University of Gezira
Analysis of the volatile oil of Vetiveria nigritana root by gas chromatography /mass spectrometry
By:
Sara Khogli Ahmed Khogli
Jan. 2016
Analysis of the Volatile oil of Vetiveria Nigritana Roots by Gas Chromatography /Mass Spectrometry
Sara Khogli Ahmed Khogli
B. Sc. in Chemistry & Mathematics, University of Khartoum (2005) Postgraduate Diploma in Chemistry, University of Khartoum (2009) Submitted to the University of Gezira in Partial Fulfillment of the Requirements for the Award of the Degree of the Master of Science in Chemistry Department of Applied Chemistry and Chemical Technology Faculty of Engineering and Technology
Jan. 2016
Analysis of the Volatile oil of Vetiveria Nigritana Root by Gas Chromatography /Mass Spectrometry
Sara Khogli Ahmed Khogli
Supervision Committee:
Name Signature Position Dr. Mubarak El siddig Elamin Main Supervisor ……… Dr. Mustafa Ohag Mohamed Co-supervisor ………
Date: Jan. 2016
Analysis of the Volatile oil of Vetiveria nigritana Root by Gas Chromatography /Mass Spectrometry
Sara Khogli Ahmed Khogli
Examination Committee:
Name Position Signature Dr. Mubarak El siddig Elamin Chair Person ...... Dr.Sakina Mohamed Ahmad Yagi External Examiner ...... Dr. Mohamed Osman Babiker Enternal Examiner ......
Date of Examination: 19/1/2016
DEDICATION
I would like to dedicate this work to: My small family: beloved husband &dear son. My big family: great Dad & Mum, sweet brothers & sisters. To all my friends & anyone who contributed to this work.
Acknowledgement
I am deeply grateful to my supervisor Dr: Mubarak El siddig for his valuable information and advice throughout The duration of my research. I send my thanks to all my teachers, friends and colleagues who helped me.
Analysis of the volatile oil of Vetiveria nigritana root by Gas chromatography
/mass spectrometry
Sara Khogli Ahmed Khogli
Abstract Vetiveria nigritana, locally known as ′ Mistica′,′ Um sikkin′ and ′ Gash Almoya′ is perennial grass species of family Poaceace. It is a very thick and tall, deeply rooted within the ground type of grass, native to Northrn, Eastern and tropical parts of Africa, Nigeria and Senegal. In Sudan the dried roots are used as a "water purifier," a disinfectant and an antiseptic and also as water flavor. The plant contains essential oil that can be distilled and used for traditional medical applications. The objective of this study was to identify the chemical composition of the essential oil of Vetiveria nigritana root by means of GC-MS method of analysis. For this purpose, Vetiveria nigritana sample (roots) was obtained from Omdurman market (origin Nyala western Sudan). The essential oil was obtained by the water distillation method (yield 0.2% ).Analysis of the oil by GC-MS Technique revealed the presence of more than 34 compounds, of which 24 compounds were identified. The major compounds were Cedr-8-en-15-ol (10.84%) followed by 4, 6, 6- trimethyl-2-(3-methylbuta-1, 3-dienyl) 3oxatricyclo [5.1.0.0(2, 4)] octane (10.50 %) which was overlapping with caryophyllene.The sesquiterpenes were the dominant compounds of V.nigritana essential oil. It is recommended that the grass could be grown on a commercial scale for production of oil for use in the fragrance, flavor and medical products.
تحليل الزيت الطيار لجزور Vetiveria nigritana باستخدام جهاز كروماتوغرافيا الغاز\مطياف عشبة الكتلة.
