Characterization and Biological Activities of Oils from Seeds of Khaya Senegalensis , Eruca Sativa and Pisum Sativum Used In

Al-Neelain University Graduate College

Characterization and Biological Activities of Oils from Seeds of Khaya senegalensis , Eruca sativa and Pisum sativum used in Sudanese Traditional Medicine توصيف واالنشطة البيلوجية لزيوت من بذور المهوقني والجرجيروالبازيال المستخدمة في الطب الشعبي السوداني

A Thesis submitted in fulfilment of the Requirements for Master Degree in Pharmacy

By: Maap Abdalla Malik Ahamed (B. Pharmacy- International University of Africa) Supervisor: Prof. Mohammed Abdel Karim Mohammed Co- supervisor: Dr. Intisar Abdel rahim Bashir

May/ 2019

DEDICATION

 To My Husband …

 To the soul of my Mother …

 To my father …

Acknowledgement

 First of all I would like to thank Almighty Allah for giving me will and health to accomplish this work.  Thanks for my supervisor Prof. Mohammed Abdel Karim Mohammed for his patience and giving me lot of time to get this work done.  I thank my co supervisor Dr. Intisar Abdelrahim Bashir for her advice and useful suggestions.  I thank the staff of Pharmacoganocy Department – Alneelain University for of all facilities.

Abstract

The Khaya senegalensis , Eruca sativa, Pisum sativum are key species in Sudanese system of medicine. The seed oils of these species were extracted to determine physical properties of oils and evaluate for antimicrobial and antioxidant activity and analyzed by GC-MS. GC-MS analysis of Khaya senegalensis oil showed the presence of 23 constituents dominated by 9-octadecenoic acid (Z) – methyl ester (47.43%). The oil from Eruca sativa showed 30 constituents and major component is 13-Docoenoic acid, methyl ester (31.92%). Pisum sativum oil revealed the presence of 33 constituents dominated by 9, 12-octadecadienoic acid (Z, Z)-, methyl ester (30.69%). Furthermore, these oils were assessed for their antimicrobial activity against pathogenic bacteria: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus Subtilis and fungal species Candida albicans. In the cup plate agar diffusion bioassay Khaya senegalensis oil showed significant activity against Staphylococcus aureus (15 mm) but it was inactive against other test microorganisms. Eruca sativa showed highly significant activity against Escherichia coli (20 mm) and Staphylococcus aureus (20 mm) but it was inactive against other test microorganisms. while Pisum sativum showed highly significant activity against all tested organisms (Pseudomonas aeruginosa 20 mm, Staphylococcus aureus 24 mm, Bacillus Subtilis 20 mm, Candida albicans 20 mm) and significant activity against Escherichia coli (14 mm). In the DPPH radical scavenging assay, Khaya senegalensis, Eruca sativa, and Pisum sativum oil showed significant antioxidant activity (90, 37, and 82%)

III

مستخلص البحث

المهوقني والجرجير والبازيال هي عبارة عن نباتات لها إستخدامات في الطب الشعبي بالسودان. تم

أستخالص الزيوت من بذور النباتات وتحليلها لمعرفه الخصائص الفيزيائيه للزيوت ولمعرفه مضادات

البكتريا ومضادات االكسده للزيوت وتحليلها عن طريق الكروموتوغرافيا الغازيه –طيف الكتله وقد اوضحت

هذه التقنيه أن زيت المهوقني يحوي 23 مكون اهمها: 9-octadecadienoic acid (Z) - , methyl.

(ester (47.43%وقد اتضح أن زيت الجرجير فيه 30 مكون اهمها: – 13-Docoenoic acid

methyl ester (31.92%)

أما زيت البازيال فيه 33 مكون اهمها: 12-octadecadienoic acid (Z, Z) - , methyl ester ,9

(%30.69). وفي أختبارمضاد البكتريا أبدي زيت المهوقني فعاليه عاليه ضد بكتريا Staphylococcus mm) aureus 15) ولكنه لم يكن فعاال ضد الميكروبات االخري قيد االختبار. كذلك اعطي زيت

الجرجير فعالية ممتازه ضد20mm).Escherichia coli , (20mm) Staphylococcus aureus) أما

زيت البازيال فقد أعطي فعالية ممتازة ضد جميع الميكروبات قيد االختبار Pseudomonas aeruginosa) 20 mm, Staphylococcus aureus 24 mm, Bacillus Subtilis 20 mm, Candida

(albicans 20 mmوفعاليه جيده ضد (Escherichia coli (14 mm.

وفي اختبار مضادات االكسده ابدي زيت المهوقني والجرجير والبازيال مقدره عاليه كمضادات اكسده

)90 و37 و 82 %(.

Table of Contents

No Title Page no. Dedication I Acknowledgment II Abstract (English) III Abstract (Arabic) IV Table of contents V List of Tables VII List of Figures VIII Introduction 1 Chapter One 1 Literature Review 5 1.1. Target species 5 1.2. Oils , Fats and waxes 12 1.3. Antimicrobials 15 1.4. Gas chromatography mass spectroscopy 18 1.5. Solvents extraction 21 Chapter Two 2. Materials and methods 25 2.1. Materials 25 2.1.1. Plant material 25 2.1.2. Instruments 25 2.1.3. Test organisms 25 2.2. Methods 26 2.2.1. Extraction of oil 26 2.2.2. Physiochemical properties of oils 26 2.2.3. GC/MS analysis 27 2.2.4. Antimicrobial test 28 2.2.4.1. Preparation of bacterial suspensions 28 2.2.4.2. Preparation of fungal suspensions 28 2.2.4.3. Testing for antibacterial activity 28 2.2.4.4 DPPH radical scavenging assay 29 Chapter Three 3 Results 30 3.1 Physiochemical properties of oils 30 3.1.1. Physiochemical properties of Khaya senegalensis oils 30 3.1.2. Physiochemical properties of Eruca sativa oil 31

3.1.3. Physiochemical properties of Pisum sativum oils 31 3.2 GC-MS analysis of oils 32 3.3 Antimicrobials activity 50 3.4 Antioxidants activity 51 Chapter Four 4 Discussion 52 Chapter Five 5.1 Conclusion 55 5.2 Recommendation 56 References 57

List of Tables

No Title Page No. 2.1 Test organisms 25 2.2 Oven temperature program 27 2.3 Chromatographic conditions 27 3.1 Physiochemical properties of Khaya sengalensis oil 30 3.2 Physiochemical properties of Eruca sativa oil 31 3.3 Physiochemical properties of Pisum sativum oil 31 3.4 Constituents of Khaya senegalensis oil 33 3.5 Constituents of Eruca sativa oil 37 3.6 Constituents of Pisum sativum oil 45 3.7 Antimicrobial activity of oils. 50 3.8 Antioxidant activity of oils. 51

VII

List of Figures

No Title Page No. 3.1 Total ion chromatograms of Khaya senegalensis 32 3.2 Mass spectrum of 9-octadecenoic acid (Z)- methyl ester 33 3.3 Mass spectrum of methyl stearate 34 3.4 Mass spectrum of hexadecanoic acid, methyl ester 35 3.5 Mass spectrum of 9,12-octadecadienoic acid (Z,Z)-methyl ester 35 3.6 Mass spectrum of eicosanoic acid, methyl ester 36 3.7 Total ion chromatograms of Eruca sativa 36 3.8 Mass spectrum of 13-docoenoic acid , methyl ester 38 3.9 Mass spectrum of cis -13-eicosenoic acid, methyl ester 39 3.10 Mass spectrum 9,12-octadecadienoic acid (Z,Z) – methyl ester 40 3.11 Mass spectrum of hexadecanoic acid methyl ester 41 3.12 Mass spectrum of 9-octadecenoicacid (Z) – methyl ester 41 3.13 Mass spectrum of 9, 12, 15-octadecatrienoic acid, methyl ester 42 3.14 Mass spectrum 15-tetracosenoic acid, methyl ester 42 3.15 Mass spectrum of methyl Stearate 43 3.16 Mass spectrum of cis -11-eicosenoic acid, methyl ester 43 3.17 Mass spectrum of docosanoic acid, methyl ester 44 3.18 Mass spectrum of eicosanoic acid, methyl ester 44 3.19 Total ion chromatograms of Pisum sativum 45 3.20 Mass spectrum of 9,12-octadecadienoic acid (Z,Z) methyl ester 47 3.21 Mass spectrum of 9-octadecenoic acid (Z)-methyl ester 48 3.22 Mass spectrum of methyl tetradecanoate 48 3.23 Mass spectrum of hexadecanoic acid, methyl ester 49 3.24 Mass spectrum of methyl stearate 49

VIII

Introduction

Khaya senegalensis belongs to family Meliaceae that is native to Africa. It is a medium-sized tree which can grow up to 15–30 m in height and 1 m in diameter. The bark is dark grey to grey-brown while the heartwood is brown with a pink-red pigment made up of coarse interlocking grains. The tree is characterized by leaves arranged in a spiral formation clustered at the end of branches. The white flowers are sweet-scented; the fruit changes from grey to black when ripening (1). Khaya senegalensis is a medicine that is used for the treatment of pain, inflammation, diarrhea, oxidative stress, ulcer, malaria and other conditions. Khaya senegalensis was also found to be bactericidal to S. aureus and S. feacalis, and fungicidal to C. albicans. One study demonstrates the potentials of K. senegalensis as a source of antimicrobials that could be harmless for use in the health care delivery process. Eruca sativa is species of tree in Brassicaceae family, is an edible annual plant. It is also called "rocket salad “rucola", "rucoli", "rugula", "colewort", and "roqpuette"(5). Eruca sativa grows 20–100 centimeters (8–39 in) in height. The pinnate leaves have four to ten small, deep, lateral lobes and a large terminal lobe. The flowers are 2–4 cm (0.8–1.6) in diameter, arranged in a corymb in typical Brassicaceae fashion; with creamy white petals veined with purple, and with yellow stamens; the sepals are shed soon after the flower opens. The fruit is a silique (pod) 12–35 millimeters (0.5–1.4 in) long with an apical beak, and containing several seeds (which are edible) (5). 1

Eruca sativa have excellent antimicrobial and antioxidant activity. it is also a great source of vitamins A, fiber, vitamin C, vitamin K, folate, iron, calcium, phosphorus, magnesium, manganese and potassium. It is also used in reducing cholesterol, maintaining blood sugar and lowers the chances of heart disease. Pisum sativum is an annual plant , it belongs to family Fabaceae .The pea is most commonly the small spherical seed or the seed-pod of the pod fruit . Each pod contains several peas, which can be green or yellow. Pea pods are botanically fruit, since they contain seeds and developed from the ovary of a (pea) flower. Pisum sativum has been shown to possess antibacterial, antidiabetic, antifungal, anti-inflammatory, anti-hypercholesterolemia, and antioxidant activities it also showed anticancer property. Health benefits of peas include weight loss and prevent anemia.

