Effect of Mixing some Fixed Oils on the Physical and Chemical

Characteristics of ( sativum L.) Bulb Essential Oil

Salma Hamed Hussien Hamed

B.Sc. (Hon.) in Biochemistry, Faculty of Applied and Industrial Science, University of Bahri, 2012.

A Dissertation

Submitted to the University of Gezira in partial Fulfillment of the Requirements for the Award of the Degree of Master of Science

in

Bioscience and Biotechnology (Biotechnology) Centre of Biosciences and Biotechnology Faculty of Engineering and Technology May 2016

Effect of Mixing some Fixed Oils on the Physical and Chemical

Characteristics of Garlic (Allium sativum L.) Bulb Essential Oil

Salma Hamed Hussien Hamed

Supervision Committee:

Name Position Signature

Dr. Mutaman Ali A. Kehail Main Supervisor ……………..

Prof. Elnour Elamin Abdelrahman Co-supervisor ……………...

Date: May 2016

i

Effect of Mixing some Fixed Oils on the Physical and Chemical Characteristics of Garlic (Allium sativum L.) Bulb Essential Oil

Salma Hamed Hussien Hamed

Examination Committee:

Name Position Signature

Dr. Mutaman Ali A. Kehail Chairperson ……………………

Prof. Elnaeim Abdalla Ali External Examiner ...………………….

Dr. Almahi Mohamed Ahmed Internal Examiner ……………………

Date of Examination: 26 May 2016

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DEDICATION

To my lovely Father and Mother,

To my Brothers and Sisters,

To my Family,

To my dear, Husband,

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ACKNOWLEDGEMENTS

My grateful thanks to ALLH who gave me the health and ability to achieve this humble study, and with his will achievement was properly completed. I would like also to express my thanks to all those who helped me to carry out this work especially Dr. Mutaman Ali, the main supervisor and Prof. Elnour Elamin Abdelrahman, co-supervisor. Thanks are also due the Technical Staff of the Microbiology Laboratory, Faculty of Engineering and Technology, University of Gezira. Great thanks are due to my family and special thanks due to my dear husband for support and encouragement.

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Effect of Mixing some Fixed Oils on the Physical and Chemical Characteristics of Garlic (Allium sativum L.) Bulb Essential Oil

Salma Hamed Hussein Hamed

Abstract

Garlic plant is native to central Asia. It contained several constituents enriching it with nutritional and pharmaceutical characteristics. This research aimed to study the stability of garlic bulb-oil when fixed with sesame oil and sunflower oil by measuring some of their physical and chemical characteristics. Garlic bulb samples were brought from Wad Medani local market, Gezira State, and were dried in the room temperature away from direct sunlight, and then the oil was extracted using cold extraction method with hexane as solvent. The physical characteristics (refractive index and density) and chemical characteristics (peroxide, iodine, saponification and acid values, and the free fatty acids) were determined for both oil samples at the Food Analysis Laboratory, Faculty of Engineering and Technology, University of Gezira. The results showed that, the color was yellow for both samples oils. The refractive index was 1.467 for the sample fixed with sesame oil, while it was 1.466 for the sample fixed with sunflower oil. The density was 1.0 for the sample fixed with sesame oil, while it was 1.2 for the sample fixed with sunflower oil. The peroxide value (mmol /kg) was 2.00 in the sample fixed with sesame oil, while it was 5.00 in the sample fixed with sunflower oil. The iodine value (g/100 g) of sample fixed with sesame oil was 65 and it was 82 in the sample fixed with sunflower oil. The saponification value (mg KOH/g) was 162 in sample fixed with sesame oil, while it was 179 in sample fixed with sunflower oil. The acid value and free fatty acids were 3.38 and 1.69% in the sample fixed with sesame oil, while they were 2.25 and 1.12% in the sample fixed with sunflower oil, respectively. Anova proved similarity in the physical and chemical characteristics for both fixed samples, although that, sesame fixed sample showed a relative better characteristics in comparison to the standards. The fractionation showed only one spot from each fixed oil sample. The most important recommendation of this study was that, garlic essential oil should not be imported, and it can be prepared locally (for saving hard currency).