سارة خوجلي احمد خوجلي مستخلص البحث
ينتمي نبات Vetiveria nigritana الذي يعرف محليا باسماء )مستيكا ( و)ام سكين( و)قش المويه( للعائله النجيليه Poaceae . وهو عشبة دائمة كثيفه , ذات جزور طويله . تعتبر مناطق شمال وشرق افريقيا والمناطق المداريه ونيجيريا والسينيغال هي اصل النبات. تستعمل جزور النبات الجافه في السودان في تنقية المياه وكمطهر لها وايضا للنكهه . يحتوي النبات علي الزيت الطيار الذي يمكن استعماله في االغراض الطبيه التقليديه . لقد كان الهدف من الدراسه هو اجراء تحليل الزيت الطيار المستخلص بطريقة التقطير بالماء باستخدام طريقة كروماتوغرافيا الغاز مطايف الكتلة ولهذا الغرض تم الحصول علي عينه جزور النبات من سوق ام درمان وذكر ان اصل العينه من منطقة نياال بغرب السودان .تم استخالص الزيت الطيار من الجزور بطريقة االستخالص بالماء حيث بلغت نسبة الزيت بالجزور ٪٠,٢. اوضح تحليل الزيت بطريقة كروماتوغرافيا الغاز مطايف الكتلة احتواء الزيت علي اكثر من ٣٤ مكونا تم التعرف علي ٢٤ مكونا منها , وقد كان المكون الرئيسي بالزيت هو سيد- ين-١٥-ول )١٠.84٪(,يليه ٦,٦,٤- ترايميثيل -٢ –)٣- ميثيل بيوتا-٣,١-داينيل (٣-أوكسا ترايسيكلو [٥.١.٠.٠)٢,٤(] )١٠.50٪( الذي خرج متزامنا مع مادة كاريوفيلين من عمود جهاز كرومتوغرافيا الغاز. كانت السيسكيتيربينات هي المكونات الغالبه بالزيت الطيار. نسبه الن الزيت الطيار لعشبة V.nigritana يسوق عالميا توصي الدراسه بان تتم زراعة العشبة بكميات تجاريه الستخالص الزيت من الجزور واستعماله في مجال العطور والنكهات والعالج . Contents Page Dedication…………………………………………………….….………...... ….i Acknowledgment ...... ii Content………………………………………………………………...... iii List of tables … ………………………….……….………...... … v List of figures…………………………………………………………………….v List of Appendix...... vi Abstract (English) ………………………………………………….………..…viii Abstract (Arabic) ………………………………………………………………...ix Chapter one 1. Introduction Introduction ……………………………………………………………….….….1 1.2. Objective ……………………………………………………….……….…..3 Chapter Tow 2. Literature Review 2.1. Classification ………………………………………………….………….…4 2.2. Description…………… ………………………………………………….…4 2.3. Habitat.. ………………………………………………………………….….5 2.4. Benefits of V.nigritana….…...... 5
2.4.1. Soil erosion and runoff prevention………...………………...………….…5 2.4.2. Landscaping……………………...... 6
2.4.3. Pest control…………... ……………………… ……………..……....…...7
2.4.4. Livestock prot…………………………………………………..….……....7 2.4.5. Traditional uses…………………………………………………....….…....8 2.4.6. Uses as water purifier……………………………………………..….…....9 2.5. Medicinal uses………………...... 10 2.6. Essential oils……...... 10 2.7. Other species of vetiv…………………………………….…………...... 12 2.8. Vetiveria nigritana VS Vetiveria zizanioides ………….………………..13 2.9. Composition……………………………………………………………..14 2.9.1. Vetiveria nigritana oil………………………….………..…………….14
Chapter Three 3.Material and Methods 3.1. Materials …………………………………………………………...…....16 3.1.1. Plant Material ………………………………………………………....16 3.2. Methods ……………………………………………………………...... 16 3.2.1. Extraction of the essential oil ……………………………….…….…..16 3.3. GC- MS analysis of the oil …………………………………………..….16 Chapter Four 4. Results and Discussion 4.1 Results ...... …...18 4.2. Discussion ...... 19 Conclusion and Recommendation...... 24 References ...... 25
List of Tables
Table (1): Oven temp. Program…………………………………………….…..16
Table (2): GC-MS analysis of Vetiver oil prepared from Vetiveria nigritana
………………………………………………………………………….....…….18 List of Figures
Figure (1). Landscaping with V. nigritana…………………….…..……….….…6
Figure (2) Vetiveria Nigritana root………………………………..……..….…....8
Figure (3) Uses as water purifie……………………………………….....….…....9
Figure (4) Vetiveria zizanioides roots………………………………….....……..12
Figure (5) V. nigritana and V.zizanioides roots……………………….....13
Figure (6) Vetiver oil ………………………………………………………...... 15
Figure (7) QP 2010 Plus EI GS/MS……………………………………...... ….17
Figure (8) Cedr-8-en-15-ol………………………………………………….…...20
Figure (9) structure of caryophyllene ……………………………………...…....21
Figure (10) 4, 6, 6-trimethyl-2-(3-methylbuta-1,3-dienyl)
3oxatricyclo [5.1.0.0(2,4)] octan…………….………………...………….…..…21
Figure (11) Hexadecanoic acid……………………….…………...... 22
Figure (12) 1,3-Dimethylbenzen……………………………………….….…...22
List of Appendixes
Appendix (1): Ethyl benzene ………………………………………….…...27
Appendix (2): m-Xylene………………………………………………….…27
Appendix (3): m-Xylen ……...………………………………………….…..28
Appendix (4): Thymol ………………………………………………………28
Appendix (5) : Biphenylene, 1,2,3,6,7,8,8a,8b-octahydro-4,5-dimethyl …..29
Appendix (6) : Cedr-8-ene ………………………….…..………………...... 29
Appendix (7) : Humulen-(VI) ……………………….…………………...... 30
Appendix(8) : Phenol, 2,4-bis(1,1-dimethylethyl) …….……………...... …..30
Appendix (9) : delta.-Cadinene………………………….…………………...31
Appendix (10) : Caryophyllene oxide …………………….…………….…...31
Appendix (11): Valencene ……………………………….…………...... 32
Appendix (12): 2-Butenal, 2-methyl-4-(2, 6, 6-trimethyl-1
-cyclohexen-1-yl)-………………………………………………..…32
Appendix (13): Humulene oxide………………………………….…..…...…33
Appendix (14): Unidentified………………………………………….…..….33
Appendix (15): Epicedrol……………………………………….…….…...…34
Appendix (16): Unidentified…………………………………….……….…..34
Appendix (17): Torreyol……………………………………….….....…....….35
Appendix (18): trans-Caryophyllene………………………….….…...…...…35 Appendix (19): Unidentified…….………………...…………..…...…....…...36
Appendix (20): Globulol……………….………………………..………...... 36