General objective: - Extraction of oil from the following species: seeds of Khaya senegalensis , Eruca sativa and Pisum sativum. Specific objectives: - To determine physiochemical properties of the oils. - To analyze the extracted oils by GC-MS. - To evaluate the antimicrobial and antioxidant activity of oils.

1- Literature Review

1.1-The target species: (i)-Khaya senegalensis

Order: Sapindales

Family: Meliaceae

Genus: Khaya Species: K. senegalensis Native range: Africa

Khaya senegalensis is belonging to family Meliaceae that is native to Africa. Common names include African mahogany, dry zone mahogany, Gambia mahogany, khaya wood, Senegal mahogany, cailcedrat, acajou, djalla, and bois rouge (1). African mahogany is a medium-sized tree which can grow up to 15–30 m in height and 1 m in diameter. The bark is dark grey to grey-brown while the heartwood is brown with a pink-red pigment made up of coarse interlocking grains. The tree is characterized by leaves arranged in a spiral formation clustered at the end of branches. The white flowers are sweet-scented; the fruit changes from grey to black when ripening. The tree is native to Sudan, Benin, Burkina Faso, Cameroon, Central African Republic, Chad, Ivory Coast, Gabon, Gambia, Ghana, Guinea, Guinea-Bissau, Mali, Niger, Nigeria, Senegal, Sierra Leone, Togo, and Uganda. It is found riparian 3

Khaya senegalensis is used for treatment of: Pain , Inflammation , Diarrhea , Oxidative stress, Ulcer, Malaria, Anemia, Cancer and Infectious diseases (2). The very bitter bark has a considerable reputation in its natural range as a fever remedy it is also used as a laxative, vermifuge, and taenicide, depurative and for treating syphilis .The bark extract is used for treating jaundice, dermatoses, scorpion bite, allergies, infection of the gums, hookworm, bleeding wounds (disinfectant). The seeds and leaves are used for treatment of fevers and headache. The roots are used as a treatment of sterility and for the treatment of mental illness. It is also used against syphilis, leprosy and as an aphrodisiac(3). Khaya senegalensis was also used as bactericidal to S. aureus and S. feacalis, and fungicidal to C. albicans. One study demonstrates the potentials of K. senegalensis as a source of antimicrobials that could be harness for use in the Health Care Delivery processes (4).

(ii)-Eruca sativa

Order: Brassicales

Family: Brassicaceae

Genus: Eruca Species: E. sativa Native range: Mediterranean region, from Morocco and Portugal in the west of Syria, Lebanon, and Turkey in the east Eruca sativa is an edible annual plant. It is also called "rocket salad “rucola", "rucoli", "rugula", "colewort", and "roquette". 4

It is sometimes conflated with Diplotaxis tenuifolia, known as perennial wall rocket, another plant of the Brassicaceae family that is used in the same manner. Eruca sativa, which is widely popular as a salad vegetable . The Latin adjective sativa in the plant's binomial is derived from satum, the supine of the verb sero, meaning "to sow", indicating that the seeds of the plant were sown in gardens. Eruca sativa differs from E. vesicaria in having early deciduous sepals. Some botanists consider it a subspecies of Eruca vesicaria: E. vesicaria subsp. sativa. Still others do not differentiate between the two species. Other common names include garden rocket, or more simply rocket (British, Australian, South African, Irish and New Zealand English), and eruca. The English common name, rocket, derives from the French roquette, a diminutive of the Latin word eruca, which designated an unspecified plant in the Brassicaceae family (probably a type of cabbage). Eruca sativa grows 20–100 centimeters (8–39 in) in height. The pinnate leaves have four to ten small, deep, lateral lobes and a large terminal lobe. The flowers are 2–4 cm (0.8–1.6) in diameter, arranged in a corymb in typical Brassicaceae fashion; with creamy white petals veined with purple, and with yellow stamens; the sepals are shed soon after the flower opens. The fruit is a silique (pod) 12–35 millimeters (0.5–1.4 in) long with an apical beak, and containing several seeds (which are edible). Eruca sativa typically grows on dry climate, disturbed ground and is also used as a food by the larvae of some moth species, including the Garden Carpet moth. Eruca sativa roots are also susceptible to nematode infestation. A pungent, leafy green vegetable resembling a longer-leaved and open lettuce, Eruca sativa is rich in vitamin C and potassium. In addition to the leaves, the flowers, young seed pods and mature seeds are all edible. Rocket was traditionally collected in the wild or grown in home gardens along with such herbs as parsley and basil. It is now grown commercially in many places, and is available for purchase. It is also naturalized as a wild plant away from its native range in temperate regions around the world, including northern Europe and North America. In India, the mature seeds are known as Gargeer. This is the same name gargīr), but used in Arab countries for the fresh leaves. Mild) جرجير ,in Arabic frost conditions hinder the plant's growth and turn the green leaves red (5). Antibacterial activity of various solvent extracts of Eruca sativa seed as well as seed oil was investigated against Gram +ve and Gram-ve bacterial strains. Maximum zone of inhibition was observed from seed oil followed by methanolic seed extracts from all bacterial strains compared with broad spectrum antibiotics gentamicin. MIC values of seed oil were within the ranges of 52-72 μg/ml as compared to 56-70 μg/ml standard antibiotics (gentamicin). Proximate and phytochemical analysis of seed of E. sativa showed presence of all essential phyto- constituents required for promising traditional medicine. Analysis of seed oil by gas chromatography revealed that there were high concentration of erucic acid (51.2%) followed by oleic acid (15.1%) and cis-11-eicosenoic acid (12.5%). In addition, minor quantities of other essential and non-essential fatty acids were also present. Therefore this study supports effectiveness of E. sativa seeds for its use in traditional medicine for various human disorders (6). Arugula is packed with nutrition and antioxidant properties. It is also a great source of vitamins A, fiber, vitamin C, vitamin K, folate, iron, calcium, phosphorus, magnesium, manganese and potassium. Arugula is rich in protein, riboflavin, thiamin, zinc, vitamin B6, pantothenic acid and copper. The flavonoids help to prevent stickiness of cholesterol to arteries, reduce blood pressure, raise the blood flow, improve the functions of blood vessels and reduce inflammation. It assists in reducing cholesterol, maintaining blood sugar and lowers the chances of heart disease. 6

Arugula is an excellent source of antioxidants which can raise the oxygen radical absorbance capacity. Antioxidants help to maintain the balance of enzyme reactions in the cells and eliminate the free radicals which damage the system. Antioxidants help to counteract the health ailments such as common cold, heart disease, cancer and premature ageing. Arugula is a great source of Vitamin A which is an antioxidant that improves the teeth, bones and eyes condition. All the leafy vegetables provide the flavonoids compounds which prevent the lung, skin and oral cancer. Arugula provides vitamin K which helps in the formation as well as strengthening of bones. The antioxidants found in Arugula exert anti-inflammatory activity in the body. The high intake of vitamin K helps to slow down the development of Alzheimer’s disease. Arugula results in low oxalate levels and allows the presence of more minerals which promotes the bone health. Arugula ensures the bone health and strength which prevents the degeneration of bones. The plant provides high amount of vitamin which prevent cancer and promotes the immune system. Vitamin C is the great defense against the free radicals and eradicates this harmful radical from the body before they interfere to any damage to the body. Arugula is packed with minerals and vitamins which provides enhance the immune system – such constituents stimulates the creation of white blood cells and enhances the durability, strength and functions of immune system. The plant has phytochemicals such as sulphoraphane and indoles and thiocyanates which helps to counteract with the cancer in the body, encounters cervical, breast, prostate and ovarian cancer. Arugula is rich in these phytochemicals which restricts the activities of cancer cells. Arugula is a great choice for mothers because it consists of folic acid which helps to reduce the development of mental defects in the newborns. Like other leafy vegetables, Arugula is rich in folates. 7

The plant possesses vitamin B complex and other eight vitamins which works together to enhance the cells metabolism and health. Vitamin B aids in production of energy, red blood cells, fat synthesis and other processes for the health of cells. It possesses considerable amount of vitamin B complex in its organic structure. Arugula is a great source of carotenoids which are the pigments that occur naturally and improves the ability of vision. Carotenoids slow down the development of macular degeneration which is caused when the venter of field of vision is compromised. This leads to cataracts. The high intake of carotenoids helps to slow down the symptoms of ageing. Arugula has low amount of oxalates in comparison to other leafy vegetables such as spinach. Oxalates prevent the mineral absorption in the body. As it does not have high amount of oxalates, the minerals such as iron and copper is easily absorbed in the body and can be used efficiently. The plant is low in calories content, rich in nutrients and vitamins which provide the positive effect on effort for weight loss. It helps to satisfy the nutritional needs and balances the system without making extreme changes in the diet (7).

