter two

2 Materials and methods 27

2.1 Materials 27

2.1.1 Plant materials 27

2.1.2 Solvents and chemicals 27

2.1.3 Glassware 27

2.1.4 Apparatus and instruments 28

2.2 Methods 28

2.2.1 Determination of essential oils content 28

2.3 Determination of physicochemical properties of C.proximus 28 and C.citratus essential oils

2.3.1 Specific gravity 28

2.3.2 Refractive index 29

VIII

2.3.3 Optical rotation 29

2.3.4 Determination of acid value 29

2.3.5 Determination of saponification value 30

2.3.6 Determination of ester value 30

2.3.7 Chromatographic method 30

2.3.7.1 GC-MS Analysis of essential oils 30

2.3.8 Determination of antioxidant activity of the prepared 31 essential oils

2.3.8.1 DPPH radical scavenging assay 31

Chapter three

3 Results and Discussion 32

3.1 Pysico-chemical properties of the prepared essential oils of 32 C. proximus and C.citratus

3.2 GC-MS Analysis of essential oils 33

Chapter four

4 Conclusion and Recommendations 49

4.1 Conclusion 49

4.2 Recommendations 50

References 51

List of Tables

1 Table (1) Showes GC-MS-profile of components of 10 C.proximus essential oil obtained by hydro-distillation in Egypt

2 Table (2) Showes GC-MS Profile Major Constituents of 12 C.proximus oil obtained by hydro distillation.

3 Table (3) Chemical composition of the essential oil of 13 Cymbopogon schoenanthus (L.) spring of Burkina Faso

4 `Table (4) Optical activity of the essential oil of 13 Cymbopogon schoenanthus (L.)Spreng of Burkina Faso.

5 Table (5) Physical properties of Cymbopogon essential oil 14

6 Table (6) Chemical composition of Cymbopogon citratus 18 essential oil identified by GC-MS analysis

7 Table (7) Chemical composition (%) of essential oils from 19 fresh leaves of Cymbopogon citratus Staph natives from Brazil and Cuba

8 Table (8) Chemical compounds of essential oil of dried C. 20 citratus obtained by steam distillation

9 Table (9) Chemical composition of Egyptian and Madinah 21 lemongrass volatile oils

10 Table (10) Antioxidant activity of Egyptian and Madinah 22 lemongrass volatile oils

11 Table (11) Physico-chemical properties of the prepared 32 essential oils of C proximus and C.citratus

12 Table (12) Chemical composition of essential oil prepared 34 from C.proximus fresh leaves

13 Table (13) Chemical composition of essential oil prepared 37 from C.proximus dry

14 Table (14) Chemical composition of essential oil prepared 41 from C.citratus fresh

15 Table (15) Chemical composition of essential oil 43 prepared from C.citratus dry

List of Figures

1 Figure (1) Some chemical structures of essential oils 7

2 Figure (2) Cymbopogon proximus plant 9

3 Figure (3) Cymbopogon citratus plant 17

4 Figure (4) Chemical structure of the major constituents 17 of lemongrass

5 Figure (5) Gas chromatogram of essential oil prepared 33 from fresh leaves of C.proximus

6 Figure (6) Gas chromatogram of essential oil prepared 37 from dried leaves of C.proximus

7 Figure (7) Gas chromatogram of essential oil prepared 40 from fresh leaves of C.citratus

8 Figure (8) Gas chromatogram of essential oil prepared 42 from dried leaves of C.citratus

XII

CHAPTER ONE Introduction and Literature Review

1. Introduction and Literature review

1.1General introduction

The term of medicinal plants include a various types of plants used in herbalism and some of these plants have a medicinal activities. These medicinal plants considered as a rich resources of ingredients which can be used in drug development and synthesis. Besides that these plants play a critical role in the development of human cultures around the whole world. Moreover, some plants considers as important source of nutrition and are recommended for their therapeutic values such as ginger, green tea, walnuts and some others plants. Other plants their derivatives consider as important source for active ingredients which are used in aspirin and toothpaste. (Rasool Hassan, 2012)

Medicinal plants frequently used as raw materials for extraction of active ingredients which used in the synthesis of different drugs. Like in case of laxative, blood thinners, antibiotics and anti-malaria medications, contain ingredients of taxol, vincristine, and morphine isolated from foxglove, periwinkle, and yew, and opium poppy, respectively. (Rasool Hassan, 2012).Medicinal plants contain physiologically active principles that over the years have been exploited in traditional medicine for the treatment of various ailments. These medicinal herbs constitute indispensable components of the traditional medicine practiced worldwide due to the low cost, easy access and ancestral experience.(Abdalla et al, 2013)

Cymbopogon is one of the most important essentialoil yielding genera of the Poacea.The genus comprises 140 species that are widely distributed in semi –temperate to tropical regions of Asia, Africa and America. Approximately 45 species have been reported to occur in India. The Cymbopogon species that produce volatile oils are called aromatic grasses. (Kumar et al, 2009)

Cymbopogon citratus, Stapf (Lemon grass) is a widely used herb in tropical countries, especially in Southeast Asia. The essential oil of the plant is used in aromatherapy. The compounds identified in Cymbopogon citratus are mainly terpenes, alcohols, ketones, aldehyde and esters. Some of the reported phytoconstituents are essential oils that contain Citral α, Citral β, Nerol, Geraniol, Citronellal, Terpinolene, Geranyl acetate, Myrecene and Terpinolmethylheptenone.(Richa shri et al, 2011)

The scientific name of lemongrass is Cymbopogon citratus. The genus Cymbopogon contains 40 species, mostly native to the old world tropics, and constituents and important proportion of savannah grass. (Abushama et al, 2013)

Although taxonomic classification is often complicated by hybridization and polyploidy, probably nine species are found in Sudan. The extracts of some of these species are widely used in folk medicine for the treatment of digestive ailments and as flavoring. (Abushama et al, 2013). Some most important species of this genus are: Cymbopogon citratus Dc staph, (West Indian lemon grass), Cymbopogon nardus (L), Cymbopogon martini roxb (palmarosa), Cymbopogon winterianus jowitt (Citronella), Cymbopogon flexuous Staph. (East Indian lemon grass). (Ganjewala, 2009)

Cymbopogon is one of the most essential oil yielding genera of the Cymbopogon word derives from the Greek words “kymbe” (boat) and “pogon”(beard), referring to the arrangement of the spike of the flower. The word Citratus derives from the old Latin, meaning lemon-scented leaves. The common name of Cymbopogon citratus in Mexico is “zacatelimón” (lemongrass) or “télimón”. It is a perennial tropical grass; is resistant to different temperatures and can grow in warm, semi-warm and temperate climates. It is from 60 to 120 centimeters high, its leaves are green, long and slats and have pleasant aroma and taste. This grass is native to India. Because of its pleasant flavor, in Mexico is consumed as infusion in water or milk just because the herbs intake is a custom in the analgesic and antipyretic properties, besides having antimicrobial effects reported that the essential oil of lemongrass inhibited the growth of Botrytis cinerea. Reported that the oil of lemongrass could suppress the growth of pedophiles and psychrophiles in fresh-cut apples. (Vazquez Briones et al, 2015)

C .proximus, family Poaceae, known as Halfa-bur, is an aromatic densely-tufted grass growing wildly in Upper Egypt. The herb ishighly reputed in folkloric medicine as an effective diuretic, renal or abdominal antispasmodic agent, and for relieving bronchial asthma as well. The herb exerts its unique pharmacological action through relaxation of the smooth muscle fibers without abolishing the propulsive movement of the tissue, thus, it is traditionally used inthe expulsion of renal and ureteric calculi. (Abou shoer et al, 2011)

The plant is highly reputed in folk medicine as an antispasmodic and urolithiasis (renal stone) removal, for gout, diuretic agent, flatulent, biotonic, analgesic for gastrointestinal

8 track (GIT), disorders and menstrual pains as well; the plant is used externally as anti rheumatic and cosmetic herb.(Abdelmonem et al, 2012)

Hypothesis and Research Question

Cymbopogon proximus and Cymbopogon citratus are claimed for their antioxidant properties in addition to other important medicinal properties such as antimicrobial and antispasmodic properties.