Appendix (21): caryophyllene………….…………………………..……..….37
Appendix (21): 4, 6, 6-trimethyl-2-(3-methyl-buta-1, 3 dienyl)-3-oxa
Tricycle [5.1.0.0 2, 4] octan ……………………………………….……..….37
Appendix (22): Unidentified……………………………………….………....38
Appendix (23): Unidentified……………………………………..…….……..38
Appendix (24): Unidentified…………………………………….…....….…...39
Appendix (25): gamma.1-cadinene aldehyde……………………………..….39
Appendix (26): Cedr-8-en-13-ol……………………………………...... …40
Appendix (27): 2-Isopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,7
Octahydronaphthalene………………………………………………...………40
Appendix (28): Cedr-8-en-15-ol……………………………….…..………….41
Appendix (29): Unidentified………………………………….….….….....…..41
Appendix (30): Unidentified…………………………………………………..42
Appendix (31) : Unidentified……………………………………..…..…...... 42
Appendix (32) Unidentified…………………………………….……….....….43
Appendix (33): n-Hexadecanoic acid………………………..……..……..…..43
Appendix (34): Oleic Acid…………………………………..……...... ……44
1. Introduction
Aromatic is defined as having a distinctive smell. "Aromatic" plants are given this title due a distinct fragrance or odor. Chemically, the organic compounds that molecules to be aromatic form flat rings which bond easily to the receptors in the nose .One of humans' most alert senses is the sense of smell .Aromatic plants have been used for thousands of years for many different purposes ranging from medicinal to religious; from protection against spirits to culinary delights. Aromatic Plants play a valuable and important role in economic, social, cultural and ecological aspects of local communities over the world . In one form or another, they benefit virtually everyone on Earth through toiletry, bodily care, incense, flavor and ritual healing. Aromatic plants grow in almost all terrestrial and some aquatic ecosystems around the world. However increasing demand on .plants and their habitats are threatening many species harvested from the wild. Cultivation is thus a feasible diversification enterprise for many small-scale farmers as demand is high, trade opportunities are increasing and the income generating potential is good. They comprise a wide range of species which have different sources, characteristics and uses. Since time immemorial these products have made a significant contribution to human health and well-being as well as contributing to farm household income generation through trade. ‘Traditional medicine’ is defined by the World Health Organization (WHO) as the sum total of the knowledge, skills, and practices based on the theories, beliefs, and experiences indigenous to different cultures, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness (Marshall, 2011). Essential oils are the highly concentrated, volatile, aromatic essences, mainly of plants or other sources, which volatilize at room temperature .They can be more or less fluid, sometimes resinous and often have a coloring which ranges from yellow to emerald green and from blue to dark brownish red. Essential oils may perform more than one function in living plants. For instance botanists believe that plants produce essential oils to attract insects for pollination or act as insects repellent. In some cases they saw to be part of plant’s immune system. In other cases, they may simply be by products of metabolism. Essential oils may be present in many different parts of the plants (wood, bark, leaves, flowers, fruits, roots, stigma, seeds ,etc.) at concentrations ranging from one or several percent to thousandths of percent .The oil is often contained in specialized secretary structures ,which include secretary cells, ducts, cavities, glandular trachoma’s , etc. The yield of essential oils from seeds is often high-in the several tens of percentage-but for the majority of other parts of the plant, the main range is 0.1-1%. Essential oils are generally fragrant volatile material consisting of complex mixture of mono- and sesquiterpene hydrocarbons, and oxygenated materials derived from them. Other common constituents include phenyl propanoids from shikimic acid pathway, and their biotransformation products, and other compounds from the metabolism of fatty acids and amino acids. In addition to these major groups of compounds, a large number of other types of chemical components are also found, including nitrogen and sulphur compounds. Within the flora of Sudan reputed to possess distinct fragrance in one or more of its parts a total number of 377 species belonging to 251 genera and 86 families have been documented.
(Elghazali et alt, 2004).
Vetiver is a common name of the grass of the genus Vetiveria (poaceae), which consists of 11 species widely distributed in tropical regions of Asia, Africa, Australia and Pacific Islands. Vetiveria nigritana is a perennial herb, with aromatic roots, and is considered the main African species found in most sub-saharan areas from Senegal to Mozambique.The roots have been used for centuries for their fragrance and are woven into fragrant-smelling mats and fans (Khalil and Ayoub, 2011). 1.2. Objective
The objective of this study is to extract and identify the chemical composition of the essential oil of Vetiveria nigritana root by means of GC-MS method of analysis.