(iii)-Pisum sativum

Order: Fabales

Family: Fabaceae

Genus: Pisum Species: P. sativum Native Range: Mediterranean basin and the near east . Pisum sativum is an annual plant, with a life cycle of one year. It is a cool-season crop grown in many parts of the world; planting can take place from winter to early summer depending on location. The average pea weighs between 0.1 and

0.36 grams. The immature pes (and in snow peas the tender pod as well) are used as a vegetable, fresh, frozen or canned; varieties of the species typically called field peas are grown to produce dry peas like the split pea. The pea is most commonly the small spherical seed or the seed-pod of the pod fruit Pisum sativum. Each pod contains several peas, which can be green or yellow. Pea pods are botanically fruit, since they contain seeds and developed from the ovary of a (pea) flower. The name is also used to describe other edible seeds from the family Fabaceae such as the pigeon pea (Cajanus cajan), the cowpea (Vigna unguiculata), and the seeds from several species of Lathyrus. The wild pea is restricted to the Mediterranean basin and the Near East. A pea is a most commonly green, occasionally golden yellow, or infrequently purple pod-shaped vegetable, widely grown as a cool season vegetable crop. The seeds may be planted as soon as the soil temperature reaches 10 °C (50 °F), with the plants growing best at temperatures of 13 to 18 °C (55 to 64 °F). They do not thrive in the summer heat of warmer temperate and lowland tropical climates, but do grow well in cooler, high altitude, tropical areas. Many cultivars reach maturity about 60 days after planting(8). Pisum sativum has been shown to possess antibacterial, antidiabetic, antifungal, anti-inflammatory, anti-hypercholesterolemia, and antioxidant activities it also showed anticancer property. Its nonnutritive biologically active components include alkaloids, flavonoids, glycosides phenols, phytosterols, phytic acid, protease inhibitors, saponins, and tannins. This plant is rich in apigenin, hydroxybenzoic, hydroxycinnamic, luteolin, and quercetin, all of which have been reported to contribute to its remedial properties including anti carcinogenesis property(9). Health benefits of peas include weight loss due to presence of fiber .Peas are rich in protein they are also low in calories and fat and that will be a great choice for managing weight. 9

Choosing peas as protein group will be healthier choice being consumed every day since they can reduce the risk of cardiovascular disease. This benefit is served by the fiber content of peas which can block the cholesterol entering blood vessels and heart resulting in several cardiovascular problems (10). Peas are not only rich in protein and fiber but it contains high amount of vitamin C which can prevent skin damage caused by free radicals or sun exposure. Consuming pea’s everyday will help to keep healthy skin as well as make it glowing (11). Peas contain antioxidants which can neutralize the effect of free radicals which are the main culprit in aging process. Peas also have anti-inflammatory properties that may help the body to prevent wrinkle. They are also a great source of vitamin C which can promote collagen production inside the skin layer. Collagen itself is a protein that keeps skin tissue formation and keeps the skin bond into structure and thus preventing wrinkle. Peas contains high amount of antioxidant and other mineral such as copper, iron, zinc and manganese that enhance the body immune system. Those substances promote the immune cells to produce antibody and response towards the pathogen or free radicals effect in the body. Alzheimer is a disease that usually happens in elderly. People who suffer from alzheimer will lose their memory and it is a symptom of dementia. It is possible to avoid this type of mental disorder by consuming several amount of peas every day. Peas contain vitamin K that can keep the nervous system inside the brain and prevents damage which results in Alzheimer. A pea contains high fiber that is important to digestive tract. Fiber will add bulk into stool and make it easier to pass the colon. This is why consuming vegetable such peas can help to get rid of constipation 10

A pea contains some amounts of calcium which is important in maintaining healthy bones. Consuming peas everyday can certainly keep you away from osteoporosis or losing mass bones. Bad cholesterol (or known as Low Density Lipoprotein) is a type of fat that can enter the blood vessels causing cardiovascular problem. Peas contain high fiber that can help in blocking the cholesterol from entering the blood vessel layer. Health benefits of peas include preventing anemia. Folate and iron are important for preventing certain disorder such as anemias. Peas contain some amount of iron which promotes the red blood cells production and maintains normal blood function. Without iron, red blood cell can’t be formed and this will result in poor nutrients and poor oxygen transport that lead to anemia A pea is a vegetable which is rich in minerals such as potassium and magnesium. There are minerals important for muscle and nerve function. Potassium can maintain healthy nerve transfer and keep the structure of nerve tissues within the body while magnesium content in peas is responsible for promoting neural signals within the body. The main function of protein is building the body. It also repairs it when it is broken. Peas are rich in protein. Protein acts as building block and it repair the damage or change the old tissues with new one. This is why consuming protein is so important. Peas contain various vitamins such as vitamin B6, folate and vitamin B12 that can promote nutrition and absorption of hair follicle inside scalp. By getting enough nutrients, hair can grow and remain strong (12). Atherosclerosis is a condition where the blood vessels or arteries are thickened by cholesterol accumulation within the inner layer of the vessels. This condition may lead to high blood pressure and heart attack. A pea contains fiber that reduces the level of cholesterol and prevents it from entering blood vessels. 11

The folate content in peas is clinically proven. Folate has ability to keep healthy pregnancy and prevent neural tube defect in newborn baby. Peas are also saving to be consumed by mother during pregnancy and it is a great source of protein that can increase digestive function in pregnant mother tummy. Peas are high composed Vitamin A as well as beta carotene. Their constituents are beneficial in keeping healthy eyes and normal eyesight. It can also prevent elderly from age macular degeneration and cataract (13).

1.2- Oils, fats and waxes An oil is any nonpolar chemical substance that is a viscous liquid at ambient temperatures and is both hydrophobic (immiscible with water, literally "water fearing") and lipophilic (miscible with other oils, literally "fat loving"). Oils have a high carbon and hydrogen content and are usually flammable and surface active. The general definition of oil includes classes of chemical compounds that may be otherwise unrelated in structure, properties, and uses. Oils may be animal, vegetable, or petrochemical in origin, and may be volatile or non-volatile. They are used for food (e.g., olive oil), fuel (e.g., heating oil), medical purposes (e.g., mineral oil), lubrication (e.g. motor oil), and the manufacture of many types of paints, plastics, and other materials. Specially prepared oils are used in some religious ceremonies and rituals as purifying agents. Organic oils are produced in remarkable diversity by plants, animals, and other organisms through natural metabolic processes. Lipid is the scientific term for the fatty acids, steroids and similar chemicals often found in the oils produced by living things, while oil refers to an overall mixture of chemicals. Organic oils may also contain chemicals other than lipids, including proteins, waxes (class of compounds with oil-like properties that are solid at common temperatures) and alkaloids. 12

Lipids can be classified by the way that they are made by an organism, their chemical structure and their limited solubility in water compared to oils. They have a high carbon and hydrogen content and are considerably lacking in oxygen compared to other organic compounds and minerals; they tend to be relatively nonpolar molecules, but may include both polar and nonpolar regions as in the case of phospholipids and steroids. Several edible vegetable and animal oils, and also fats, are used for various purposes in cooking and food preparation. In particular, many foods are fried in oil much hotter than boiling water. Oils are also used for flavoring and for modifying the texture of foods (e.g. Stir Fry). Cooking oils are derived either from animal fat, as butter, lard and other types, or plant oils from the olive, maize, sunflower and many other species(14). Butter is a water-in-oil emulsion resulting from an inversion of the cream; in a water-in-oil emulsion, the milk proteins are the emulsifiers. Butter remains a solid when refrigerated, but softens to a spreadable consistency at room temperature, and melts to a thin liquid consistency at 32–35 °C (90–95 °F). The density of butter is 911 g/L (0.950 lb per US pint). It generally has a pale yellow color, but varies from deep yellow to nearly white. Its unmodified color is dependent on the animals' feed and genetics but is commonly manipulated with food colorings in the commercial manufacturing process, most commonly annatto or carotene (15). Fat is one of the three main macronutrients, along with carbohydrate and protein. Fats, also known as triglycerides, are esters of three fatty acid chains and the alcohol glycerol. The terms "lipid", "oil" and "fat" are often confused. "Lipid" is the general term, though a lipid is not necessarily a triglyceride. "Oil" normally refers to a lipid with short or unsaturated fatty acid chains that is liquid at room temperature, while "fat" (in the strict sense) may specifically refer to lipids that are solids at room temperature – however, "fat" (in the broad sense) may be used in

food science as a synonym for lipid. Fats, like other lipids, are generally hydrophobic, and are soluble in organic solvents and insoluble in water. Fat is an important foodstuff for many forms of life and fats serve both structural and metabolic functions. They are a necessary part of the diet of most heterotrophs (including humans). Some fatty acids that are set free by the digestion of fats are called essential because they cannot be synthesized in the body from simpler constituents. There are two essential fatty acids (EFAs) in human nutrition: alpha- linolenic acid ( omega-3 fatty acid) and linoleic acid ( omega-6 fatty acid). Other lipids needed by the body can be synthesized from these and other fats. Fats and other lipids are broken down in the body by enzymes called lipases produced in the pancreas. Fats and oils are categorized according to the number and bonding of the carbon atoms in the aliphatic chain. Fats that are saturated having no double bonds between the carbons in the chain. Unsaturated fats have one or more double bonded carbons in the chain. The nomenclature is based on the non-acid (non- carbonyl) end of the chain. This end is called the omega end or the n-end. Thus alpha-linolenic acid is called an omega-3 fatty acid because the 3rd carbon from that end is the first double bonded carbon in the chain counting from that end. Some oils and fats have multiple double bonds and are therefore called polyunsaturated fats. Unsaturated fats can be further divided into cis fats, which are the most common in nature, and trans fats, which are rare in nature. Unsaturated fats can be altered by reaction with hydrogen affected by a catalyst. This action, called hydrogenation, tends to break all the double bonds and makes a fully saturated fat. To make vegetable shortening, then, liquid cis-unsaturated fats such as vegetable oils are hydrogenated to produce saturated fats, which have more desirable physical properties e.g., they melt at a desirable temperature (30–40 °C), and stored well, whereas polyunsaturated oils go rancid when they react with

oxygen in the air. However, Trans fats are generated during hydrogenation as contaminants created by an unwanted side reaction on the catalyst during partial hydrogenation (16). Waxes are a diverse class of organic compounds that are lipophilic, malleable solids near ambient temperatures. They include higher alkanes and lipids, typically with melting points above about 40 °C (104 °F), melting to give low viscosity liquids. Waxes are insoluble in water but soluble in organic, nonpolar solvents. Natural waxes of different types are produced by plants and animals and occur in petroleum. Waxes are organic compounds that characteristically consist of long alkyl chains. They may also include various functional groups such as fatty acids, primary and secondary long chain alcohols, unsaturated bonds, aromatics, amides, ketones, and aldehydes. They frequently contain fatty acid esters as well. Synthetic waxes are often long-chain hydrocarbons (alkanes or paraffin) that lack functional groups. Waxes are synthesized by many plants and animals. Those of animal origin typically consist of wax esters derived from a variety of carboxylic acids and fatty alcohols. In waxes of plant origin, characteristic mixtures of un esterified hydrocarbons may predominate over esters. The composition depends not only on species, but also on geographic location of the organism (17). 1.3-Antimicrobials: An antimicrobial is an agent that kills microorganisms or stops their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibiotics are used against bacteria and antifungals are used against fungi. They can also be classified according to their function. Agents that kill microbes are called microbicide, while those that merely inhibit their growth are called biostatic. The use of antimicrobial medicines to treat infection is known as antimicrobial chemotherapy, while the use of antimicrobial medicines to prevent infection is known as antimicrobial prophylaxis.