The research question deals with the effect of drying on their antioxidant activities and chemical composition of the two species.

Objective

General Objectives

To investigate physico-chemical properties of essential oils prepared from fresh and dried leaves of C. proximus and C.citratus and to determine their antioxidant activities and chemical composition.

Specific Objectives

To collect of fresh leaves of C. proximus and C.citratus and drying the fresh by shade procedure.

To dry the collected leaves by shade drying procedure.

To determine the oil yield of the fresh and dried leaves.

To determine physico-chemical properties of the prepared oils.

To assess the antioxidant activities of the prepared oils.

To determine the chemical composition of the prepared oils by GC-MS analysis.

To discuss the effect of drying on oils yield; their physicochemical properties; their chemical composition and their antioxidant activities.

1.2 Literature review

1.2.1 Essential oils

Essential oils are defined as volatile substances of a complex mixture of chemical components (terpenes, monoterpenes, terpenoids, alcohols, aldehyde, and ketones) which evaporate when contact with air and are biosynthesized by plants. They can be obtained from different parts of plants and are generally recognized as safe (GRAS). Attention is now given to natural antimicrobial substances of plant origin since they could be a rich source of bioactive compounds; and they might replace synthetic additives. The essential oils of basil, garlic, cinnamon, lemongrass, oregano and rosemary have been added as active chemical compounds in edible coatings. (Vazquez Briones et al, 2015)

1.2.2 Methods of preparation of essential oils

1.2.2.1 Distillation

Distillation is one of the separation processes. Distillation is defined as a process in which a liquid or vapor mixture of two or more substances is separated into its component fractions of desired purity, extraction processes can accommodate changes in flow rates and the solvent can be recovered and recycled for reuse. Amount of solvent and operating temperature can be varied. (Ibrahim, 2006)

1.2.2.2.1Hydro- distillation (HD)

HD has become the standard method of essential oil extraction from plant material. The process involves the complete immersion ofplant material in water, followed by boiling.(Tongnuanchan and Benjakul., 2014).

1.2.2.3 Hydro- diffusion

This method is used when the plant material has been dried and is not damage at boiling temperature. Hydro diffusion extraction is a type of steam distillation, which is only different in the inlet way of steam into the container of still. Hydro diffusion method is superior to steam distillation because of a shorter processing time and a higher oil yield with less steam used. (Tongnuanchan and Benjakul, 2014)

1.2.2.4 Solvent extraction

Different solvents including acetone, hexane, petroleum ether, methanol or ethanol can be used for extraction, for general practice the solvent is mixed with the plant material and then heated to extract the essential oil, followed filtration.(Tongnuanchan and Benjakul., 2014)

1.2.2.5 Super critical carbon dioxide

Carbon dioxide (CO2) is the most commonly used super critical fluids because of its modest critical conditions. Under high pressure, Co2 turns into liquid which can be used as avery inert and safe medium to extract the aromatic molecules from raw material. (Tongnuanchan and Benjakul., 2014)

1.2.3 Chemical constituents of essential oils

Several plants contain essential oils, however, parts of plants, which serve as the major source of essential oil can be different. Those include roots, peels, leaves, seeds, fruits, barks, and so on. Plant essential oils are usually the complex mixture of natural compounds, both polar and non-polar compounds. Dominant compounds in various essential oils are presented in general, the constituents in essential oils are terpenes, (monoterpenes and sesquterpenes), aromatic compounds (aldehyde, alcohol, phenol, methoxy derivative, and so on), and terpenoids (isoprenoids). Compounds and aroma of essential oils can be divided into 2 major groups: terpene hydrocarbons and oxygenated compound. (Tongnuanchan and Benjakul, 2014)

1.2.3.1Some chemical structures of essential oil are shown in Figure(1)

CH3

O O

O H3C CH3

Safrol Neral

CH2

CH3

CH2

CH3 H C CH 3 3 Carvone

Beta Myrcene

1.2.4 Uses of essential oils

Essential oils have been traditionally used for treatment of infections and diseases all over the world for centuries .Essential oils were used in ancient Rome, Greece and Egypt andthroughout the Middle and Far East as perfumes, food, flavors, deodorants and pharmaceuticals. Essential oils (EO), also known as volatile oils, are concentrated natural plant products which contain volatile aroma compounds. Essential oils are generally liquid, aromatic and possess pleasant odors and essence. The term “essential oil” is often used in cosmetics and perfume industries as synonymous with perfume oil, base or compound.(Kahsay and Unithan, 2016)

1.2.5Cymbopogon proximus plant

Cymbopogon proximus (Family Poaceae) is a traditional medicinal Sudanese plant commonly known as” Mahareb”, which is used in some parts of the folk medicine in Sudan for gout, renal colic, helmenthiasis, diuresis, inflammation of the prostate, and antipyretic. In the Egyptian folk medicine, it is famous as an effective diuretic and renal antispasmodic, decoction of the entire dried herb has been used for centuries by certain tribes in South Egypt as a diuretic, colic pain killer, aid for removal of small stones from the urinary tract, and antipyretic. The plant has been found to possess antispasmodic, hypotensive , antiemetic anticonvulsant ,hypoglycemic , antioxidant , antibacterial , fungicidal ,ovicidal and larvicidal properties . The study was undertaken to assess the effectiveness of C.proximus as a prophylactic agent against experimentally-induce nephrolithiasis in rats. (Warrag et al, 2014).

C. proximus Stapf is a weed known as Halfa bur that grows in the Egyptian desert. It is highly reputed in Egyptian folk medicine as an effective renal antispasmodic and diuretic agent .C.proximus is an ascending densely tufted perennial grass, common in the hills and rocky grounds of Elba and the sandy coast of the Red Sea on the southern boundaries of Egypt. The entire dried herb has been used for centuries by the Bisharin and Ababda tribes of the Aswan Province in the form of a decoction to produce diuresis, to relieve colicky pains, to help the removal of small stones from the urinary tracts, and as an antipyretic in fevers.(Selim, 2011).

1.2.6Taxonomy

Kingdom: Plantae

Order : Poales

Family : Poaceae

Genus : Cymbopogon

Species : proximus (C.shcoenanthus (L.) spring)

Fig (2) Cymbopogon proximus

1.2.7 Botanical characteristics

The Cymbopogon proximus is a herbal plant. Common name iscamel‟s hay and is known locally as (Maharaib). It is a perennial herb, erect, tufted 9 cm long, culms slender, glabrous and 3 – 4 nodes. Leaf simple, alternate, linear 5-7 cm long, 1cm wide, sheathed apex spiny entire, and‟ Inflorescence spikelets highly branched 5 cm log (Eltahir and Ereish, 2010).

1.2.8 Geographical distribution

The plant is widely distributed in Africa (northwest tropical, northeast tropical and east tropical), temperate Asia (western Asia and Arabia) and tropical Asia (Indian and Indo- China). In addition, Cymbopogon proximus is found in northern and Central Sudan (Clayton et al., 2005).

1.3 Chemistry of the C. proximus

The analysis of the chemical composition is carried out by Gas chromatography. It shows that the 16 made up ones identified account for 65.2% of the essential oil composition these compounds belong to the two classes „regulary met in essential oil: the mono ones and sesquiterpenes. However, proporotion of mono terpenes (53.2%) is higher that of sesquiterpenes (12%). Among the identified compounds two monoterpenes (the piperitone and gama- careene) remain the principal components in the essential oil. (Yentema. et al, 2007)

1.3.1 Chemical composition of C. proximus

Table (1) Showes GC-MS- profile of components of C.proximus essential oil obtained by hydro distillation in Egypt.