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تأثير استخدام بعض الزيوت الثابتة على الخصائص الفيزيائية والكيميائية لزيت الثوم الطيار

سلمى حامد حسين حامد

ملخص الدراسة

الثوم نبات موطنه األصلي أسيا الوسطي. يحتوي علي عدة مكونات جعلته غني بالمغذايات والخصائص العالجية.

هدف هذا البحث لدراسة مدي ثبات زيت ابصال الثوم المثبت بزيت السمسم وزيت زهرة الشمس التجارية وذللك بدراسة بعض

الخصائص الفيزيائية والكيميائية. تم إحضار عينات ابصال الثوم من السوق المحلي لمدينة ود مدني، والية الجزيرة, وتم

تجفيفها في حرارة الغرفة بعيدا عن ضوء الشمس المباشر، ثم تم أستخال ص الزيت بإستخدام طريقة اإلستخالص البارد ومذيب

الهكسين. تم تحديد الخصائص الفيزيائية )معامل االنكسار والكثافة( والكيميائية )الرقم البيروكسيدي، اليودي، التصبن,

والحامضي، واألحماض الدهنية الحرة( لعينتي الزيت في معمل تحليل األغذية، كلية الهندسة والتكنولوجيا، جامعة الجزيرة.

أوضحت النتائج ان اللون أصفر لكال عينتي الزيت. معامل االنكسار كان )1.467( للعينة المثبتة بزيت السمسم و )1.466(

للعينة المثبتة بزيت زهرة الشمس. كانت الكثافة )1.0( للعينة المثبتة بزيت السمسم و )1.2( للعينة المثبتة بزيت زهرة الشمس.

كان الرقم البيروكسيدي 2.0 )مل مول/كجم( للعينة المثبتة بزيت السمسم, بينما كانت 5 للعينة المثبتة بزيت زهرة الشمس. كان

الرقم اليودي 65 )جم/100جم( للعينة المثبتة بزيت السمسم, و كانت 82 للعينة المثبتة بزيت زهرة الشمس. كان رقم التصبن

162 )مجم KOH /جم( للعينة المثبتة بزيت السمسم, بينما كانت 179 للعينة المثبتة بزيت زهرة الشمس. كان الرقم الحامضي

واألحماض الدهنية الحرة 3.38 و 1.69% للعينة المثبتة بزيت السمسم, بينما كانت 2.25 و 1.12% للعينة المثبتة بزيت

زهرة الشمس, علي التوالي. أثبت إختبار تحليل التباين تماثل في الخصائص الفيزيائية والكيميائية بين العينتين , علي الرغم من

أن العينة المثبتة بزيت السمسم قد أظهرت صفات جيدة نسبياً مقارنة مع المدي القياسي. أظهرت التجزئة وجود بقعة واحدة لكل

من عينتي الزيت. التوصية األكثر أهمية هي: عدم إستي ارد زيت الثوم وأنه يمكن تحضيره محلياً )بغرض توفير العمالت

الصعبة(.

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LIST OF CONTENT

Content Page Detection Iii Acknowledgements Iv Abstract V Abstract Arabic Vi List of Content Vii List of Table Viii CHAPTER ONE: INTRODUCTION CHAPTER TWO: LITERATURE REVIEW 2.1 Garlic 3 2.1.1 Scientific Classification 3 2.1.2 Uses 4 2.1.3 Medicinal and Pharmaceutical Uses 5 2.1.4 Nutritional Contents 6 2.1.5 Adverse Effects and Toxicology 6 2.1.6 Seasme oil 10 2.1.7 Sunflower oil 10

CHAPTER THREE: MATERIALS AND METHODS 3.1. Samples 12 3.2. Methods 12 3.2.1. Physical Tests 12 3.2.1.1. Determination of Refractive Index 12 3.2.1.2. Determination of Density 12