2.Literature review 2.1. Classification Family: Poaceae Genus: Vetiveria Species: V. Nigritana (Linn.) Synonym: Chrysopogon nigritanus Common names: Vetiver grass Vernacular name: Mistika,Um Skkin
2.2. Description Vetiveria Nigritana is a perennial grass species of the family Poaceae and therefore is also a monocotyledon. It is a very thick and tall type of grass that is deeply rooted within the ground and is usually used to protect crops and deter soil erosion. V. nigritana is a species native to Africa and is most commonly seen in Nigeria, Northern and Eastern Africa and tropical parts of Southern Africa. The plant, like other vetiver grasses, has been used in these regions due to its extreme drought tolerance, ability to grow in infertile soil and the fact that it can live under complete submergence. In fact, V. nigritana can thrive in a very diverse range of environmental and climatic conditions. V. nigritana is a very beneficial plant within subsistence agriculture, especially in Africa, due to its ability to preserve soils and reduce water runoff, which ultimately is correlated with higher crop yields. In addition, the plant is also beneficial in protecting a farmers stored crop harvests and plants while in the field because the plant can be used as a repellent or a means of destroying pest larvae before they have the ability to affect a farmer’s cash crop. V. nigritana also has various cost-effective medical applications for subsistence farmers and can be used as feed to maintain livestock in absence of other more common feeds. The species V .nigritana is found predominately in the semi-arid belt where traditionally its roots are prized for their property to give drinking water a good taste, and for medicinal purposes (Dressler, 2014). 2.3. Habitat Vetiver grows in any type of soil but a rich and fairly well drained loam is considered best. The loamy soils, which are loose in texture, are ideal for root growth and harvesting as well. Vetiver plant also grows on a variety of problematic soils like waterlogged soils, sandy soils and areas with high water table and flood prone. A luxuriant growth of healthier root is obtained from plants growing under warm and damp conditions on rich, temporary inundated, marshy land. It grows best in areas with warm summer and well-distributed rainfall (Dressler, 2014). 2.4. Benefits of V.nigritana
2.4.1 Soil erosion and runoff prevention
One of the main benefits of using V. nigritana, especially in subsistence agriculture, is that it reduces soil erosion and nutrient loss associated with water runoff and wind displacement. Essentially, when V. nigritana is planted in hedgerows surrounding a crop or in multiple spaced rows, it creates a barrier that prevents soil run-off. This is because the plant’s thick grass structure is able to accumulate soil and nutrients, such as nitrogen, phosphorus and carbon that would otherwise have been lost due to runoff. In fact, studies have shown that planting V. nigritana hedgerows reduces soil loss by around 70%, which allows farmers to retain the nutrients within their soil. Secondly, the use of V. nigritana also reduces water runoff, in some cases by 90%, which is beneficial because in areas where irrigation is not present, conserving natural rainfall is essential for the survival of a farmer’s crop. In addition, the use of the type of vetiver grass is ideal in places like the African subtropics to prevent soil erosion and runoff because V. nigritana has been shown to be highly effective on a variety of different slopes in preventing the degradation of soil. Therefore, V. nigritana would benefit subsistence farmers because the increased nutrients, retention of soil and the accumulation of water that it causes would help improve crop yields (Dressler, 2014). 2.4.2 Landscaping: Being a grass with a beautiful form and aesthetic value, vetiver is sometimes used as an ornamental plant in landscaping, or as a decorative potted plant. Vetiver is a beautiful ornamental plant for gardens, patios, decks, etc. The bush of the vetiver plant is so large that it hides unsightly structures. Grown as a hedgerow, it forms a dense, uniform, and attractive hedge under tropical and subtropical climates. It also forms an aesthetically beautiful barrier to unsightly view (Chomchalow, 2001) . (Figure 1).
Figure 1. Landscaping with V. nigritana
2.4.3 Pest control
Another benefit of V. nigritana is its ability to deter pests from damaging crops while in the field during the growing season and from ruining the quality of plants while in storage. In Africa it was discovered that by planting V. nigritana in hedgerows around maize, that maize stem borers would attach to the plant more frequently than to maize and when the larvae hatched the potential borers died. In addition, V. nigritana is also beneficial in protecting certain crops after harvest, such as rice crops, because when the leaves of the plant are boiled in sea salt and then placed below and above the crop in a storage environment they act as a repellent for insects, while also preventing mold. Therefore, the use of V. nigritana would be beneficial to subsistence farmers because it would protect their crops and help ensure their survival (Dressler, 2014).
2.4.4 Livestock protection
Additionally, V. nigritana, like other forms of vetiver grasses, is beneficial in protecting the livestock of subsistence farmers. Even though vetiver grasses usually have a low protein ratio of about 8% they can be continuously harvested and fed to livestock if there is a shortage of other feeds for the animals due to poor harvests, diseased crops or contaminated storages. This is valuable to subsistence farmers because a lack of feed usually leads to animals being slaughtered. Therefore, as a result of the use of vetiver grasses, the fertilizer production and grazing benefits of livestock are not lost, which is another reason why they are ideal for subsistence farmers (Dressler, 2014).