Peak No Compound Area (%)

1 Limonene 2.45

2 α- Pinene o.74

3 Piperitone 72.44

4 δ-3-carene 0.77

5 α-Terpinene 0.25

6 P-Cymene 0.68

7 1,8-Cineole 0.27

8 (Z)-β-Ocimene 0.48

9 (E)-β-Ocimene 0.25

10 δ-Terpinene 0.12

11 α-Terpinolene 0.07

12 Linalool 0.07

13 Linalyl acetate 0.35

14 β-Bourbobene 0.14

15 β-Elemene 0.82

16 Elemol 9.43

17 δ-Eudesmole 0.54

18 β-Eudesmol 1.26

19 α-Eudesmol 4.34

Oil yield 0.58

Table (2) Showes GC-MS Profile Major Constituents of C.proximus oil obtained by hydro distillation.

Peak No Compound Area %

1 Piperitone 72.44

2 Elemol 9.43

3 Eudesmol 4.34

4 Limonene 2.45

5 β-Eudesmol 1.26

The hydro distillation of dried C. proximus gave a brownish (yields 0.58%, w/w). The identified compounds, qualitative and quantitative analytical results by GC and GC/MS are shown in Table 1.

According to their elution order on a ZB-1 capillary column. The GC–MS analysis led to the identification of 19 different components, representing 95.47% of the total oil constituents (Table 1). A total of 19 constituents representing 95.47% of the oil were identified; piperitone (72.44%), elemol (9.43%), α-eudesmol (4.34%), limonene (2.45%) and β-eudesmol (1.26%) were the main components comprising 88.92% of the oil. A portion (4.53%) of the total composition was not identified. The oil yield from the whole plants of C. proximus prepared by the hydro- distillation method was 0.58% (v/w).GC/MS analysis revealed.

That the oil contained 19 components in the oil containing piperitone (72.44%).(Selim, 2011).

Table (3) Chemical composition of the essential oil of Cymbopogon schoenanthus (L.) spring of Burkina Faso

`Table (4) Optical activity of the essential oil of Cymbopogon schoenanthus (L.) Spreng of Burkina Faso.

The study concerns the characterization of the essential oil of Cymbopogon schoenanthus of Burkina Faso, extracted by drive with the water vapor in a distillation operation. The analysis of the chemical composition is carried out by Gas chromatography. It shows that the 16 made up ones identified account for 65.2% of the essential oil composition. These compounds belong to the two classes regularly met in essential oils: the mono ones and sesquiterpenes. However, proportion of monoterpenes (53. 2%) is higher than that of sesquiterpenes (12%). Among the identified compounds two monoterpenes (the piperitone and δ-2-carene) remain the principal components in the essential oil. Then the authors determine the density d = 0.9057 by double weighing, the optical activity α =

+28.175 by polarimetry and the refractive index n = 1.465 by an interferometric method which they describe. (Yentema et al, 2007)

The essential oils were analyzed for identification of their chemical composition using GC-MS technique. Piperitone (43.2, 45.8%), elemol (13.45, 14.43%), 4-carene (7.55, 9. 75%), β- eudesmol (5.41, 4.32%), limonene (2.45, 4.03%) and α- eudesmol (2.61, 4.66%), β-elemene (2.24, 1.30%) were the main components identified in the essential oil of C. proximus leaves and inflorescence, respectively. The essential oil content of C. citratus leaves, C.nervatus inflorescences, C. proximus leaves and C.proximus inflorescence were found to be (1.7±0.08), (2.0±0.09), (0.9±0.02) and (3±0.2%), respectively. Their physical constants are shown in Table (5). (Elhassan. et al, 2016)

Table (5) Physical properties of Cymbopogon essential oil

1.4 Medicinal uses of C.Proximus

Cymbopogon proximus is commonly known as Maharieb, which is used in some part of Sudan to treat intestinal spasm and kidney stones, the plant inflorescence decoction is used to treat kidney pains and arthritis. Also this plant is intensively used in the folk medicine in Sudan for gout, renal colic, and helmenthiasis, dieresis, inflammation of prostate and as antipyretic.(Khalafalla et al, 2015).

1.4.1 Antimicrobial activity

The antibacterial activity of the oil of C. proximus could, in part, be associated with major constituents such as α-terpinene, linalool,-δ-terpinene. These components have been reported. To display antibacterial effects. Terpenes were active against bacteria, as described previously by other authors, EO containing terpenoids are more active against Gram positive bacteria than against Gram negative bacteria In addition, and thecomponents in lower amounts may also contribute to the antibacterial activity of the oil, probably involving some type of synergism with other active compounds. The

NCCLS method (NCCLS, 2000) was used to determine the antibiotic-resistance and sensitivity pattern profile of the antibiotic- resistant bacteria.(Selim, 2011)

1.4.2Antioxidant activity

The total antioxidant activity of the C. proximus was expressed as the number of equivalents of ascorbic acid (ASE). The antioxidant capacity was estimated from the regression equation derived from concentration versus optical density of the sample and ascorbic acid. The extraction by methanol showed equivalents 48.66±3.1 μg dried C. proximus. The color of the reaction mixture changes from purple to yellow, and its absorbance at wavelength 517 nm decreases. Shows the DPPH radical scavenging activities of the essential oil and methanol extract of C.proximus, the IC50values were compared with the IC50 values of butylatedhydroxyanisole (BHA) and ascorbic acid. A lower IC50value indicates greater antioxidant activity. The IC50values of the essential oil and methanol extract were found to be 998.47±67.65 and 48.66±3.1, respectively. The scavenging effects of BHA and ascorbic acid were found to be 2.77 and 3.3 times greater than the methanol extract of C. proximus respectively. The methanol extracts of C. proximus showed a highly effective free radical scavenging in the DPPH assay. These extracts exhibited a remarkable antioxidant effect at low concentration. The essential oil of C proximus was only slightly active.(Selim , 2011).

1.5 Cymbopogon citratus plant

Cymbopogon citratus (lemongrass/oil grass) is a perennial grass that grows spontaneously around the world, mainly in the tropical and savannah regions.(Adesegun et al,2013)

Cymbopogon citratus is an herb worldwide known as lemongrass. Theprefix „lemon‟ owes to its typical lemon like odor, which is mainly due to the presence of citral, a cyclic monoterpene. Cymbopogon citratus a fast growing, perennial aromatic grass is native to South India and Srilanka, now widely cultivated in the tropical areas of America and Asia. Freshly cut and partially dried leaves are used medicinally and are the source of the essential oil. The plant is used extensively in ayurvedic medicine. (Richashri et al, 2011)

C.citratusis an economically important aromatic perennial plant of the Poaceae family that has been used to extract essential oils. It is grown around the world and has a century -long record of extensive therapeutic applications in traditional and ayurvedic medicine in a number of countries.(Ekpenyong et al, 2014)

Studies indicate that Cymbopogon citratus possesses various pharmacological activities such as anti-amoebic, antibacterial, antidiarrheal, antifilarial, antifungal and anti- inflammatory properties. Various other effects like anti-malarial, anit mutagenicity, Antimycobacterial, antioxidants, hypoglycemic and neurobehavioralhave also been studied. (Manvitha and Bidya, 2014)

Cymbopogon citratus (D C.)Stapf. (Lemongrass) is a plant in the Poaceae family that contains 1 to 2% essential oil on a dry basis. C. citratus is of West Indian origin and yields an essential oil with high content of citral (>70%). (Farhang et al, 2013)

Shows the classification of C. citratus. (Richa shri et al, 2011)

1.6 Taxonomy

Kingdoms : Plantae

Order : Poales

Family : Poaceae

Genus : Cymbopogon

Species : Citratus

1.7 Botanical characteristics

Lemongrass is an aromatic plant belonging to the Gramineae family. It is a tall, clumped perennial grass growing to a height of 1 m. The leaf-blade is linear, tapered at both ends and can grow to a length of 50 cmand width of 1.5 cm. The leaf-sheath is tubular in shape and acts as a pseudo stem. This plant produces flowers at matured stages of growth.(Tajidin et al, 2012)