The main classes of antimicrobial agents are disinfectants ("nonselective antimicrobials" such as bleach), which kill a wide range of microbes on non-living surfaces to prevent the spread of illness. Antiseptics are applied to living tissue and help reduce infection during surgery while antibiotics destroy microorganisms within the body. The term "antibiotic" originally described only those formulations derived from living microorganisms but is now also applied to synthetic antimicrobials, such as the sulphonamides, or fluoroquinolones. The term was initially restricted to antibacterials but its context has broadened to include all antimicrobials. Antibacterial agents can be further subdivided into bactericidal agents, who kill bacteria, and bacteriostatic agents, which slow down or stall bacterial growth. Antimicrobial use is known to have been common practice for at least 2000 years. Ancient Egyptians and ancient Greeks used specific molds and plant extracts to treat infection. In the 19th century, microbiologists such as Louis Pasteur and Jules Francois Joubert observed antagonism between some bacteria and discussed the merits of controlling these interactions in medicine. In 1928, Alexander Fleming became the first to discover a natural antimicrobial fungus known as Penicillium rubens and named the extracted substance penicillin which in 1942 was successfully used to treat a Streptococcus infection. Antibacterials are used to treat bacterial infections. The drug toxicity to humans and other animals from antibacterials is generally considered low. Prolonged use of certain antibacterials can decrease the number of gut flora, which may have a negative impact on health. Consumption of probiotics and reasonable eating can help to replace destroyed gut flora. Stool transplants may be considered for patients who are having difficulty in recovering after prolonged antibiotic treatment. 16

The discovery, development and use of antibacterial during the 20th century have reduced mortality from bacterial infections. The antibiotic began with the pneumatic application of nitroglycerine drugs, followed by a "golden" period of discovery from about 1945 to 1970, when a number of structurally diverse and highly effective agents were discovered and developed. Since 1980 the introduction of new antimicrobial agents for clinical use has declined, in part because of the enormous expense of developing and testing new drugs. In parallel there has been an alarming increase in antimicrobial resistance of bacteria, fungi, parasites and some viruses to multiple existing agents. Antibacterials are among the most commonly used drugs and among the drugs commonly misused by physicians, for example, in viral respiratory tract infections. As a consequence of widespread and injudicious use of antibacterial, there has been an accelerated emergence of antibiotic-resistant pathogens, resulting in a serious threat to global public health. The resistance problem demands that a renewed effort be made to seek antibacterial agents effective against pathogenic bacteria resistant to current antibacterials. Possible strategies towards this objective include increased sampling from diverse environments and application of metagenomics to identify bioactive compounds produced by currently unknown and uncultured microorganisms as well as the development of small-molecule libraries customized for bacterial targets. Antifungals are used to kill or prevent further growth of fungi. In medicine, they are used as a treatment for infections such as athlete's foot, ringworm and thrush and work by exploiting differences between mammalian and fungal cells. They kill off the fungal organism without dangerous effects on the host. Unlike bacteria, both fungi and humans are eukaryotes. Thus, fungal and human cells are similar at the molecular level, making it more difficult to find a target for an antifungal drug to attack that does not also exist in the infected organism. Consequently, there are often side effects to

some of these drugs. Some of these side effects can be life-threatening if the drug is not used properly. As well as their use in medicine, antifungals are frequently sought after to control mold growth in damp or wet home materials. Sodium bicarbonate (baking soda) blasted on to surfaces acts as an antifungal. Another antifungal serum applied after or without blasting by soda is a mix of hydrogen peroxide and a thin surface coating that neutralizes mold and encapsulates the surface to prevent spore release. Some paints are also manufactured with an added antifungal agent for use in high humidity areas such as bathrooms or kitchens. Other antifungal surface treatments typically contain variants of metals known to suppress mold growth e.g. pigments or solutions containing copper, silver or zinc. These solutions are not usually available to the general public because of their toxicity. Antiviral drugs are a class of medication used specifically for treating viral infections. Like antibiotics, specific antivirals are used for specific viruses. They are relatively harmless to the host and therefore can be used to treat infections. They should be distinguished from viricides, which actively deactivate virus particles outside the body. Traditional herbalists used plants to treat infectious disease. Many of these plants have been investigated scientifically for antimicrobial activity, and some plant products have been shown to inhibit the growth of pathogenic microorganisms. A number of these agents appear to have structures and modes of action that are distinct from those of the antibiotics in current use, suggesting that cross-resistance with agents already in use may be minimal(18). 1.4- Gas Chromatography-Mass Spectrometry (GC-MS): Gas chromatography–mass spectrometry (GC-MS) is an analytical method that combines the features of gas-chromatography and mass spectrometry to identify

different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation, and identification of unknown samples, including that of material samples obtained from plant Mars during probe missions as early as the 1970s. GC-MS can also be used in airport security to detect substances in luggage or on human beings. Additionally, it can identify trace elements in materials that were previously thought to have disintegrated beyond identification. Like liquid chromatography– mass spectrometry, it allows analysis and detection even of tiny amounts of a substance. The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer. The gas chromatograph utilizes a capillary column which depends on the column's dimensions (length, diameter, film thickness) as well as the phase properties (e.g. 5% phenyl polysiloxane). The difference in the chemical properties between different molecules in a mixture and their relative affinity for the stationary phase of the column will promote separation of the molecules as the sample travels the length of the column. The molecules are retained by the column and then elute (come off) from the column at different times (called the retention time), and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass-to- charge ratio (19). These two components, used together, allow a much finer degree of substance identification than either unit used separately. It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame ionization detector) cannot differentiate between multiple molecules that happen to take the same amount of time to travel through the column (i.e. have the same retention time), which results in two or more molecules that co-elute. Sometimes two different molecules can also have a similar pattern of ionized fragments in a mass spectrometer (mass spectrum). Combining the two processes reduces the possibility of error, as it is extremely unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer. Therefore, when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it typically increases certainty that the analyze of interest is in the sample. The most common type of mass spectrometer (MS) associated with a gas chromatograph (GC) is the quadrupole mass spectrometer, sometimes referred to by the Hewlett-Packard (now Agilent) trade name "Mass Selective Detector" (MSD). Another relatively common detector is the ion trap mass spectrometer. Additionally one may find a magnetic sector mass spectrometer, however these particular instruments are expensive and bulky and not typically found in high- throughput service laboratories. Other detectors may be encountered such as time of flight (TOF) and tandem quadrupoles (MS-MS). After the molecules travel the length of the column they pass through the transfer line and enter into the mass spectrometer where they are ionized by various methods with typically only one method being used at any given time. Once the sample is fragmented it will then be detected, usually by an electron multiplier diode, which essentially turns the ionized mass fragment into an electrical signal which is then detected. A mass spectrometer is typically utilized in one of two ways: full scan or selective ion monitoring (SIM). The typical GC-MS instrument is capable of performing both functions either individually or concomitantly, depending on the setup of the particular instrument. 20

The primary goal of GC-MS is to identify and quantify an amount of substance. This is done by comparing the relative concentrations among the atomic masses in the generated spectrum. Two kinds of analysis are possible, comparative and original. Comparative analysis essentially compares the given spectrum to a spectrum library to see if its characteristics are present for some sample in the library. This is best performed by a computer because there are a myriad of visual distortions that can take place due to variations in scale. Computers can also simultaneously correlate more data (such as the retention times identified by GC), to more accurately related data (20). Another method of analysis measures the peaks in relation to one another. In this method, the tallest peak is assigned 100% of the value, and the other peaks being assigned proportionate values. All values above 3% are assigned. The total mass of the unknown compound is normally indicated by the parent peak. The value of this parent peak can be used to fit with a chemical formula containing the various elements which are believed to be in the compound. The isotope pattern in the spectrum, which is unique for elements that have many natural isotopes, can also be used to identify the various elements present. Once a chemical formula has been matched to the spectrum, the molecular structure and bonding can be identified, and must be consistent with the characteristics recorded by GC-MS. Typically; this identification is done automatically by programs which come with the instrument, given a list of the elements which could be present in the sample. A “full spectrum” analysis considers all the “peaks” within a spectrum. Conversely, selective ion monitoring (SIM) only monitors selected ions associated with a specific substance. This is done on the assumption that at a given retention time, a set of ions is characteristic of a certain compound. This is a fast and efficient analysis, especially if the analyst has previous information about a sample or is only looking for a few specific substances. When the amount of information collected about the ions in a given gas chromatographic peak decreases, the sensitivity of the analysis increases. So, SIM analysis allows for a smaller quantity of a compound to be detected and measured, but the degree of certainty about the identity of that compound is reduced. GC-MS is used for the analysis of unknown organic compound mixtures. One critical use of this technology is the use of GC-MS to determine the composition of bio-oils processed from raw biomass. GC-MS is becoming the tool of choice for tracking organic pollutants in the environment. The cost of GC-MS equipment has decreased significantly, and the reliability has increased at the same time, which has contributed to its increased adoption in environmental studies. GC-MS is the main tool used in sports anti-doping laboratories to test athletes' urine samples for prohibited performance-enhancing drugs, for example anabolic steroids (21). 1-5-Solvent Extraction Solvent extraction, also known as Liquid–liquid extraction or partitioning, is a method to separate a compound based on the solubility of its parts. This is done using two liquids that don't mix, for example water and an organic solvent. Solvent extraction is used in the processing of perfumes, vegetable oil, or biodiesel. It is also used to recover plutonium from irradiated nuclear fuel, a process which is usually called nuclear reprocessing. The recovered plutonium can then be re-used as nuclear fuel. In this process one of the components of a mixture dissolves in a particular liquid and the other component is separated as a residue by filtration. Solvent extraction involves crushing of oil seeds and oil seed cakes. From ancient time, vegetable oils were obtained by crushing oil seeds in village ghanis / kolhus / chekkus in India. At the beginning of the 20th century the vegetable oils industry was based on some 500, 00 bullock-driven ghanis producing about 800,000 tons of