2.4.5 Traditional uses
It has been well known since ancient times that vetiver possesses aromatic roots. Pleasant aroma is released from vetiver root dug from the soil and hanged in the shade. In India the dried roots are used to give fragrance to linen clothes while the root mass is used as a blind to cool down the heat of the summer, especially in Northern India. The vetiver blind is continually doused with water throughout the day, turning the hot wind into a scented cooling breeze,which passes through the soaked vetiver blind, releasing a sweet aroma. The scented vetiver roots are also used for making fans, clothes hangers, and are mixed with other kind of flower scents and leaves for making sachets .(Sastry 1998) (Figure 2).
(Figure 2). Vetiveria Nigritana root
2.4.6 Uses as water purifier The most common use of V. nigritana in Senegal and The Gambia is as a "water purifier," a disinfectant and an antiseptic. It apparently helps eliminate pathogens bacteria and also improves the taste of water. It is said that water found near vetiver is safe for drinking. These uses are widespread in Senegal and Gambia. Even If we don’t know exactly when these uses started, we can hypothesize in accordance with what we observed and heard that vetiver usage could go back several generations (Figure 3).
(Figure 3). Uses as water purifie
2.5. Medicinal uses
There are many medical uses that have been derived from V. nigritana because the plant contains very high levels of essential oils that can be distilled and used for traditional medical applications. Firstly, V. nigritana can and has been used to increase the quality of the taste of drinking water in less developed nations such as Africa. In addition, the plant has also traditionally been used to eliminate pathogenic bacteria that are present. Lastly, V. nigritana is also beneficial at reducing diarrhea and has been used historically to treat children with a great deal of success. Therefore, V.nigritana is beneficial to poor farmers because it provides traditional cost effective ways to fight infections and disease
2.6. Essential oils
An essential oil is a mixture of chemical compounds, commonly containing 40 to 80 monoterpenoids, sesquiterpenoids and diterpenoids, and many in relatively small proportions. The composition of essential oils, which are produced by specialized plant cells ʻin vivoʼ and the quality of the extracted products, differ in the majority of cases. The chemical composition of the essential oils which are commercialized depends on their method of production. In the course of the isolation procedure some of the compounds disappear, some change in structure, or suffer decomposition. The most widely used procedure for extracting essential oils from the plant is steam, or water distillation. The distillation of volatile oils by means of water or steam has long been practiced, but modern plants possess many advantages over the older stills, in which charring and undesirable decomposition of the oil often took place. Solvent extraction processes may be used for plants which contain small amounts of essential oil, particularly those which dissolve in water very easily, or for plants accumulating aromatic compounds which are partly volatile. For extraction different solvents, like alcohol, hexane, benzene, toluene, petrol, etc. can be used. Enfleurage is a special form of extraction. In this procedure glass plates are covered with a thin layer of fixed oil or fat upon which the fresh flowers are spread. The volatile oil gradually passes into the fat and the exhausted flowers are removed and replaced by a fresh supply. In the digestion process the flowers are gently heated in melted fat until exhausted. The volatile oil is obtained from the fat by three successive extractions with alcohol. More recently Supercritical Fluid Extraction (SFE) is used for separation of volatile oil. The method is based on the good solubility of volatile compounds in CO2, kept in supercritical fluid phase using special conditions of pressure and temperature. Cold pressing is a well-known procedure for extraction of scent materials. This method can be used for extracting the essential oils from the fresh fruits of plants belonging to the genus Citrus (Chomchalow, 2001). 2.7. Other species of vetiver
The genus Vetiveria contains another species known as Vetiveria zizanoides commonly known as the Khas-Khas, Khas or Khus grass in India , synonymously known as “Chrysopogon zizanioides”. It is also a perennial grass with thick fibrous adventitious roots which are aromatic and highly valued. This tufted grass grows throughout the plains of India ascending up to an elevation of 1200 m. Having wide ecological amplitude, this grass grows in a wide variety of ecological habitats covering all biogeographic provinces of India. No wonder that this is one grass which has been extensively used by almost all the tribes. V.zizanioides is a densely tufted grass with the culms arising from an aromatic rhizome up to 2m high; the roots are stout, dense and aromatic; leaves are narrow, erect, keeled with scabrid margins. It is found throughout the plains and lower hills of India, particularly on the river banks and in rich marshy soil. (Anon, 2014) (Figure 4).
(Figure 4). Vetiveria zizanioides roots
2.8. Vetiveria nigritana VS Vetiveria zizanioides Whereas V zizanioides type roots are thick and smooth (with little fibrous secondary roots), the ones from V nigritana are thin and more fibrous. Also, the slips in V zizanioides have axillary buds close to nodes imparting spreading plant habit, but in V nigritana the nodes are sparse with little axillary buds.The thicker and smooth roots are preferred for perfume quality oil ( Juliard. C.) (Figure 5).