Fig (3) Cymbopogon citratus plant

1.8 Chemistry of Cymbopogon citratus

1.8.1 Chemical composition of C.citratus

The chemical composition of the essential oil of Cymbopogon citratus varies according to the geographical origin, the compounds as hydrocarbon terpenes, alcohols, ketones, esters and mainly aldehyde have constantly been registered. The essential oil (0.2–0.5%, West Indian lemon grass oil) consists of, mainly, citral. Citral is a mixture of two stereo isomeric monoterpene aldehyde; the Trans isomer geranial (40–62%) dominates over the cis isomer neral (25–38%).Chemical structure of the major constituents of lemongrass essential oil [Figure 4]. (Richa shri et al, 2011)

Fig (4) Chemical structure of the major constituents of lemongrass essential oil

Table (6) Chemical composition of Cymbopogon citratus essential oil identified by GC-MS analysis

α- citral (39.16 %), Z- citral (30.95 %), limonene (5.83 %), Caryophyllene (3.44) and geranyle acetate (3.1%) identified as the main components in Cymbopogon citratus essential oil. (Table6)

Fresh leaves of Cymbopogon citratus at flowering stage were harvested, air-dried under the shade and stored at room temperature in darkness until distillation. (Farhang et al, 2013)

Compounds identified in C. citratus essential oils from Brazil and Cuba are presented in Table (7) GC/MS analysis allowed the identification of 13 and 12 main chemical components for Brazilian and Cuban oils, respectively. In both of them, the major components were the isomers geranial with 53.2 and 51.14% and neral with 36.37 and 35.21% for Brazilian and Cuban samples, respectively. (Teixeira pinto et al, 2015)

Table (7) Chemical composition (%) of essential oils from fresh leaves of Cymbopogon citratus (DC) Staph natives from Brazil and Cuba.

Table (8) Chemical compounds of essential oil of dried C. citratus obtained by steam distillation

The essential oil (EO) of lemongrass (Cymbopogon citratus) or lemon tea” leaves were studied. The EO was obtained by the steaming (0.75 ± 0.05%) and distillation (1.5 ± 0.07%), assisted by microwaves, Methods. The EO had a refractive index of 1.483 ± 0.001 (20 °C) and a density of 0.873 ± 0.005 g/mL (27°C). Color parameters of the oil corroborate the yellow hue observed by the naked eye. (Vazquez-Briones et al, 2015)

Table (9) Chemical composition of Egyptian and Madinah lemongrass volatile oils

Table (10) Antioxidant activity of Egyptian and Madinah lemongrass volatile oils

GCMS analysis of Egyptian Lemongrass (Cymbopogon citratus) essential Oil showed the identification of 49 compounds accounting for (92.6%) of the total amount Table 9 .The major compounds which identified were Geranial (20.9%), neral (16.2), geraniol (8.3%) and linalool (5.6%). (Mansour et al, 2015)

The volatile oil obtained from the leaves of Cymbopogon citratus cultivated in Madinamonawara, Saudi Arabia was pale yellow with pleasant and distinct odour. The percentage of constituents is shown in Table 9 Eighteen compounds could be identified in the oil accounting for about (95.0%) of it. The major compounds which identified by GC MS were geranial (37.8%), neral (33.6%), α -myrecene (8.4%) and geranial acetate (3.5%) In Madinah, neral, geranial and myrecene detected in higher concentrations in comparison to the Egyptian lemongrass, while other compounds found in lower concentrations e.g. linalool (0.7%) and carvone (0.4%) However, other constituents are found only in the essential oil Egyptian of Cymbopogon citratus e.g., terpin-4-ol (2.1%), cineole (0.6%) and carveol (0.5%).The antioxidant activity of Lemongrass (Cymbopogon citratus) essential oil cultivated in both Egypt and Madinamonawara tested by DPPH radical scavenging and β-Carotene-linoleate bleaching assays.

Recorded activities are presented in (Table 9), where lower IC50values indicate higher activity. Both essential oils of lemon grass under the study activity in agreements with However, Egyptian herb essential oil exhibited a higher scavenging ability for DPPH -1 -1 (IC501.0mg ml ) in comparison to Madinah Lemongrass volatile oil, (IC50 6.9mg ml ). Again, the inhibiting effect for linoleic acid oxidation and the subsequent bleaching of β- carotene was higher for Egyptian Lemon grass essential oil(55.1%) than Madinah one (36.3%), which assured the DPPH radical scavenging assay results.( Mansour et al, 2015)

Physico chemical properties of C.citratus oil such as specific gravity, optical rotation , refractive index, saponification value, acid value, ester value and density were determined

88 at room temperature respectively ,(0.9104± 0.0003, + 23, 1.4689± 0.004, 44.2 ± 1.1, 4.85, 39.15). (Kahsay and Unnithan, 2016)

1.9 Pharmacological Activity

Although a lot of pharmacological investigations have been carried out based on the ingredients present, but a lot more can still be explored, exploited and utilized. A summary of the findings of these studies is presented below. (Richa shri et al, 2011)

1.9.1 Anti-amebic Effect

The essential oil in broth culture was active on Entamoeba histolytica. (Richa shri et al, 2011)

1.9.2 Antibacterial Activity

The chromatographic fraction of the essential oil in agar plate was active on Bacillus subtilis, Escherichia coli, Staphylococusaureu. And Salmonellaparatyphi and Shigellaflexneri.These activities are shown in two of the three main components of the oil identified through chromatographic and mass spectrometric methods. While the α-citral (geranial) and β-citral (neral) components individually elicit an antibacterial action on gram-negative and gram-positive organisms, the third component, myrcene, did not show any observable antibacterial activity on its own. The extract was also active when the volatile oil extract was oxidized via the active oxygen method.(Richashri et al, 2011)

1.9.3 Antidiarrheal Activity

Cymbopogon citratus stalk decoction reduced the fecal output in a dose-dependent manner. (Richashri et al, 2011)

1.9.4Antifilarial Activity

Fresh leaves were active on Set aria digitations.(Richashri et al, 2011)

1.9.5Antifungal Activity

Lemon grass oil is active against such dermatophytes such as Trichophyton mentagrophytes, T. rubrum, Epidermophyton floccosum and Microsporum gypseum and is among the most active agents against human dermatophytes. Other studies reported that lemon grass oil is active against keratinophilic fungi, 32 ringworm fungi and food storage

83 fungi. Lemongrass oil is also effective as a herbicide and as an insecticide because of these naturally occurring antimicrobial effects.(Richashri et al, 2011)

1.9.6 Anti-inflammatory Activity

The hot water extract of the dried leaves administered intragastrically to rats was active when compared with carrageen in-induced pedal edem.(Richa shri et al, 2011)

1.9.7 Antimalarial Activity

The essential oils of Cymbopogon citratus were found to produce 86.6% suppression in the growth of Plasmodium berghei when compared with chloroquine (taking inhibition by chloroquine as 100%). (Richa shri et al, 2011)

1.9.8 Antimutagenicity

The ethanol extract of lemon grass extract exhibits an anti-mutagenic activity in various models and retards the growth of fibro sarcoma cells transplanted in mice in association with the prevention of lung metastasis. The plant extract is known to show inhibition on the formation of azoxymethane-induced DNA adducts and aberrant crypt foci in the rat colon. Inhibitory effects of the plant extract on the early phase of hepatocarcinogenesis after initiation with diethyl nitrosamine were seen in 344 male Fischer rats.(Richa shri et al, 2011)

1.9.9 Antimycobacterial Activity

The essential oil in agar plate was active on Mycobacterium smegaris.(Richa shri et al, 2011)

1.9.10 Antinociceptive Effect

The essential oil of Cymbopogon citratus possesses a significant anti nociceptive activity. Comparing the results obtained with three different experimental models of nociception (hot-plate, acetic acid-induced writhing's and formalin test), we can speculate that the essential oil acts both at the peripheral and at the central levels. (Richa shri et al, 2011)