oils. Slowly, in addition to these ghanis, power-driven ghanis (rotary ghanis made indigenously) imported expeller and imported hydraulic press plants started crushing oilseeds. Around this time many European countries and United States of America had established huge solvent extraction plants for recovering directly almost all the available oil in the oilseeds like Cottonseed and Soybean (22). There is a net transfer of one or more species from one liquid into another liquid phase, generally from aqueous to organic. The transfer is driven by chemical potential, i.e. once the transfer is complete, the overall system of protons and electrons that make up the solutes and the solvents are in a more stable configuration (lower free energy). The solvent that is enriched in solute(s) is called extract. The feed solution that is depleted in solute(s) is called the raffinate. LLE is a basic technique in chemical laboratories, where it is performed using a variety of apparatus, from separatory funnels to countercurrent distribution equipment called as mixer settlers. This type of process is commonly performed after a chemical reaction as part of the work-up, often including an acidic work-up (23). The term partitioning is commonly used to refer to the underlying chemical and physical processes involved in liquid–liquid extraction, but on another reading may be fully synonymous with it. The term solvent extraction can also refer to the separation of a substance from a mixture by preferentially dissolving that substance in a suitable solvent. In that case, a soluble compound is separated from an insoluble compound or a complex matrix (24). Solvent extraction is exclusively used in separation and purification of uranium and plutonium, zirconium and hafnium, separation of cobalt and nickel, separation and purification of rare earth elements etc., its greatest advantage being its ability to selectively separate out even very similar metals. One obtains high-purity single metal streams on 'stripping' out the metal value from the 'loaded' organic wherein

one can precipitate or deposit the metal value. Stripping is the opposite of extraction: Transfer of mass from organic to aqueous phase (25). In solvent extraction, a distribution ratio is often quoted as a measure of how well- extracted a species is. The distribution ratio is equal to the concentration of a solute in the organic phase divided by its concentration in the aqueous phase. Depending on the system, the distribution ratio can be a function of temperature, the concentration of chemical species in the system, and a large number of other parameters. Sometimes, the distribution ratio is referred to as the partition coefficient, which is often expressed as the logarithm. Note that a distribution ratio for uranium and neptunium between two inorganic solids (zirconolite and perovskite) has been reported. In solvent extraction, two immiscible liquids are shaken together. The more polar solutes dissolve preferentially in the more polar solvent, and the less polar solutes in the less polar solvent. Although the distribution ratio and partition coefficient are often used synonymously, they are not necessarily so. Solutes may exist in more than one form in any particular phase, which would mean that the partition coefficient (Kd) and distribution ratio (D) will have different values. This is an important distinction to make as whilst the partition coefficient has a fixed value for the partitioning of a solute between two phases, the distribution ratio changes with differing conditions in the solvent. After performing liquid–liquid extraction, a quantitative measure must be taken to determine the ratio of the solution’s total concentration in each phase of the extraction. This quantitative measure is known as the distribution ratio or distribution coefficient (26).

2-Materials and Methods

2-1-Materials 2-1-1- Plant material Seeds of Khaya senegalensis ,Eruca sativa and Pisum sativum were collected from Khartoum state (Sudan) and authenticated by the Department of Phytochemistry and Taxonomy, Institute of Medicinal and Aromatic Plants , Khartoum – Sudan .Natural region center ,Heaborium specimen were deposited. 2-1-2- Instruments - GC-MS analysis was conducted on a Shimadzo GC-MS –QP2010 Ultra instrument with a RTX-5MS column (30 m , length ;0.25mm diameter ; 0.25μm, thickness) . - UV-visible light spectrometer. 2-1-3- Test organisms The standard microorganisms shown in table (2.1)

Table 2.1: Test microorganisms

No. Microorganism Type source

1 Bacillus subtilis G +ve NCTC8236 2 Staphylococcus aureus G +ve ATCC 25923 3 Pseudomonas aeruginosa G –ve NCTC 6750 4 Escherichia coli G –ve ATCC 9736 5 Candida albicans Fungi NCTC 10716  NCTC .national collection of type culture,Colindale.England.  ATCC.American type culture collection,Maryland,USA.

2-2- Methods 2.2.1-Extraction of oil Dry powdered plant material (300g) was exhaustively extracted with n-hexane at room temperature for 72h.The solvent was removed under reduced pressure and the oil was kept in the fridge at 4oC for further manipulation. The oil (2ml) was placed in a test tube and 7ml of alcoholic sodium hydroxide were added followed by 7ml of alcoholic sulphuric acid. The tube was stoppered and shaken vigorously for five minutes and then left overnight.(2ml) of supersaturated sodium chloride were added, then (2ml) of normal hexane were added and the tube was vigorously shaken for five minutes. The hexane layer was then separated.(5μl) of the hexane extract were mixed with 5ml diethyl ether . The solution was filtered and the filtrate (1μl) was injected in the GC-MS vial (27). 2-2-2-Physiochemical properties of oils Specific gravity: Specific gravity was determined according to A.O.A.C (2000) (28). Refractive index: The refractive index of the oil was determined by (AOAC, 1990) (29). Determination of color: Colour was determined according to Hand book of Food Analysis (30). Viscosity: Viscosity was determined according to Diamante and Lan (31). Acid value: Acid value was determined according to Handbook of Food Analysis (32). Saponification value: Saponification value were determined according to ISO 3657: 2002 (33). Un saponification value: Un saponification matters were determined according to (34) British Standard . 26

Peroxide value: Peroxide value was determined according to ISO 3960: 2007 (35). Iodine value: Iodine value was determined according to ISO 3961:1996 (36).

2.2.3- GC-MS analysis The target oils were analyzed by gas chromatography – mass spectrometry. A Shimadzo GC-MS-QP2010 Ultra instrument with a RTX-5MS column (30m length ; 0.25mm diameter ; 0.25 μm, thickness) was used. Helium (purity; 99.99 %) was used as carrier gas. Oven temperature program is given in Table 2.2, while other chromatographic conditions are depicted in Table 2.3.

Table 2.2: Oven temperature program Rate Temperature(oC) Hold Time (min.-1)

- 150.0 1.00 4.00 300.0 0.00

Table 2.3: Chromatographic conditions

Column oven temperature 150.0oC Injection temperature 300.0oC Injection mode Split Flow control mode Linear velocity Pressure 139.3KPa Total flow 50.0ml/ min Column flow 1.54ml/sec. Linear velocity 47.2cm/sec. Purge flow 3.0ml/min. Spilt ratio - 1.0

2.2.4-Antimicrobial test 2.2.4.1-Preparation of bacterial suspensions One ml aliquots of 24 hours broth culture of the test organisms was aseptically distributed onto nutrient agar slopes and incubated at 37°C for 24 hours. The bacterial growth was harvested and washed off with sterile normal saline, and finally suspended in 100 ml of normal saline to produce a suspension containing about 108-109colony forming units per ml. The suspension was stored in the refrigerator at 4°C until used. The average number of viable organism per ml of the stock suspension was determined by means of the surface viable counting technique. Serial dilutions of the stock suspension were made in sterile normal saline in tubes and one drop volumes (0.02 ml) of the appropriate dilutions were transferred by adjustable volume micropipette onto the surface of dried nutrient agar plates. The plates were allowed to stand for two hours at room temperature for the drop to dry, and then incubated at 37°C for 24 hours (37). 2.2.4.2-Preparation of fungal suspensions Fungal cultures were maintained on sabouraud dextrose agar incubated at 25°C for four days. The fungal growth was harvested and washed with sterile normal saline, and the suspension was stored in the refrigerator until used (38). 2.2.4.3-Testing for antibacterial activity The cup-plate agar diffusion method was adopted with some minor modifications, to assess the antibacterial activity of the oil. (2ml) of the standardized bacterial stock suspension were mixed with 200 ml of sterile molten nutrient agar which was maintained at 45°C in a water bath. (20 ml) Aliquots of the incubated nutrient agar

were distributed into sterile Petri dishes, the agar was left to settle and in each of these plates which were divided into two halves, two cups in each half (10 mm in diameter) were cut using sterile cork borer (No 4), each one of the halves was designed for one of the compounds. Separate Petri dishes were designed for standard antibacterial chemotherapeutic, (ampicillin and gentamycin). The agar discs were removed, alternate cup were filled with 0.1 ml samples of each compound using adjustable volume micrometer pipette and allowed to diffuse at room temperature for two hours. The plates were then incubated in the upright position at 37°C for 24 hours. The above procedure was repeated for different concentrations of the test compounds and the standard antibacterial chemotherapeutics. After incubation, the diameters of the resultant growth inhibition zones were measured in triplicates and averaged (39). 2.2.4.4- DPPH radical scavenging assay 4.3 mg of DPPH (1, 1-Diphenyl –2-picrylhydrazyl) was dissolved in 3.3 ml methanol; it was protected from light by covering the test tubes with aluminum foil. 150 µl DPPH solutions was added to 3ml methanol and absorbance was taken immediately at 517nm for control reading. 50 µl of various concentrations of Propyl Gallate compound as well as standard compound were taken and the volume was made uniformly to150 µl using methanol. Each of the samples was then further diluted with methanol up to 3ml and to each 150 µl DPPH was added. Absorbance was taken after 15 min. at 517nm using methanol as blank on UV- visible spectrometer. The percentage scavenging values for each drug compounds as well as standard preparation were calculated. The DPPH free radical scavenging activity was calculated using the following formula: % scavenging = [Absorbance of control - Absorbance of test sample/Absorbance (40) of control] X 100 . 29

3- Results 3.1-Physiochemical properties of oils The physical properties of the seed oil including color, iodine, saponification, acid, peroxide values, refractive index, density, Refractive viscosity, and unsaponifiable matter.

3.1.1- Physiochemical properties of Khaya senegalensis oils Table 3.1. Physiochemical properties of seed oil of Khaya senegalensis Properties of Oil Value Colour Golden yellow Refractive index (210C) 1.458 ± 0.001 Relative density 0.053 ± 0.002 Iodine value (g/100g) 88.40 ± 0.058 Saponification value (mg KOH) 195.58 ± 0.044 Acid value (mg KOH) 2.69 ± 0.015 Unsaponifiable matter (%) 1.50 % Peroxide value 12% Refractive viscosity 32cp

3-1-2- Physiochemical properties of Eruca sativa oil Table 3.2. Physiochemical properties of seed oil of Eruca sativa oil Properties of Oil Value Colour (red 5.4–yellow 3.1–blue 0.4) Refractive index (21 0C) 1.469±0.0738 Relative density 0.9077±0.0632 Iodine value (g/100g) 63.63±0.076 Saponification value (mg KOH) 165.495±1.96 Acid value (mg KOH) 2.24±0.056 Unsaponifiable matter (%) 2.648%

Peroxide value 10% Refractive viscosity 38cp

3.1.3- Physiochemical properties of Pisum sativum oils Table 3.3. Physiochemical properties of seed oil of Pisum sativum oil Properties of Oil Value Colour Green or yellow Refractive index (21 0C) 1.465±0.067 Relative density 0.794±0.0476 Iodine value (g/100g) 185.30±1.53 Saponification value (mg KOH) 198.05±1.68 Acid value (mg KOH) 4.5± 0.265 Unsaponifiable matter (%) 0.53% Peroxide value 15%

Refractive viscosity 35cp

3.2- GC-MS analysis of oils The oils of Khaya senegalensis, Eruca sativa and Pisum sativum were analyzed by GC-MS and furthermore evaluated for their antimicrobial activity. 3-2-1-GC-MS analysis of Khaya senegalensis oil GC-MS analysis of Khaya senegalensis oil was carried out. Identification of the constituents was based on the MS library (NIST) .Also the observed fragmentation pattern was discussed. 23 constituents were detected by GC-MS analysis. The typical total ion chromatogram (TIC) is displayed Fig (1)- and Table 3.4.