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3.2.2. Chemical Tests 12 3.2.2.1. Determination of Saponificarion Value 13 3.2.2.2. Determination of Peroxide Value 13 3.2.2.3. Determination of Iodine Value 13 3.2.2.4. Determination of Free Fatty Acid 14 3.2.2.5. Determination of Acid Value 14 3.3. Thin Layer Chromatography Test 15 3.4. Statistical Analysis 15 CHAPTER FOUR: RESULTS AND DISCUSSION 4.1 Physical Characteristics of Garlic bulb-oil fixed with sesame and 16 sunflower oils 4.2 Chemical Characteristics of Garlic bulb-oil fixed with sesame and 18 sunflower oils 4.3 TLC Test of the fixed garlic bulb-oil samples 20 CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions 22 5.2 Recommendations 22 Reference 23

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LIST OF TABLES

Table No. Title Page

2.1 The nutritional values in 100 g of Garlic 9

4.1 Some Physical Characteristics of Garlic Bulb-Oil fixed by 17 sunflower and sesame oils

4.2 Some chemical characteristics of Garlic Bulb-Oil fixed by 19 sunflower and sesame oils

4.3 TLC Test of Garlic Bulb- Oil fixed by sunflower and sesame 21 oils using solvent system (hexane: acetone; 2:1)

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CHAPTER ONE INTRODUCTION

Garlic (Allium sativum L.) is a commonly used in food and herbal supplement. It has been reported that, some of it compounds are active in reversing cancer development, effective in the treatment of arthritis, prevent breast cancer, it can also be used as mosquito repellant when mixed with olive oil and applied on the body and it is an immune enhancer. A. sativum is a bulbous plant. It grows up to 1.2 m (4 ft) in height. It produces hermaphrodite flowers. Pollination occurs by bees and other insects (Ted and Avram, 2008). As a medicinal and a pharmaceutical plant, garlic has an anthelmintic, anti-bacterial, antibiotic, anti-cancerous activities, in addition to anticoagulant, antioxidant, antiseptic, antispasmodic, anti-tumor, anti- viral, blood thinner, carminative, cholagogue, diaphoretic, aids, the digestive system, diuretic, expectorant, febrifuge, stimulant, and as an agent that strengthens, stimulates or tones the stomach (Global Herbal Supplies, 2009). Garlic is an excellent source of minerals and vitamins that are essential for optimum health. The bulb is one of the richest sources of potassium, iron, calcium, magnesium, manganese, zinc and selenium. Selenium is a heart-healthy mineral, and is important factor for antioxidant enzymes within the body (USDA, 2006). is volatile oil, also called as essential oil. Extraction of garlic bulb-oil must be run by adding non-volatile oil or fixed oil. Essential oil is oil that comes from a plant that smells like the plant it comes from, and that is used in perfumes and flavorings. Any of large class of volatile odoriferous oils of vegetable origin that give plants their characteristics odors and often other properties, that are obtained from various parts of the plants (as flowers, leaves, or bark) by steam distillation, expression or extraction, that are usually mixtures of compounds (as aldehydes or esters), and that are used often in the form of essences in perfumes, flavorings, and pharmaceutical preparations-called also ethereal oil, volatile oil, compare fatty oil, fixed (Amazon.com, 2016). Fixed oils do not evaporate at room temperature, while volatile can evaporate when placed under room temperature. Fixed oils require some specific techniques for extraction but volatile oils can be extracted easily by the distillation process, some type of spot (permanent

1 stain) left after evaporation in fixed oils other than volatile oils. Fixed oils can easily be saponified but volatile oils unable to undergo saponification. Esters of higher fatty acids and glycerin are called as fixed oils, but volatiles oils are mixtures of cleoptenes and stearoptenes. Fixed oils are optically inactive, possess low refractive index and their major source is seeds of the plant, on the other hand, volatile oils are optically active, posses high refractive index and their primary source is leaves, roots, in petals and bark (Medimoon.com, 2013). The Study Objective This research aimed to study the effects of addition or mixing of Sunflower or Sesame oil on some physical and chemical characteristics (oil stability) of Garlic bulb essential oil.