1.9.11 Antiprotozoan Activity

A dose-dependent Antiprotozoan effect of the essential oil of Cymbopogon citratus could be observed on two strains of Crithida deanei.(Richa shri et al, 2011)

1-9-12 Ascaricidal Activity

The fresh leaf essential oil has an ascaricidal activity. ((Richa shri et al, 2011)

1.9.13 Antioxidant Capacity

The role of phenolic acid and flavonoids as natural anti-oxidants and free radical scavenger has been of interest due to their pharmacological behavior. Phenolic acids present in the plant showed the anti-oxidant profile.(Manvitha and Bidya, 2014)

1.9.14 Hypocholesterolemic Effect

The elevated cholesterol concentration was significantly lowered in the animals given the plant extract. This reduction was found to be dose dependent. This result shows that the extract possesses a hypocholesterolemic potential.(Richa shri et al, 2011)

1.9.15 Hypoglycemic and Hypolipidemic Effects

A fresh leaf aqueous extract of Cymbopogon citratus administered in normal rats lowered the fasting plasma glucose and total cholesterol, triglycerides, low-density lipoproteins and very low-density lipoprotein dose dependently while raising the plasma high-density lipoprotein level in the same dose-related fashion, but with no effect on the plasma triglyceride levels.(Richa shri et al, 2011).

1.9.16 Larvicidal Activity

The fresh leaf essential oil has a larvicidal activity.(Richa shri et al, 2011)

1.9.17 Neurobehavioral Effect

The essential oil was evaluated for sedative hypnotic activity through pentobarbital sleeping time, anxiolytic activity by elevated plus maze and light/dark box procedures and anticonvulsant activity through seizures induced by pentylenetetrazole and maximal electroshock. The essential oil was effective in increasing the sleeping time, the percentage of entries and time spent in the open arms of the elevated plus maze as well as

85 the time spent in the light compartment of the light/dark box. In addition, the essential oil delayed clonic seizures induced by pentylenetetrazole and blocked the tonic extensions induced by maximal electroshock, indicating the elevation of the seizure threshold and/or blockage of the seizure The fresh leaf essential oil has a larvicidal activity.(Richa shri et al, 2011)

1.10 Traditional uses of Cymbopogon citratus

Cymbopogon citratus is a great interest due to its commercially valuable essential oils and widely used in food technology as well as in traditional medicine. People now days are more aware on health issue due to the emergence of new diseases. Treatment using plant- based medicine appears to be an alternative approach due to the adverse effects associated with the use of synthetic drugs.

Lemongrass is a folk remedy for coughs, elephantiasis, flu, gingivitis, headache, leprosy, malaria, ophthalmic, pneumonia and vascular disorders. (Manvitha and Bidya, 2014).

CHAPTER TWO Materials and Methods

2. Materials and methods

2.1Materials

2.1.1Plant materials

Fresh leaves of Cymbopogon proximus and Cymbopogon citratus were collected in Almugran, Khartoum and shade dried. Voucher specimens were identified at the Medicinal and Aromatic Plants Research Institute (MAPRI) National Center for Research, Khartoum, Sudan.

2.1.2 Solvent and chemicals

Distilled water

Ethanol 96%

Hexane

Hydrochloric acid conc.

.Petrolieum ether (60-80%)

Phenolaphtaline

Potassium hydroxide

Sodium sulphate anhydrous

2.1.3 Glassware:

Beaker

Burette

Conical flask

Measuring cylinder

Round bottom flask

2.1.4 Apparatus and Instruments

Clevenger apparatus

GC/MS. Shimadzu (Japan)

Heating mantle.

Polarimeter

Refractometer

Sensitive balance

Spectro photometer

2.2 Methods

2.2.1 Determination of essential oils content

Plants materials (Fresh and air-dried) leaves of Cymbopogon proximus sand Cymbopogon citratus were hydro-distilled using Clevenger apparatus for four hours. The weight of each sample was 100g. The prepared essential oils were dried over anhydrous sodium sulphate and stored in vials at 40C for analysis .The yields of the oils were calculated based on the weight of each sample. Results were presented in table (11).

2.3Determination of physicochemical properties of C.proximus and C.citratus essential oils

The physicochemical properties of the oils were determined according to the British standards method.(British Standards , 1976)

2.3.1 Specific gravity

The specific gravity was determined at room temp (300 C) using pycnometer (density bottle), and the specific gravity was calculated according to the following equation:

Specific gravity m2-m/m1-m

Where: m = mass of the empty pycnometer

88 m1 = mass of pycnometer filled with water m2 = mass of pycnometer filled with essential oil

2.3.2Refractive index

Refractive index of plant essential oil was determine using Abee Refractometer at(300C) and the reading was corrected to be at (200C) according to the follow equation: ndt=ndt"+0.0004(t-t") ndt”= reading taking at working temperature t" ndt= refractive index at specific temperature.

2.3.3 Optical rotation

The optical rotation is the angle through which the plane of polarization is rotated when polarized light passes through a layer of a liquid. Substances are described as dextrorotatory or levorotatory according to whether the plane of polarization is rotated clockwise or counterclockwise, respectively, as determined by viewing towards the light source. Dextrorotation is designated (+) and laevorotation is designated (-).

2.3.4 Determination of Acid value

The essential oil (2g) a conical flask and dissolved in 50 ml of distilled alcohol by gentle warming, and then titrated against 0.1N KOH using phenolphthalein as indicator until Slight pink color is appeared, and then the acid value calculated by following equation:

Acid value= V×N×56/W

Where:

V: Volume of KOH added in ml.

N: Normality of KOH.

W: Weight of oil sample taken in grams.

2.3.5 Determination of saponification value:

Two g of oil was transferred into a round bottle flask and was dissolved in 5ml of distilled ethyl alcohol and 25 ml of 0.5N alcoholic KOH was added. The flask was fitted with reflux condenser and refluxed for about one hour until the reaction was complete and the liquid became clear. A blank experiment simultaneously was conducted in the same way without oil, and the two flasks were cooled. Then 2 drops of phenolphthalein were added and titrated against a standard solution of 0.5N hydrochloric acid until the pink color disappeared. The saponification value was calculated by using following equation:

Saponification value of oil= (B-A) ×0.5×56/W

B =Volume (ml) of N/2 HCl in blank experiment

A = Volume (ml) of N/2 HCl in first test.

56 = Molecular weight of KOH

W =Weight of oil sample taken in grams

0.5= Normality of HCl

2.3.6 Determination of e ester Value

The ester value was calculated by the difference betweensaponification and acid values.

Ester value = saponification value- acid value.

2.3.7 Chromatographic Method

2.3.7.1 GC-MSanalysis ofessential oils

The analysis of the essential oils was performed at Dr Alwia Imam‟s pharmaceutical Development Centre, UMST, Sudan using a GC-MS-QP2010 Ultra, Shimadzu, Japan. The essential oils constituents were identified by comparison of their retention time (RT) with the mass spectral library of the GC-MS data software system (NIST / Library) under the following conditions:

Column : Rtx-5MS… Length (30m)…Diameter (0.25mm)…Thickness (0.25 μm)

Total flow : 50.0ml/min Column flow : 1.55ml/min Purge flow : 3.0ml/min Column Oven Temp : 600C Carrier gas : Helium 2.3.8 Determination of antioxidant activity of the prepared essential oils

2.3.8.1 DPPH radical scavenging assay:

The antioxidant activity of the prepared essential oils of C.proximus and C.citratus were assessed on the basis of the radical scavenging effect of 2,2-Di phenyl-1-picrylhydrazyl stable free radical activity by modified method of shimada et.al (1992). The diluted working solutions of the test sample were prepared in dimethylesulfoxide (DMSO). Solution of DPPH was prepared in ethanol and 90µl of this solution was mixed with 10µl of sample solution. These solution mixtures were kept in the dark for 30 minutes and optical density was measured at 517nm using a spectrophotometer.

DMSO (10µL) with DPPH solution (90µl) was used as a blank

The optical density was recorded and percentage of inhibition was calculated using the formula given below.