Fig 3.1: Total ion chromatograms

Table 3.4:Constituents of Khaya senegalensis oil peak R.Time Area Area % Name 1 10.982 84935 0.03 1,3-cyclohexadiene,5-(1,5-dimethyl) 2 11.086 26025 0.01 .alpha-Farnesene 3 11.149 21233 0.01 .beta-Bisabolene 4 11.250 40610 0.01 Dodecanoic acid, methyl ester 5 11.351 88180 0.03 Cyclohexane,3-(1,5-dimethyl-4-hexenyl 6 13.563 391421 0.14 Methyl tetradecanoate 7 15.425 185135 0.07 9-Hexadecenoic acid,methyl ester (Z) 8 15.469 976477 0.35 7-Hexadecenoic acid,methyl ester (Z) 9 15.671 39910747 14.19 Hexadeconic acid,methyl ester 10 16.025 1392405 0.50 n-Hexadecanoic acid 11 16.371 175501 0.06 Hexadecanoic acid,14-methyl-,methyl 12 16.432 344537 0.12 Cis-10-Heptadecenoic acid , methyl est 13 16.640 983761 0.35 Heptadecenoic acid , methyl ester 14 17.326 23236280 8.26 9,12-Octadecenoic acid (Z,Z) methylest 15 17.413 1333372701 47.43 9-Octadecaoicacid (Z),methyl ester 16 17.598 53042557 18.86 Methyl stearate 17 17.740 3376633 1.20 Oleic Acid 18 19.132 1324280 0.47 11-Eicosanoic acid ,methyl ester 19 19.333 9693446 3.45 Eicosanoic acid , methyl ester 20 20.591 5827462 2.07 9-Octadecanoic acid,1,2,3-propanetely 21 20.951 3166935 1.13 Docosanoic acid ,methyl ester 22 21.092 1345536 0.48 Cholest-5-en-3-ol(3,beta),carbonyl 23 22.455 2176128 0.77 Tetracosanoic acid ,methyl ester 281182925 100.00 33

Main constituents of the oil are discussed below: 9-Octadecenoic acid (Z) - methyl ester (47.43%) The mass spectrum of 9-octadecenoic acid (Z) - methyl ester is shown in Fig 3.2. The peak at m/z296, which appeared at R.T. 17.415 in total ion chromatogram, + + corresponds M { C19H36O2} . The peak at m/z266 corresponds to loss of methoxyl function.

Fig 3.2: mass spectrum of 9-octadecenoic acid (Z)- methyl ester Methyl Stearate (18.86%) The mass spectrum of methyl stearate is shown in Fig 3.3. The peak at m/z 298, which appeared at R.T.17.595 in total ion chromatogram, corresponds M + + {C19H38O2} . The peak at m/z 267 correspond to loss of a methoxyl function.

Fig 3.3: mass spectrum of methyl stearate Hexadecanoic acid, methylester (14.19%) The mass spectrum of hexadecanoic acid, methyl ester is shown in Fig 3.4. The peak at m/z 270, which appeared at R.T. 15.670 in total ion chromatogram + + .corresponds M { C17H34O2} . The peak at m/z 239 corresponds to loss of methoxyl function.

Fig 3.4: mass spectrum of hexadecanoic acid, methyl ester 9,12-Octadecadienoic acid (Z,Z)-methyl ester (8.26%) The mass spectrum of 9, 12-octadecadienoic acid (Z, Z)-methyl ester is shown in Fig 3.5. The peak at m/z294, which appeared at R.T. 17.325 in total ion + + chromatogram, corresponds M { C19H34O2} . The peaks at m/z 263 correspond to loss of methoxyl function.

Fig 3.5: mass spectrum of 9,12-octadecadienoic acid (Z,Z)-methyl ester Eicosanoic acid, methyl ester (3.45%) The mass spectrum of eicosanoic acid, methyl ester is shown in Fig 3.6. The peak at m/z 326 which appeared at R.T. 19.335 in total ion chromatogram, corresponds + + M { C21H42O2 } . The peak at m/z 295 corresponds to loss of methoxyl function.

Fig 3.6: mass spectrum of eicosanoic acid, methyl ester

3.2.2- GC-MS analysis of Eruca sativa oil GC-MS analysis of Eruca sativa oil was carried out. Thirty constituents were detected by GC-MS analysis. The typical total ion chromatogram (TIC) is displayed Fig 3.7 - see also Table 3.5. 36

Fig3.7: Total ion chromatograms of Eruca sativa Table 3.5: Constituents of Eruca sativa oil peak R.Time Area Area % Name 1 11.257 132787 0.02 Dodecanoic acid ,methyl ester 2 13.564 1658575 0.30 Methyl tetradecenoate 3 14.372 116630 0.02 5-Octadecenoic acid,methyl ester 4 14.472 103617 0.02 Cis -5-Dodecenoic acid ,methyl ester 5 14.637 426459 0.08 Pentadecenoic acid,methyl ester 6 15.366 796456 0.14 7,10-Hexadecenoic acid ,methylester 7 15.431 1349066 0.24 7-Hexadecenoc acid,methyl ester(Z) 8 15.471 3724465 0.67 9-Hexadecenoic acid ,methyl ester (Z) 9 15.564 442463 0.08 Trans-13-Octadecenoic acid,methyl es 10 15.672 41419220 7.47 Hexadecanoic acid,methyl ester 11 16.434 877232 0.16 Cis-10-Heptadecenoic acid , methyl est 12 16.640 796120 0.14 Heptadecenoic acid ,methyl ester 13 17.337 67363418 12.16 9,12-Octaecadecadienoic acid (Z,Z)met 14 17.412 40702089 7.34 9-Octadecenoic acid , methyl ester 15 17.430 30004137 5.41 9.12,15-Octadecatrienoic acid,methyl

16 17.583 18302069 3.30 Methyl stearate 17 18.288 429294 0.08 Cis -10-Nonadecenoic acid, methyl este 18 18.472 209651 0.04 Nonadecenoic acid , methyl ester 19 18.988 363736 0.66 Gamma-Linolenic acid , methyl ester 20 19.159 75674626 13.66 Cis -13- Eicosenoic acid ,, methyl ester 21 19.198 17402194 3.14 Cis -11-Eicosenoic acid ,methyl ester 22 19.334 14773965 2.67 Eicosanic acid ,methyl ester 23 19.979 3352508 0.60 11-Octadecenoic acid ,methyl ester 24 20.158 450308 0.08 Heneicosanoic acid,methyl ester 25 20.844 176903758 31.92 13-Docosnoic acid,methyl ester 26 20.961 16865354 3.04 Docosanoic acid, methyl ester 27 21.553 2373074 0.43 Cyclopropaneoctanoic acid,2-octyl,met 28 21.717 1021723 0.18 Tricosanoic acid, methyl ester 29 22.305 23804564 4.30 15-tetracosenoic acid , methyl ester 30 22.455 9065888 1.64 Tetracosanoic acid , methyl ester 554178746 100.00

Main Constituents of Eruca sativa oil is discussed below: 13-Docoenoic acid, methyl ester (31.92%) The mass spectrum of 13-docoenoic acid, methyl ester is shown in Fig 3.8. The peak at m/z 352 which appeared at R.T. 20.854 in total ion chromatogram, + + corresponds M {C23H44O} . The peak at m/z 322 corresponds to loss of methoxyl function.

Fig 3.8: mass spectrum of 13-docoenoic acid , methyl ester

Cis -13-eicosenoic acid, methyl ester (13.66%) The mass spectrum of cis -13-eicosenoic acid, methyl ester is shown in Fig3.9. The peak at m/z 324 which appeared at R.T. 19.160 in total ion chromatogram, + + corresponds M { C21H40O2 } . The peak at m/z 294 corresponds to loss of methoxyl function.

Fig 3.9: mass spectrum of cis -13-eicosenoic acid, methyl ester

9,12-Octadecadienoic acid (Z,Z) – methyl ester (12.16%) The mass spectrum 9, 12-octadecadienoic acid (Z, Z) – methyl ester is shown in Fig3.10. The peak at m/z 294 which appeared at R.T. 17.355 in total ion + + chromatogram, corresponds M { C19H34O2 } . The peak at m/z 263 corresponds to loss of methoxyl function.

Fig 3.10: mass spectrum 9,12-octadecadienoic acid (Z,Z) – methyl ester

Hexadecanoic acid, methyl ester (7.47%) The mass spectrum of hexadecanoic acid methyl ester is shown in Fig 3.11. The peak at m/z 270 which appeared in R.T. 15.670 in total ion chromatogram, + + corresponds M { C17H34O2 } . The peak at m/z 239 corresponds to loss of methoxyl function.

Fig 3.11: mass spectrum of hexadecanoic acid methyl ester 9-Octadecenoicacid (Z) – methyl ester (7.34%) The mass spectrum of 9-octadecenoicacid (Z) – methyl ester is shown in Fig 3.12. The peak at m/z 296 which appeared at R.T. 17.410 in total ion chromatogram, + + corresponds M { C19H36O2 } . The peak at m/z 264 corresponds to loss of methoxyl function.

Fig 3.12 :mass spectrum of 9-octadecenoicacid (Z) – methyl ester 9, 12,15-Octadecatrienoic acid , methyl ester (5.41%) The mass spectrum of 9, 12, 15-octadecatrienoic acid, methyl ester is shown in Fig 3.13. The peak at m/z 292 which appeared at R.T .17.430 in total ion + + chromatogram, corresponds M { C19H32O2 } . The peak at m/z 261 corresponds to loss of methoxyl function.