( Figure 5). V. nigritana and V.zizanioides roots
2.9. Composition
2.9.1 Vetiveria nigritana oil
Vetiver is mainly cultivated for the fragrant essential oil distilled from its roots. Vetiver oil is viscous light-brown oil with a rich green-woody earthy and nut- like fragrance. In its diluted form, vetiver oil is used to provide sweet note and soothing cool effect. It has been utilized as raw material for various fragrant products such as perfumes, deodorants, lotions, soaps, cosmetics, etc. Having complex chemical composition and oil odor, high solubility in alcohol that improves it miscibility with other perfumery material, vetiver oil is a unique perfumery resource. Worldwide production is estimated at about 250 tons per annum. Due to its excellent fixative properties, vetiver is used widely in high end perfumes. It is contained in 90% of all western perfumes. Haiti is one of the leading producers of vetiver in the world, along with Java, China, India, Brazil, and Japan. The United States, Europe, India, and Japan are the main consumers. In perfumery, the older French spelling, vetiver, is often used (Demole et al. 1995).
Vetiver oil is amber brown and rather thick. Its odor is described as deep, sweet, woody, smoky, earthy, amber, and balsam. The best quality oil is obtained from 18- to 24-month-old roots. The roots are dug up, cleaned, and then dried. Before the distillation, the roots are chopped and soaked in water. The distillation process can take up to 24 hours. After the distillate separates into the essential oil and hydrosol, the oil is skimmed off and allowed to age for a few months to allow some undesirable notes forming during the distillation to dissipate. Like patchouli and sandalwood essential oils, the odor of vetiver develops and improves with aging. The characteristics of the oil can vary significantly depending on where the grass is grown and the climate and soil conditions. The chemical composition of the oil is extremely complex and consists of sesquiterpenes and their derivatives, belonging to 11 structural classes. The sesquiterpenes α-vetivone, β-vetivone and khusimol are considered as the "finger print" of vetiver oil. The different and complex composition of vetiver oils from different geographic locations has been reported in literature. Longifolene D, 2-hydroxycyperol and aromadendrene oxide (1) have been reported as major components (Khalil, and Ayoub 2011). Other published data showed prezizanoic acid, preziza-7(15)-en- 12-ol, cedren-8-en- 15- ol, preziza-7(15)-en-3α-ol, and zizanoic acid as major components (Sidibeˊ et al. 2012) (Figure 6).
(Figure 6). Vetiver oil
3. Materials and Methods 3.1. Materials 3.1.1. Plant Material
The plant material (dry roots) was obtained from Omdurman market 200g ( origin: Niyala,western Sudan) and was identified as V. nigritana (Benth) Stapf at the Medicinal and Aromatic Plants Research Institute (MAPRI), the National Center for Research (NCR) ,Khartoum, The roots were pulverized into fine powder. 3.2. Methods
3.2.1. Extraction of the essential oil
Dry and powdered V.nigritana roots (50 g) were water - distilled in a Clevenger apparatus for essential oil for 12 h using a heating mantle ( Electrothermal). 3.3. GC- MS analysis of the oil
The GC-MS analysis of the oil was performed on a QP 2010 Plus EI GS/MS instrument , using a capillary column 25 m × 0.22 mm internal diameter and film thickness 0.33 mm, carrier gas was helium with flow rate : 1.2 ml/min, column oven temp: 35.0 oC, injection temp. : 250 oC, injection mode: split. Oven temp. Program Table (1):-
Rate(oC/ min) Temperature (oC) Hold time (min) - 35.0 3.00 5.00 240.0 0.00 3.00 280.0 4.00
The identification of components was based on comparison of their mass spectra with those of (Mc Lafferty and Stauffer 1989),(Adams 2001) and (Joulain 1998) libraries. The results are presented in Table (2).
(Figure 7). QP 2010 plus EI GS/MS
4. Results and Discussion 4.1. Results
The composition of the oil from the roots of V. nigritana is summarized in Table (2).