Percent (%) inhibition of DPPH activity=

(100- )* 100

Where A= Absorbance of the sample solution

B = Absorbance of the blank solution.

CHAPTER THREE Results and Discussion

3. Results and Discussion

The yields of volatile oils of two plants (fresh and dried samples) prepared by hydro- distillation in Clevenger apparatus of the fresh and dried leaves were: 1% and 2.87% for C.proximus and 0.5% and 1.5% for C.citratus.

Physico-chemical properties of the prepared oils from the fresh and dried samples of the two plants are reported in the following table:

3.1Table (11): Physico-chemical properties of the prepared essential oils of C.proximus and C.citratus

No Physico-chemical- Cymbopogon proximus Cymbopogon citratus property Fresh leaves Dried leaves Fresh Dried leaves leaves

1 Colour Faint yellow Dark yellow Yellow Brown

2 Solubility Soluble in Soluble in Soluble in 85% ethanol 80% ethanol 85% ethanol

3 Odour Pleasant Pleasant

4 Specific gravity 0.916 0.914 0.870 0.862 (Density)

5 Refractive index 1.479 1.471 1.479 1.483

6 Optical rotation +25.4 +28.4 +50.00 +50.00

7 Acid value 3.64 2.24 17.90 24.36

8 Ester value 206.36 235.76 149.91 126.91

9 Saponification value 210 238 167.814 151.275

The different values reported about the physico-chemical properties of the prepared essential oils of C. proximus and C.citratus (Yentema et al, 2007, Elhassan et al, 2016 and Kahsay and Unithan, 2016) were not in agreement with our findings. This may due to different climatic conditions and geographical locations.

3.2 GC-MS Analysis of essential oils

The prepared essential oils from fresh and dried leaves of C. proximus and C.citratus were subjected to GC-MS analysis using a GC-MS-QP-2010 Ultra, Shimadzu, Japan. The oils constituents were identified by comparison of their retention time with the mass spectral library of the GC-MS data software system (NIST). Results are reported in Table (12) and Table (13) for fresh and dried samples of C.proximus leaves, and in Table (14) and Table (15) for fresh and dried samples of C.citratus leaves. The Gas chromatograms of the four samples are displayed in Figures 5, 6, 7 and 8 respectively.

Fig (5) Gas chromatogram of essential oil prepared from fresh leaves of C.proximus

Table (12) Chemical composition of essential oil prepared from C.proximus fresh leaves

No Compound Name R.Time Area% Molcular formula

1. Hexanol 3.206 0.02 C6H14O

2. Cyclo hexane, (2-methylpropyl) 4.090 0.01 C10H20

3. Alpha- pinene 4.172 0.02 C10H16

4. 1-Hexanol, 4-methyle. (s), 4.254 0.03 C7H16O

5. Camphene 4.423 0.01 C10H16

6. 1,3,5-Cyclo heptatriene 3,7,7tri methyl 4.756 0.64 C10H14

7. β-Myrecene 4.911 0.12 C10H16

8. 2,3-Dehydro-1,8-cineole 5.088 0.13 C10H16O

9. 2-Carene 5.256 16.33 C10H16

Alpha-phellandrene 5.317 0.14 C10H16 10.

11. ( 2-Carene 5.532 0.06 C10H16

12. P-cymen 5.676 0.27 C10H14

13. D-Limonene 5.756 4.81 C10H16

14. Trans-beta-Ocimene 5.853 0.15 C10H16

15. Beta-Ocimene 6.056 0. 13 C10H16

16. (+)-4-Carene 6.903 0.07 C10H16

17. L-Fenchone 6.950 0.19 C10H16

18. Fenchol, exo- 7.449 0.04 C10H18O

19. Beta linalool 7.581 1.74 C10H18O

20. Carveol 7.848 0.08 C10H16O`

21. Cis-beta-Terpineol 7.943 1.08 C10H18O

22. P-Mentha-1,5-dien-8-ol 8.509 1.06 C10H6O

23. Trans -2-caren-4-ol 8.711 0.34 C10H16O

24. P-Cymen-8-ol 8.800 0.03 C10H14O

25. Carvacrol 8.869 0.78 C10H14O

26. Alpha –Terpinol 9.004 2.20 C10H18O

27. Trans-piperitol 9.104 0.38 C10H18O

28. 2-Sec-butylecyclopentanone 9.208 0.05 C9H16O

29. Trans piperitol 9.344 0.68 C10H18O

30. Carvone 9.411 0.02 C10H14O

31. 6-Octen-1-ol,7-methyl-3-methylene 9.514 0.14 C10H18O

32. Trans-Carveol 9.581 0.04 C10H16O

33. 3-Thujen-2-one 9.681 0.02 C10H14O

34. Tricyclo(5.4.0.0(1,3)undecane 9.800 0.20 C11H18

35. (-)-Carvone 10.109 0.05 C10H14O

36. Geraniol 10.250 0.32 C10H18O

37. Pipertone 10.393 59.74 C10H16O

38. 1,3-cyclo hexa diene-1-methanol,4-(1- 10.526 0.15 C10H16O methylethyl)

39. 2-Caren-10-al 10.772 0.13 C10H14O

40. Thymol 11.022 0.04 C10H14O

41. Barosma camphor 11.251 0.03 C10H16O2

42. 2-oxabicyclo(2 .2.2) octan-1,3,3tri methyl 11.367 0.05 C10H16O2

43. Cyclo hexane, 1-ethenyl-1-methyl-2,4-bis 13.058 0.18 C15H24

44. Camphenol, 6- 13.529 0.04 C10H16O

45. Caryophyllene 13.667 0.40 C15H24

46. α- Bulnesene 14.179 0.14 C15H24

47. Humulene 14.316 0.03 C15H24

48. β-Chamigrene 14.797 0.07 C15H24

49. Dihydro-beta-agaro furan 14.985 0.42 C15H26O

50. Eremophilene 15.040 0.02 C15H24

51. Beta – bisabolene 15.199 0.02 C15H24

52. (R)-Cuparene 15.291 0.33 C15H22

53. Isocaryophillene 15.519 0.09 C15H24

54. 2-(1-cyclopent-1-enyl-1- 15.598 0.05 C13H20O methylethyl)cyclopentanone

55. Tricyclo(7.2.0.0(3,8)undec-4- 15.650 0.04 C15H24 ene,4,8,11,11-tetramethyl-

56. 2,2-Dimethyl-3 -vinyl- 15.715 0.25 C11H18 bicyclo(2.2.1)heptanes

57. α-Longipinene 15.761 0.8 C15H24

58. Cyclo hexane methanol, 4-ethenyl 16.017 2.61 C15H24

59. δ-eudesmol 17.525 0.33 C15H26O

60. Tetra cyclo(6.3.2.0(2,5).0(1,8)tridecan-9- 17.634 0.08 C15H24O ol,4,4-dimethyl-

61. β-Eudesmol 17.867 0.58 C15H26O

62. α-Eudesmol 17.904 0.40 C15H26O

63. Azulene,1,2,3,3a,4,5,6,7-octahydro-1,4-din 18.029 1.34 C15H24

100.00

Fig (6) Gas chromatogram of essential oil prepared from the dried leaves of C.proximus