Fig 3.13: mass spectrum of 9, 12, 15-octadecatrienoic acid, methyl ester 15-Tetracosenoic acid, methyl ester (4.30%) The mass spectrum 15-tetracosenoic acid, methyl ester is shown in Fig 3.14. The peak at m/z 380 which appeared at R.T. 22.305 in total ion chromatogram, + + corresponds M { C25H48O2 } . The peak at m/z 350 corresponds to loss of methoxyl function .

Fig 3.14: Mass spectrum 15-tetracosenoic acid, methyl ester Methyl Stearate (3.30%) The mass spectrum of methyl Stearate is shown in Fig 3.15. The peak at m/z 298 which appeared at R.T. 17.585 in total ion chromatogram, corresponds + + M { C19H38O2 } . The peak at m/z 267 corresponds to loss of methoxyl function.

Fig 3.15: mass spectrum of methyl Stearate

Cis -11-eicosenoic acid, methyl ester (3.14%) The mass spectrum of cis -11-eicosenoic acid, methyl ester is shown in Fig 3.16. The peak at m/z 324 which appeared in R.T.19.200 in total ion chromatogram, + + corresponds M { C21H40O2 } . The peak at m/z 292 corresponds to loss of methoxyl function.

Fig 3.16: mass spectrum of cis -11-eicosenoic acid, methyl ester

Docosanoic acid, methyl ester (3.04%) The mass spectrum of docosanoic acid, methyl ester is shown in Fig 3.17. The peak at m/z 354 which appeared at R.T. 20.960 in total ion chromatogram, + + corresponds M { C23H46O2 } . The peak at m/z 323 corresponds to loss of methoxyl function

Fig 3.17: mass spectrum of docosanoic acid, methyl ester Eicosanoic acid, methyl ester (2.67%) The mass spectrum of eicosanoic acid, methyl ester is shown in Fig 3.18. The peak at m/z 326 which appeared at R.T. 19.335 in total ion chromatogram, corresponds + + M { C21H42O2 } . The peak at m/z 295 corresponds to loss of methoxyl function.

Fig 3.18: mass spectrum of eicosanoic acid, methyl ester 44

3.2.3- GC-MS analysis of Pisum sativum oil Pisum sativum oil was analyzed by GC-MS which revealed the presence of 33 constituents. The typical total ion chromatogram (TIC) is displayed Fig (3.19). Constituents of the oil are shown in Table 3.6.

Fig 3.19: Total ion chromatograms of Pisum sativum oil

Table 3.6: Constituents of Pisum sativum oil peak R.Time Area Area % Name 1 3.412 79087 0.05 Alph.phellandrene 2 3.973 112489 0.06 Bicycle(3.1.0)hexane,4-methylene 3 4.511 39204 0.02 Cyclohexane,1-methyl-4-(1-methy 4 5.067 58167 0.03 Gamma-Terpinene 5 5.592 120151 0.07 Cis-4-methoxy thujane 6 5.918 93299 0.05 Bicycle(4.1.0)heptan-3-ol,4,7,7-tri 7 6.771 19388 0.01 Terpinen-4-ol 8 8.661 38768 0.02 Undecanoic acid ,2-methyl 9 9.789 347117 0.20 Methyleugenol 10 10.107 223403 0.13 Caryophyllene 11 11.220 1033280 0.59 Dodecanoic acid ,methyl ester 12 11.341 548072 0.31 1,3-benzodioxole ,4-methoxy -6- 13 11.674 10985007 0.63 Asarone 14 13.541 23760215 13.62 Methyl tetradecanoate 15 14.609 395609 0.23 Pentadecanoic acid ,methyl ester 16 15.402 87005 0.05 7-Hexadecanoic acid,methyl ester 17 15.442 212209 0.12 9-Hexadecanoic acid ,methyl ester 18 15.641 22919345 13.13 Hexadecanoic aid methyl ester 19 16.613 425659 0.24 Heptadecanoic acid methyl ester 20 17.306 53558482 30.69 9,12-octadecanoic acid (Z,Z)methy 21 17.354 47845620 27.42 9-octadecanoic acid ,methyl ester 22 17.551 8852668 5.07 Methyl stearate 23 18.909 4037685 2.31 Tricycle(20.8.0.0(7.16)triaconatane 24 18.953 1065128 0.61 9,12,15-octadecatrienoic acid ,meth 25 19.106 1261062 0.72 Cis -11-Eicosanoic acid,methyl es

26 19.305 1564178 0.90 Eicosanoic acid,methyl ester 27 20.748 294568 0.17 13-Docosenoic acid ,methyl ester 28 20.924 503141 0.29 Docsanoic acid ,methyl ester 29 21.690 167836 0.10 Tricosanoic acid ,methyl ester 30 21.894 1821984 1.04 Gamma-sitrosterol 31 22.427 528775 0.30 Tetracosanoic acid ,methyl ester 32 22.524 632307 0.36 Thiazole (3,2-a)benzimidazole 33 24.871 765409 0.44 Gamma-tocopherol 174509901 100.00

Some major constituents of Pisum sativum oil are discussed below: 9,12-Octadecadienoic acid (Z,Z) – methyl ester (30.69%) The mass spectrum of 9,12-octadecadienoic acid (Z,Z) methyl ester is shown in Fig 3.20. The peak at m/z 294 which appeared at R.T.17.305 in total ion + + chromatogram, corresponds M {C19H34O2 } . The peak at m/z 263 corresponds to loss of methoxyl function.

Fig 3.20: mass spectrum of 9,12-octadecadienoic acid (Z,Z) methyl ester

9-Octadecenoic acid (Z)-methyl ester (27.42%) 47

The mass spectrum of 9-octadecenoic acid (Z)-methyl ester is shown in Fig 3.21. The peak at m/z 296 which appeared at R.T. 17.355 in total ion chromatogram, + + corresponds M { C19H36O2 } . The peak at m/z 266 corresponds to loss of methoxyl function.

Fig 3.21: mass spectrum of 9-octadecenoic acid (Z)-methyl ester Methyl tetradecanoate (13.62%) The mass spectrum of methyl tetradecanoate is shown in Fig 3.22. The peak at m/z 242 which appeared at R.T. 13.540 in total ion chromatogram, corresponds M + { + C15H30O2 } . The peak at m/z 211 corresponds to loss of methoxyl function.

Fig 3.22: mass spectrum of methyl tetradecanoate 48

Hexadecanoic acid, methyl ester (13.13%) The mass spectrum of hexadecanoic acid, methyl ester is shown in Fig 3.23. The peak at m/z 270 which appeared at R.T. 15.640 in total ion chromatogram, + + corresponds M { C17H34O2} . The peak at m/z 239 corresponds to loss of methoxyl function.

Fig 3.23: mass spectrum of hexadecanoic acid, methyl ester Methyl Stearate (5.07%) The mass spectrum of methyl stearate is shown in Fig 3.24. The peak at m/z 298 which appeared at R.T. 17.550 in total ion chromatogram, corresponds M + { + C19H38O2 } . The peak at m/z 267 corresponds to loss of methoxyl function.

Fig3.24: Mass spectrum of methyl stearate 3.3-Antimicrobial activity of oils Khaya senegalensis ,Eruca sativa and Pisum sativum oils were assessed for antimicrobial activity against five standard human pathogens. The diameters of the growth of inhibition zones are shown in table 3.7. Results were interpreted as follows :(< 9mm: inactive; 9-12 mm : partially active ; 13- 18 mm : active ;>18mm : very active ).

Table 3.7: Antimicrobial activity of oils

Oils Conc. (mg/ml) Ec Pa Sa Bs Ca

Khaya senegalensis 100 - - 15 - -

Eruca sativa 100 20 - 20 - -

Pisum sativum 100 14 20 24 20 20

Ec: Escherichia coli Pa: Pseudomonas aeruginosa Sa: Staphylococcus aureus Bs: Bacillus Subtilis Ca: Candida albicans

Khaya senegalensis oil showed significant activity against Staphylococcus aureus but it was inactive against other pathogens. Eruca sativa showed highly significant activity against Escherichia coli and Staphylococcus aureus. Pisum sativum showed excellent activity against all tested organisms and good activity against Escherichia coli.

3.4-Antioxidants activity Khaya senegalensis ,Eruca sativa and Pisum sativum oil was assessed for antioxidant activity. The DPPH radical scavenging assay, the oil showed significant antioxidant activity (Table 3.8).

Table 3.8: Antioxidant activity of oils

No. Sample %RSA ±SD (DPPH) standard Propyl Gallate 93± 0.01

1 Khaya senegalensis 90±0.08 2 Eruca sativa 37±0.8 3 Pisum sativum 82± 0.09

4- Discussion Khaya senegalensis, Eruca sativa and Pisum sativum are a group of plants used in traditional municipal treatment in a group of African countries , it belong to a member of the family Meliaceae, Brassicaceae, and Fabaceae consecutively. These plants are contain a number of antimicrobials and antioxidants; this assumption was the basis of the theory on which this research was based. Oils from the seeds were extracted, analyzed to determine physical properties of oils and screened by GC-MS and then evaluated for antimicrobial and antioxidant activity. In the cup plate agar diffusion bioassay Khaya senegalensis oil showed significant activity against Staphylococcus aureus (15 mm zone of inhibition) but it was inactive against other test pathogens. The activity against Staphylococcus aureus assumed due to presence of 9- octadecenoic acid (Z) – methyl ester which is major component (47.43%) among the other 23 constituents. In 2007 a study In Phytochemical screening and antimicrobial activity of the ethanolic and methanolic extracts of the leaf and bark of Khaya senegalensis was done in Nasarawa State University, Nigeria which showed similar result against Staphylococcus. aureus (15 , 20 mm zone of inhibition for the leaf and bark consecutively) and an antimicrobial susceptibility test against Staphylococcus feacalis and Candida albicans were susceptible to both the leaf and bark extracts, while Escherichia. coli was not. The extracts were also found to be bactericidal to Staphylococcus. aureus and Staphylococcus feacalis, and fungicidal to Candida. albicans. Results of the phytochemical screening showed that saponins, tannins, alkaloids, glycosides, steriods, terpenoids and flavonoids were the active

compounds present in the leaves and bark of the plant and the activity of leaves and bark of the plant due to the presence of this compounds in plant (40). Eruca sativa showed highly significant activity against Escherichia coli (20 mm zone of inhibition) and Staphylococcus aureus (20 mm zone of inhibition). This activity against Escherichia coli and Staphylococcus aureus assumed due to the presence of 13-Docoenoic acid, methyl ester which is the major component (31.92%) among other 30 constituents. In comparison to a study in phytochemical analysis and antibacterial activity of Eruca sativa seed done in Arid Agriculture University , Pakistan , in 2011, This study showed that a maximum zone of inhibition was observed from seed oil followed by methanolic seed extracts for Gram+ve and Gram-ve bacterial strains compared with broad spectrum antibiotics gentamicine MIC values of seed oil were within the ranges of 52-72 µg/ml as compared to 56-70 µg/ml standard antibiotic (Gentamicine), the zone of inhibition for Escherichia coli and Staphylococcus aureus are 28mm, 24mm consecutively. Analysis of seed oil by gas chromatography revealed that there was high concentration of Erucic acid (51.2%) followed by oleic acid (15.1%) and cis-11- eicosenoic acid (12.5%). In addition, minor quantities of other essential and non- essential fatty acids were also present (41). Pisum sativum showed highly significant activity against P.aeruginosa(20mm) , S.aurous (24mm) , B.Subtilis (20mm) , C. albicans (20mm) and significant activity against Escherichia coli (14mm). Previous study in 2005 was done in University of Karachi, Pakistan about the study of antibacterial activity of P. sativum leave Which exhibited significant antibacterial activity against Gram+ve and Gram-ve tested organisms with average zone of inhibition about 16 mm for both (42). 53