Table (2): GC-MS analysis of Vetiver oil prepared from Vetiveria nigritana
Peak R. T area% Compound No. 1 8.49 0.12 Ethyl benzene 2 8.77 0.46 m-Xylene 3 9.55 0.20 o-Xylene 4 22.37 0.22 Thymol 5 24.22 0.98 Biphenylene,1,2,3,6,7,8,8а,8b-octahydro-4,5-dimethyl- 6 25.43 0.34 Cedr-8ene 7 26.46 0.30 Humulen-(VI) 8 27.95 0.20 Phenol, 2,4-bis(1,1-dimethylethyl) 9 28.16 0.52 delta.-Cadinene 10 29.69 1.41 (-)-Caryophyllene oxide 11 29.90 1.31 Valencene 12 30.10 2.70 2-Butenal, 2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl) 13 30.31 0.32 Humulene oxide 14 30.42 0.23 Unidentified 15 30.52 2.86 Epicedrol 16 30.96 1.64 Unidentified 17 31.14 2.46 Torreyol 18 31.26 2.05 trans-Caryophyllene
19 31.42 0.74 Unidentified
20 31.49 0.79 Globulol 21 31.66 12.55 4,6,6-trimethyl-2-(3-methyl-buta-1,3 dienyl)-3-oxa tricyclo [5.1.0.0 2,4]octan Overlapping with caryophyllene 22 31.82 3.21 Unidentified 23 32.02 1.90 Unidentified 24 32.32 8.92 Unidentified 25 33.25 2.37 gamma.1-cadinene aldehyde 26 33.35 3.40 Cedr-8-en-13-ol 27 33.48 8.44 2-Isopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,7 octahydronaphthalene 28 34.90 10.84 Cedr-8-en-15-ol 29 35.21 8.37 Unidentified 30 35.51 6.41 Unidentified 31 36.51 1.61 Unidentified 32 37.31 1.11 Unidentified 33 37.60 2.84 n-Hexadecanoic acid 34 40.88 0.89 Oleic acid
4.2. Discussion There are distinct geographical differences in the quality and yield of vetiver oil obtained from species growing in different regions of the world. The yield of the oil prepared from the Sudanese V. nigritana root was hydrodistillation to reach 0.2% at the end of distillation process lasted for 12 h. The prepared oil was screened primarily by solvent systems, followed by complete analysis with GC MS (model QP 2010 Plus EI). The apparatus was equipped with an advanced library which enabled a reliable identification of the sample components. The main components of the oil were 52.77% sesquiterpene hydrocarbons and 31.77% their oxygenated derivatives. From the thirty four compounds detected, twenty four were identified (Table 1). The major constituent was Cedr-8-en-15-ol (10.84%) and 4, 6, 6-trimethyl-2-(3-methylbuta-1, 3-dienyl) 3oxatricyclo [5.1.0.0(2, 4)] octane (10.50%) overlapping with caryophyllenethe, and the two comprising (12.55%) of the oil. It is known that the chemical composition of vetiver oil is extremely complex and contain sesquiterpene compounds. This result is new addition to the current literature which specifies the different composition of the oil. The major component (10.84%) in V. nigritana oil was Cedr-8-en-15-ol (MW 220, item No. 28, retention time 34.90 min.). The peak at m/e (202)
(Appendix 28) is due to the loss of water molecule (H2O) (Fig.8).
(Fig.8). Cedr-8-en-15-ol
The presence of Cedr-8-en-15-ol in V.nigritana oil has been reported in literature (Sidibé et al,2012)
The GC / MS analysis of Vetiver oil showed the presence of compound 4, 6, 6 trimethyl-2-(3-methylbuta-1,3-dienyl) 3 oxatricyclo[5.1.0.0(2,4)] octane (MW 218) overlapping with caryophyllene (MW 204) (Appendix 21) (item No 21, retention time 31.66 min) , representing 12.55 % by weight of the total compounds .
Figure 9: Structure of caryophyllene Figure 10: Structure of 4, 6, 6- trimethyl-2-(3-methylbuta-1,3-dienyl) 3-oxatricyclo [5.1.0.0(2,4)] octane
The presence of both 4, 6, 6-trimethyl-2-(3-methylbuta-1,3-dienyl) 3 oxatricyclo [5.1.0.0(2,4)] octane and caryophyllene has not been reported before in literature in vetiver oil.
The mass spectrum of compound n-Hexadecanoic acid (palmitic acid) (MW 256) with retention time 37.60 min. and percentage of 2.84 % showed a base + peak at m/e 43 (Appendix 33 ) is due to the alkyl group ion (CH3CH2CH2- ) . As shown in the following formula. (Fig.11) 213 43 3
Fig.11 .Hexadecanoic acid
The mass spectrum of compound m-xylene (MW106) with retention time 8.77 min and percentage of 0.46 % showed a base peak at m/e 91 (Appendix 2 ) due to the loss of a methyl group (CH3) as shown in the following formula .Fig.12
15 91
Fig.12. m-Xylene
It has been shown that in most in cases the ion of mass 91 is a tropylium rather than a benzylic cation. This explains the ready loss of a methyl group from xylenes although toluene dose not easily lose a methyl group.The incipient molecular ion rearranges to the parent tropylium radical ion, which then cleaves to the simple + tropylium ion (C7 H7 )
The frequently observed peak at m /e 65 results from elimination of a neutral acetylene molecule from the tropylium ion:
Conclusion and Recommendations
Conclusion:
The main components of the oil were sesquiterpene hydrocarbons and their oxygenated derivatives. This result is in agreement with all the results reported in literature. From the thirty four compounds detected, twenty four were identified. The major constituent was cedr-8-en-15-ol (10.84%), followed by 4, 6, 6- trimethyl-2-(3-methylbuta-1, 3-dienyl) 3oxatricyclo [5.1.0.0(2, 4)] octane (10.50%) which was overlapping with caryophyllene, the two comprising 12.55% of the oil. It is known that the chemical composition of vetiver oil is extremely complex and contain sesquiterpene compounds. The presence of cedr-8-en-15-ol among the major components has been reported by Sidibeˊ et al. (2012). The difference between the chemical composition of the analyzed sample and other samples reported in literature is due to difference location and climate. This result is new addition to the current literature which specifies the different composition of the oil. Recommendation:
Since vetiver oil is an article of international trade as a pure fragrance and in composite perfumes and flavors, and also in aromatherapy, it is recommended that the Vetiveria nigritana grass be grown in a commercial scale for production of oil.