Table (13) Chemical composition of essential oil prepared from C.proximus dry

No Compound Name R.Time Area% Molecular formula

1. 1- Hexanol 3.143 0.02 C6H14O

2. Trans-3-caren-2-ol 3.425 0.02 C10H16O

3. Alpha-pinene 4.077 0.03 C10H16

4. (S)-4-methyl-1-hexanol 4.156 0.02 C7H16O

5. Camphene 4.319 0.03 C10H16

6. 1,3,5-Cycloheptatriene,3,7,7 trimethyl- 4.643 0.48 C10H14

7. Trans-iso limonene 4.792 0.11 C10H16

8. Beta,-Myrcene 4.899 0.01 C10H16

9. 2,3-Dehydro-1,8-Cineole 4.961 0.08 C10H16O

10. (±)-2-Carene 5.128 20.22 C10H16

11. Alpha.-Phellandrene 5.183 0.11 C10H16

12. (+)-2-Carene 5.389 0.06 C10H16

13. P-Cymene 5.527 0.17 C10H14

14. D-Limonene 5.606 4.28 C10H16

15. Trans-.beta-Ocimene 5.700 0.14 C10H16

16. Beta., Ocimene 5.895 0.12 C10H16

17. (+)-4-Carene 6.703 0.07 C10H16

18. L-Fenchone 6.743 0.12 C10H16O

19. Beta-Linalool 6.858 0.01 C10H18O

20. Fenchol, exo- 7.216 0.02 C10H18O

21. 2-Cyclohexen-1-ol, 1-methyl-4-(1-methylet) 7.346 1.07 C10H18O

22. 1,4-Hexadiene,5-methyl-3-(1- 7.422 0.02 C10H16 methylethylidene)

23. Iso Carveol 7.598 0.06 C10H16O

24. 4-Thujanol 7.691 0.67 C10H18O

25. α-Phellandren-8-ol 8.229 0.51 C10H16O

26. Trans-2-Carene-4-ol 8.421 0.24 C10H16O

27. P-Cymen-8-ol 8.505 0.01 C10H14O

28. 3,9-Epoxy-p-mentha-1,8(10)-diene 8.570 0.45 C10H14O

29. Alpha-terpineol 8.700 1.36 C10H18O

30. Trans-piperitol 8.796 0.25 C10H18O

31. 1-Methyl propyl cyclopentanone 9.025 0.43 C9H16O

32. 6-octen-1-ol,7-methyl-3-methylene- 9.191 0.01 C10H18O.

33. Trans-carveol 9.246 0.02 C10H16O

34. Tricyclo(5.4.0.0(1,3)undecane 9.454 0.13 C11H18

35. (-)-Carvone 9.750 0.02 C10H14O

36. Geraniol 9.903 0.32 C10H18O

37. Pipertone 10.025 58.98 C10H16O

38. 1,3-Cyclohexadiene-1-methanol,4-(1- 10.150 0.05 C10H16O methylethyl)

39. 2-caren-10-al 10.376 0.06 C10H14O

40. Thymol 10.600 0.06 C10H14O

41. Barosma Camphor 10.833 0.08 C10H16O2

42. 2-Oxabicyclo(2.2.2)octan-6-one,1,3,3- 10.938 0.08 C10H16O2 trimethyl

43. Cyclohexene,1-ethenyl-1-methyl-2,4bis(1- 12.423 0.03 C15H24 methylethenyl)-{1-alpha,2beta,4,beta)}

44. 8-Isopropenyl-1,5dimethyl-cyclodeca- 12.549 0.28 C15H24 1,5diene

45. Spiro(2.4)heptane,1,2,4,5tetramethyl-6- 12.983 0.07 C12H20 methylene

46. Caryophyllene 13.118 0.72 C15H24

47. O-Mentha-1(7),8-diene-3-ol 13.594 0.14 C10H16O

48. α-Humulene 13.732 0.05 C15H24

49. β-Chamigrene 14.187 0.20 C15H24

50 α-Selinene 14.258 0.05 C15H24

51 Dihydro-beta-agarofuran 14.367 0.24 C15H26O

52 Eremophilene 14.423 0.07 C15H24

53 (R)-Cuparene 14.658 0.52 C15H22

54 δ-Eudesmol 14.875 0.08 C15H26O

55 2-(1-Cyclopent-1-enyl-1- 14.943 0.05 C13H20O methylethyl)cyclopentanone

56 Bicyclo(3.1.1)hept-3-ene, 4,6,6-trimethyl- 15.013 0.12 C12H18O 2vinyloxy-

57 2,2Dimethyl-3-vinyl-bicyclo(2.2.1)heptanes 15.056 0.07 C11H18

5 8 (+) α-Longipinene 15.104 0.15 C15H24

59 Cyclohexane methanol,4-ethenyl-alpha,alpha, 15.353 2.56 C15H26O 4-trimethyl-3-(1-methylethenyl)-,{1R- (1.alpha,3.alpha,4beta)}

60 Methyl(z)-5,11,14,17-eicosatetraenoate 16.015 0.49 C21H34O2

61 6,10-Dodecadien-1-yn-3-ol,3,7,11-trimethyl 16.113 0.03 C15H24O

62 1H-3a,7-methanoazulene,2,3,6,7,8,8a-alpha- 16.601 0.04 C15H24 hexan

63 δ-eudesmol 16.778 0.39 C15H26O

64 Tetra cyclo(6.3.2.0(2,5).0(1,8)tridecan-9- 16.877 0.10 C15H24O ol,4,4-dimethyl-

65 β-Eudesmol 17.108 0.59 C15H26O

66 α-Eudesmol 17.149 0.42 C15H26O

67 1. Beta, 4.beta.H, 10beta, H-Guaia-, 11-diene. 17.268 1.47 C15H24

68 α-Terpineol acetate 21,476 0.02 C12H20O2

100.00

Fig (7) Gas chromatograme of essential oil prepared from fresh leaves of C.citratus

Table (14) chemical composition of essential oil prepared from C.citratus fresh

Peak No Compound Name R.Time Area% Molcular formula

1 Sulcatone 4.960 0.8 C8H14O

2 Beta-Myrcene 5.022 8.58 C10H16

3 Trans-beta-ocimene 5.858 0.13 C10H16

4 Beta-ocimene 6.063 0.10 C10H16

5 1,6-Octadiene 2,5dimethyl- 6.189 0.16 C10H18 ,(E)

6 5-Tetradecen-3-yne,(Z)- 6.953 0.09 C14H24

7 Cinerone 7.019 0.04 C10H14O

8 Linalool 7.076 0.88 C10H18O

9 Verbenol 7.272 0.10 C10H16O

10 8-Decen-2-one,9-methyl-5- 7.903 0.08 C12H20O methylene-

11 2-Octen,2-methyl-6- 7.989 1.56 C10H18 methylen-

12 Isogeraniol 8.158 0.31 C10H18O

13 3-Cyclohexene-1- 8.384 2.44 C10H16O carboxaldehyde,2,4,6- trimethyl

14 3-Cyclohexene-1- 8.752 3.84 C10H16O carboxaldehyde,1,3,4- trimethyl

15 3-decyn-2-ol 9.073 0.36 C10H18O

16 Di-menthol 9.670 0.69 C10H20O

17 Lavandulol 9.708 1.03 C10H18O

18 Oxiranecarboxaldehyde,3- 9.814 0.16 C10H16O2 methyl-3-(4-methyl-3-

pentenyl)

19 β-Citral 9.989 31.26 C10H16O

20 Geraniol 10.218 6.17 C10H18O

21 Citral 10.578 40.72 C10H16O

22 Geranyl acetate 12.756 0.55 C12H20O2

23 Caryophyllene 13.662 0.13 C15H24

24 α-Bergamoten 13.863 0.14 C15H24

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Fig (8) Gas chromatogram of essential oil prepared from the dried leaves of C.citratus

Table (15) chemical composition of essential oil prepared from C.citratus dry

Peak Compound Name R.Time Area% Molcular No formula

1 Beta-myrcene 5.255 0.80 C10H16

2 Furan 5.389 0.26 C9H18O

3 2-Propanol,1-chloro-3-ethoxy 5.891 0.06 C5H11CLO2

4 2-octen,2-methyl-6-methylene 6.234 0.17 C10H18

5 2-Cyclohexene-1-one, 3-methyl- 6.345 0.14 C7H10O

6 6-methyl-2-(2-oxiranyl)-5- 6.567 0.28 C10H18O2 hepten-2-ol

7 Trans-Linalool oxide 6.814 0.26 C10H18O2

8 Linalool 6.942 0.35 C10H18O

9 (E)-2-(2-Butoxyethoxy)ethyl-2- 7.475 0.63 C13H24 O4 methylbut-2-enoate

10 3,5-Dimethyl-2-cyclohexen-1- 7.680 5.10 C8H12O one

11 Citronellol 8.907 0.32 C10H20O

12 2-Octen-1-ol,3,7-dimethyl 8.946 0.69 C10H20O

13 Methyl-3-methyl-3-hexanote 9.026 0.56 C8H14O2

14 Beta Citral 9.158 19.42 C10H16O

15 Geraniol 9.314 1.60 C10H18O

16 Citral 9.586 25.02 C10H16O

17 (+)-3-(2-Hydroxy-2- 9.765 10.84 C10H18O methylpropyl)-2,2- dimethylcyclopropane-trans-1- carboxylic acid