In the DPPH radical scavenging assay, Pisum sativum oil showed significant free radical scavenging capacity ( 82± 0.09). Pisum sativum oil revealed the presence of 33 constituents dominated 9,12- octadecadienoic acid ( Z,Z)-,methyl ester( 30.69%) . A research achieved by Haymanti Saha et al in india (2014) about Evaluation of antioxidant activity of Pisum sativum (pod and grain) and detection of its bioactive compounds by GC -MS analysis which showed that Pisum sativum contained ascorbic acid and butoxy acetic acid, edoheptulosan , Sucrose and Fructose 1, 3, 6-trideoxy, 3, 6-epitio , Lactose , Hexanoic acid, 5,oxo-trimethyl silyl ester and Mannosamine and 2-thiozolamine,4,5-di hydro and Goitrin , Inositol and Mannitol 1-thio heptyl -1-deoxy and Methionine which are responsible for the antioxidant value of pod and grain of Pisum sativum(43) .

5. Conclusion and Recommendations 5.1. Conclusion Khaya senegalensis oil showed significant activity against Gram +ve bacteria (Staphylococcus aureus) but it was inactive against Gram –ve bacteria. This activity is due to the presence of 9-octadecenoic acid (Z) – methyl ester (47.43%) among other 23 constituents. Eruca sativa showed highly significant activity against Gram -ve bacteria(Escherichia coli) and Gram +ve bacteria (Staphylococcus aureus) .This activity is due to the presence of 13-docoenoic acid, methyl ester (31.92%) among other 30 constituents . Pisum sativum showed highly significant activity against all test organisms (Gram +ve and Gram –ve bacteria) . In the DPPH radical scavenging assay, Khaya senegalennsis , Eruca sativa Pisum sativum oil showed significant free radical scavenging capacity .

5.2. Recommendation

Further studies may include:. - Screening the extracted oils for other biological activities (antiviral, antimalarial, anti-inflammatory, antileishmenial ……. Activities ) - Isolation and characterization of other phytochemicals in the target plants.

References

1. World Agroforestry Centre. Scientific classification of Khaya senegalensis. http://www.worldagroforestry.org/.published April 1990. Accessed April 2018.

2. Ishaq. F, Zezi. A, Olurishe. T. Avicenna Journal of Phytomedicine. 4(6):377- 84 (2014).

3. World Agroforestry Centre. Khaya senegalensis. http://www.worldagroforestry.org/. published April 7 1990. Accessed May 3 2018.

4. Makut .S, Gyar.D, Pennap .G . African Journal of Biotechnology,7(2) 9 (2008) .

5. USDA. Plants Profile of Eruca sativa. https://www.USDA.org\ Eruca sativa. Published January 4 2018. Accessed April 3 2018.

6. Gulefraz M, Sidig. S, Tarig. H .Phytochemical analysis and antibacterial activity of Eruca sativa seed . Dissertations thesis. Arid Agriculture University, Pakistan ;2011.

7. Yoshinari B, Iwakuma M, Nagami H. African Journal of Health Benefit.; 41(23): 5835–5841 (2002).

8. World Agroforestry Centre. Scientific classification of Pisum sativum. https://www. World agroforestry.org/pisum sativum. Published March 7 2015. Accessed April 3 2018.

9. Rungruangmaitree R, Jiraungkoorskul W. Pharmacognasy Review.; 11(21): 39–42 (2017).

10. Valverde C, Frias J, Hernandex A, Martin-Alvarez P. Assessment of nutritional compounds and antinutritional factors in pea (Pisum sativum) seeds. J Sci. Food Agric.; 83:298–306 (2003).

11. Chon SU. Total polyphenols and bioactivity of seeds and sprouts in several legumes. Curr Pharm Des..; 19: 12–24 (2013).

12. Majeed H, Safdar W, Ali B, Mohannad A, Ahmad I, Mumtaz A. Genetic assessment of the genus Pisum L. based on sequence specific amplification polymorphism data. J Med Plants Res. 6: 959–67 (2012).

13. Dian. Health benefits of pea. Dr.Health Benefits.Com. http://Proven Health benefits of pea.org/.Published March 2018.Accessed June 15 2018.

14. Alberts, B; Johnson, A; Lewis, J; Raff, M; Roberts, K; Walter, P. Molecular Biology of the Cell. New York: Garland Science, 62, 118-119 (2002).

15. Hunziker, O F; D. Fay Hosman "Tallowy Butter—its Causes and Prevention" Journal of Dairy Science. American Dairy Science Association. 1(4): 320–346 (2008).

16. Mozaffarian, D, Katan, B. Ascherio, A, Stampfer, J. and Willett C. "Trans Fatty Acids and Cardiovascular Disease". New England Journal of Medicine. 354 (15): 1601–1613 (2006).

17. Petersson, V.; Gustafsson, M.; Nordblad, M; Börjesson,p Mattiasson, Bo; Adlercreutz, P. "Wax esters produced by solvent-free energy-efficient enzymatic synthesis and their applicability as wood coatings". Green Chemistry. 7(12); 1463- 9270 (2005).

18. Kingston W. "Irish contributions to the origins of antibiotics". Irish Journal of Medical Science. 177(2): 87–92. (2008).

19. David S, Zelda Fulton G. Kitson. Gas Chromatography and Mass Spectrometry: A Practical Guide. Academic Press. (2011).

20. Ministry of Agriculture Government of India. Solvent Extraction. The Solvent Extractors Association of India. http://www.seaofindia.com/. Published 2015. Accessed July 2018.

21. Wang, T. Lenahan, R. "Determination of volatile halocarbons in water by purge-closed loop gas chromatography". Bulletin of Environmental Contamination and Toxicology 32(1): 429–438 (1984).

22. Tekin .K, Karagöz . S, Bektaş .S. "A review of hydrothermal biomass processing". Renewable and Sustainable Energy Reviews. 40: 673–687 (2014).

23. Alon, T.; Amirav, A. "Isotope Abundance Analysis Method and Software for Improved Sample Identification with the Supersonic GC-MS". Rapid Communications in Mass Spectrometry. 20 (17): 2579–2588 (2006).

24. Labarta .J, Grossmann. I. African Journal of Liquid –Liquid Extraction. 47 (10): 2243-2252 (2011).

25. Reyes J. Olaya, M. Velasco, R.; Serrano, M.D.; Marcilla, A. "Correlation of the Liquid-Liquid Equilibrium Data for Specific Ternary Systems with One or Two Partially Miscible Binary Subsystems". Fluid Phase Equilibria. 278: 9-14 (2008).

26. Marcilla, A.; Reyes , J. .; Serrano, M.D.; Olaya, M.M. "GE Models and Algorithms for Condensed Phase Equilibrium Data Regression in Ternary Systems: Limitations and Proposals". The Open Thermodynamics Journal. 5: 48- 62 (2011).

27. Aly .M , Shahira. M , Maha .M. Pharmacognosy Journal. 2 (9):12. (2011).

28. AOAC Hand book of Food analysis. Specific gravity (Apparent) of Oils, Pycnometer method, Official method 920.212 (17thed), AOAC International, Gaithersburg,MD, USA, p: 72 (2000).

29. AOAC Handbook of Food analysis: Index of refraction of oils and fats. Official method 921.08 (17th ed), AOAC International, Gaithersburg, MD, USA, p: 70 (2000).

30. ISI. Hand book of food analysis: Methods of sampling and test for oils and fat (Part XIII) (1984).

31. Diamante LM, Lan T. Absolute viscosities of vegetable oils at different temperatures and shear rate range of 64.5 to 4835 s−1. Journal of Food Processing. 1-6 (2014).

32. ISO. Acid value. 660: (1996).

33. ISO. Animal and vegetable fats and oils.Determination of saponification value. 3657: (2002).

34. SI. Handbook of food analysis: (Part XIII)-1984, page 67/A.O.A.C 17th (ed.) (2000).

35. ISO. Animal and vegetable fats and oils – Determination of peroxide. 3960: (2007).

36. ISO. Animal and vegetable fats and oils – Determination of iodine value. 3961: (1996).

37. Robert ,T. Essential Oil Safety: A Guide for Health Care Professionals, Churchill Livingstone, London (1995).

38. Brand-Williams W, Cuvelier ME, Berset C. Use of free radica method to evaluate antioxidant activity. Lebensm Wiss Technology 28:25-30 (1995).

39. Makut, M., Gyar, S., Pennap, G. African Journal of Biotechnology. 7(9), pp. 1216-1219 (2008).

40. Muhammd G., Alla , S., Hira , T. Phytochemical analysis and antibacterial activity of Eruca sativa seed . Pak. J. Bot. 43(2): 1351-1359 (2011).

41. Sabahat S., Perween, T. Antibacterial activities of Mentha Piperita, Pisum sativum and Momordica Charanti. Pak. J. Bot. 37(4): 997-1001 (2011).

42. Haymanti , S., Alok ,p ., Venkat , S. Scholars Research Library. 6(6): 359- 365 (2014).

43. Gilman A., Rall T., Alan S. Essential oil. The Pharmacological Basis of Therapeutics. https://en.wikipedia.org/wiki/Goodman_%26_Gilman%27s_The_Pharmacological _Basis_of_Therapeutics. Published 2010 .Accessed 2018.