References:
Adams, R.P, 2001. Identification of Essential Oil Components by Gas
Chromatography/Quadrupole Mass Spectroscopy Allured Publishing Corp.,
Carol Stream, I.L.
Anon, 2014. Chrysopogon zizanioides .From Wikipedia, the free encyclopedia.
Chomchalow, N. (2001). The Utilization of Vetiver as Medicinal and Aromatic Plants. Technical Bulletin No. 2001/1. Pacific Rim Vetiver Network. 27 pp.
Demole, E.P., Holzner, G.W., Youssefi, M.J. 1995. Malodor formation in alcoholic perfumes containing vetiveryl acetate and vetiver oil. Perfum. Flav. 20: 35-40.
Dressler, S., Schmidt, M., Zizka, G. (2014). "Chrysopogon nigritanus". African plants – a Photo Guide. Wikipedia. Elghazali, G.E., Al-Khalifa, K.S., Salem, G.A., Abdullah, E.M, 2010. Traditional medicinal plants indigenous to Al-Rass province, Saudi Arabia. J. Med. Plants Res., 4 (24) (2010), 2680–2683.
Juliard . C. Application of Vetiver in Western Africa.
www.vetiver.com/KUW_WORKSHOP_papers/KUW_4CJ.pdf Joulain D, König, W.A1998.The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. E.B. Verlag, Hamburg. Khalil, M. A., Ayoub, S .M. H. 2011. Analysis of the essential oil of Vetiveria nigritana (Benth.) Stapf root growing in Sudan. Journal of Medicinal Plants Research Vol. 5(32), 7006-7010, 30 December, 2011.
Marshall, E. 2011. Health and wealth from medicinal plants. Rural Infrastructure and Agro-Industries Division Food and Agriculture Organization of the United Nations. Rome 2011.
Mc Lafferty. F, Stauffer. D.B.1989. the Wiley NBS registry of Mass Spectral Data, 2nd Edition, J.Wiley and Sons, N.Y. Sastry KNR. 1998. Socio-economic dimensions of vetiver in rainfed areas of Karnataka, India. Proc. ICV-1, Chiang Rai, Thailand. 243-248. Sidibeˊ. L, Fofana . B , Traoré. N, Tolofoundyé. A, Figuiredo. G, Chalchat . J . C., Bessière. J. M. 2012. Chemical composition of Vetiveria nigritana from Koulikoro Area, Pelagia Research Library, Der Chemica Sinica, 2012, 3(5):1276-1279 .
Appendix (1): Ethyl benzene
Appendix (2): m-Xylene
Appendix (3): o-Xylene
Appendix (4): Thymol
Appendix (5) : Biphenylene, 1,2,3,6,7,8,8a,8b-octahydro-4,5-dimethyl
Appendix (6): Cedr-8-ene
Appendix (7): Humulen-(VI)
Appendix (8): Phenol, 2,4-bis(1,1-dimethylethyl)
Appendix (9): delta.-Cadinene
Appendix (10): Caryophyllene oxide
Appendix (11): Valencene
Appendix (12): 2-Butenal, 2-methyl-4-(2, 6, 6-trimethyl-1-cyclohexen-1- yl)-
Appendix (13): Humulene oxide
Appendix (14): Unidentified
Appendix (15): Epicedrol
Appendix (16): Unidentified
Appendix (17): Torreyol
Appendix (18): trans-Caryophyllene
Appendix (19): Unidentified
Appendix (20): Globulol
Appendix (21): caryophyllene
Appendix (21) 4, 6, 6-trimethyl-2-(3-methyl-buta-1, 3 dienyl)-3-oxa tricyclo [5.1.0.0 2, 4] octan
Appendix (22): Unidentified
Appendix (23): Unidentified
Appendix (24): Unidentified
Appendix (25): gamma.1-cadinene aldehyde
Appendix (26): Cedr-8-en-13-ol
Appendix (27): 2-Isopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,7 octahydronaphthalene
Appendix (28): Cedr-8-en-15-ol
Appendix (29) Unidentified
Appendix (30) Unidentified
Appendix (31) Unidentified
Appendix (32) Unidentified
Appendix (33): n-Hexadecanoic acid
Appendix (34): Oleic Acid