18 2-2-Dimethyl-1-oxa-spiro(2.4) 9.850 15.08 C8H14O heptanes

19 Ethyl-2(5-methyl-5- 10.146 0.62 C13H22O4 vinyltetrahydrofuran-2- yl)propan-2-yl carbonate

20 D-Nerolidol 10.223 0.33 C15H26O

21 Neric acid 10.296 1.12 C10H16O2

22 13-Heptadecyn-1-ol 10.595 0.55 C17H32O

23 Geranic acid 10.753 5.37 C10H16O2

24 2,6-Octadien-1-ol,3,7 dimethyl 11.150 0.36 C12H20O2 acetate

25 Oxirane(7-octenyl) 11.230 0.83 C10H18O

26 6-Methyl-2-(2-oxiranyl)-2- 13.109 0.86 C10H20O2 heptanol

27 Cyclooctanone 13.342 1.91 C8H14O

28 Linalool oxide 13.501 1.27 C10H18O2

29 4,4,6-Trimethyl-cyclohex-2-en-1- 13.849 3.31 C9H16O ol

30 11,11.Dimethyl- 14.095 0.51 C14H22 spiro(2,9)dodeca.3,7-dien

31 3,7-Nonadien-2-ol,4,8 dimethyl 20.054 0.88 C11H20O

32 Bicyclo(3.1.1)heptan-3one-2- 20.133 0.52 C13H20O (but-3-enyl-6-6dimethyl

100.00

Cymbopogon proximus is widely distributed in Africa and Asia in addition to northern and Central Sudan. Analysis of its essential oil was carried out mainly by Gas chromatography. The GC-MS profile of the oil components was discussed in the literature (Yentema et al, 2007; Selim, 2011; Elhassan et al, 2016). The uses of the plant decoction in Sudanese folk medicine for treatment of intestinal spasms and kidney stones were reported by (Khalafalla et al, 2015) in addition to treatment of gout, renal colic and prostate inflammations. In order to spot more light on the chemical composition of the plant essential oil prepared by water distillation and its uses in treatment of the said ailments, the present work attempted to investigate the effect of drying on the chemical composition of the oil and its activity. Most of the compounds detected by GC-MS analysis belong to mono and sesquiterpene hydrocarbons and their oxygenated derivatives. Sixty three compounds were detected in fresh leaf sample with the principal components: Pipertone (59.74%), 2-Carene (16.33%) and D-Limonene (4.81%). In the dried leaf sample Sixty eight compounds were identified and the three monoterpenes remained the principal components of the essential oil (58.98%; 20.22% and 4.28%) respectively. The proportion of monoterpenes was higher compared to the sesquiterpenes, almost 2:1 but was not compatible with published data in the literature (Yentemaet al, 2007 and Elhassan et al, 2016). The antioxidant activity of the dried leaf oil sample was significantly increased, compared to the fresh leaf oil sample (from 08± 0.03 to 28± 01%) bearing in mind that the two oil samples did not exceed the barrier of 50% to antioxidant activity.

Cymbopogon citratus grows spontaneously around the world, mainly in the tropical and Savannah regions and used traditional in treatment due to its various pharmacological activities. The chemical composition of its essential oil varies to the geographical location and seasonal variations. The major components are geranial and neral in addition to α- myrcene and geranyle acetate (Vazquez-Briones et al, 2015; Mansour et al, 2015). GC- MS analysis of the fresh leaf oil sample revealed the presence of 24 compounds of which β-Citral was 31.26%; Citral (40.72%) and β-myrcene (8.58%). The dried leaf oil sample gave 32 compounds of which β-Citral (19.42%); Citral (25.02%) and 5.37% of the essential oil was geranic acid. The antioxidant activities of the fresh and dried leaf oil samples were 43± 0.03% and 66± 0.0%. It is noteworthy that the drying effect on the chemical composition of the oil was significant. The drop in content of geranial and neral in the dried leaf sample in addition to the formed geranic acid has increased the

antioxidant potential from 43to 66%. Two new compounds: (+)-3-(2-Hydroxy-2- methylpropyl)-2, 2-dimethylcyclopropanone-trans-1-carboxylic acid (10.84) and 2, 2- Dimethyl-1-oxa-spiro (2.4) heptanes (15.08%) were formed upon drying of the fresh leaves. Based on these findings, it is possible that the increased antioxidant activity of the dried leaf sample oil is relevant to these major changes in the chemical composition of the oil.

Chemical structures of the components of the essential oil of C.proximus dry and fresh

A- Mono terpen hydrocarbon

CH3

CH3 camphene 2-carene CH3

Alpha-Pinene

CH2

H3C CH3 Beta- Myrcene Beta-Ocimene

B- Oxygenated monoterpen hydrocarbon OH CH3 CH3

H3C CH3 OH

Thymol Pipertone Alpha-Terpinol

Geraniol Carveol

C- Sesquiterpen Hydrocarbon

CH3 CH2 H3C CH3

CH3 CH2 H H

H3C CH3

Eremophelin Caryophllene Chamigrene

Chemical structures of the components of the essential oil of C.citratus fresh and dry

CH2

H3C CH3 Beta- Myrcene Trans-Beta-Ocimene Beta-Ocimene

B- Mono terpen oxygenated

CHO

2-Cyclopenten-1-One,2-(butenyl)-3-methyl-,(Z) Beta-Citral

OH OH

Geraniol Verbenol Linalool

C- Sesquiterpen Hydrocarbon

CH3

H3C

CH3 Apha-Bergamoten

CHAPTER FOUR Conclusion and Recommendations References

4. Conclusion and recommendations

4.1 Conclusions

1. The essential oils were prepared from fresh and dried samples by water distillation using Clevenger apparatus. The yields were 1%and2.87% for C.proximus and 0.5% and1.5% for C.citratus fresh and dried leaves, respectively.

2. Results of physico-chemical properties of samples were not compatible with the current literature due to different climatic conditions and geographical locations.

3. The antioxidant activity of the oil samples prepared from fresh and dried leaves of C.proximus did not exceed the barrier of 50% (08±0.03 and 28± 01%) respectively.

4. The antioxidant activity of the oil samples prepared from fresh and dried leaves of C.citratus were (43± 0.03% and 66± 0.0%) respectively.

5. The prepared four samples of the two plants were analyzed by GC-MS technique and their profiles were assessed.

6. The monoterpenes/sesquiterpen proporotion was almost 2:1 in essential oils of C.proximus and was not compatible with the literature.

7. The chemical composition of C.citratus fresh and dried leaves samples was different (24 compounds and 32 compounds) respectively.

8. The drop in content of some major components and formation of new compounds led to changes in the chemical composition hence increased antioxidant activity of the dried leaf essential oil.

4.2 Recommendations

New starting points for future studies have emerged and based on findings of the present research. The followings are recommended:

1. Search and research of differences in physical and chemical properties of samples prepared from fresh and dried leaves of C.proximus and C.citratus.

2. Investigation of effect of drying on oils prepared from fresh and dried samples of C.proximus and C.citratus on other activities such as antimicrobial and other possible activities.

3. Study of the effect of methods of preparation of essential oils of fresh and dried samples of C.proximus and C.citratus and investigation of their chemical composition by other chromatographic methods.

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