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CHAPTER TWO LITERATURE REVIEW

2.1 Garlic Allium sativum, commonly known as garlic, is a species in the genus, Allium. Its close relatives include the onion, , , chive, and rakkyo. Garlic is native to central Asia (Ensminger, 1994), and has long been a staple in the Mediterranean region, as well as a frequent seasoning in Asia, Africa, and Europe. It was known to Ancient Egyptians, and has been used for both culinary and medicinal purposes (Simonetti, 1990). Garlic is a commonly used as food and herbal supplement. It has been reported that, some of its compounds are active in reversing cancer development, effective in the treatment of arthritis, prevent breast cancer, it can also be used as mosquito repellant when mixed with olive oil and applied on the body and it is an immune enhancer. A. sativum is a bulbous plant. It grows up to 1.2 m (4ft) in height. It produces hermaphrodite flowers. Pollination occurs by bees and other insects (Ted and Avram, 2008). 2.1.1 Scientific Classification Kingdom: Plantae Order: Asparagales Family: Amaryllidaceae Subfamily: Allioideae Genus: Allium Species: A. sativum, (McGee, 2004) According to Zohary and Hopf (2002) “A difficulty in the identification of its wild progenitor is the sterility of the cultivars”, thought to be descended from the species Allium longicuspis, which grows wild in central and southwestern Asia. A. sativum grows in the wild in areas where it has become naturalized. The” wild garlic”, “crow garlic”, and “field garlic” of Britain are members of the species A. ursinum, A. vineale, and A. oleraceum, respectively. In America, A. vineale (known as “wild garlic” or “crow garlic”) and A. canadense, known as “meadow garlic” or “wild garlic” and “wild onion”, are common weeds in fields. One of the

3 best-known “garlic”, the so-called , is actually a wild leek (A. ampelorasum), and not a true garlic. Single clove garlic (also called pearl or ) originated in the Yunnan province of China (Salunkhe and Kadam, 1998). 2.1.2. Uses Garlic is widely used around the world for its pungent flavor as a seasoning or . The garlic plant’s blub is the most commonly used part of the plant. With the exception of the single clove types, garlic bulbs are normally divided into numerous fleshly sections called cloves. Garlic cloves are used for consumption (raw or cooked) or for medicinal purposes. They have a characteristic pungent, spicy flavor that mellows and sweetens considerably with cooking (Katzer, 2009). Other parts of the garlic plant are also edible. The leaves and flowers (bulbils) on the head (spathe) are sometimes eaten. They are milder in flavor than the bulbs, and most often consumed while immature and still tender. Immature garlic is sometimes pulled, rather like a , and sold as “green garlic”. When green garlic is allowed to grow past the “scallion” stage, but not permitted to fully mature, it may produce a garlic “round”, a bulb like a boiling onion, but not separated into cloves like a mature bulb. Additionally, the immature flower stalks (scapes) of the hard-neck and (Thompson, 1995). In edible or rarely eaten parts of the garlic plant include the “skin” and root cluster. The papery, protective layers of “skin” over various parts of the plant are generally discarded during preparation for most culinary uses, thought in Korea immature whole heads are sometimes prepared with the tender skins intact. The root cluster attached to the basal plate of the bulb is taste than the cloves. They are often used in stir frying or braised like asparagus the only part not typically considered palatable in any form (Block, 2010). Garlic is a fundamental component in many or most dishes of various regions, including eastern Asia, southeast Asia, the middle east, northern Africa, southern Europe, and parts of south and central America. The flavor varies in intensity and aroma with the different cooking methods. It is often paired with onion, tomato, or ginger. The parchment-like skin is much like the skin of an onion, and is typically removed before using in raw or cooked form. An alternative is to cut the top off the bulb, coat the cloves by dribbling olive oil (or other oil-based seasoning) over them, and roast them in an oven. Garlic softens and can be extracted from the cloves by squeezing the (root) end of the bulb, or individually by squeezing one end of the clove. In Korea, heads of garlic are fermented at high

4 temperature; the resulting product, called , is sweet and syrupy, and is now being sold in the United –States, United Kingdom and Australia. Garlic may be applied to different kinds of bread to create a variety of classic dishes, such as , garlic toast, bruschetta, crostini and canapé. Oils can be flavored with garlic cloves. These infused oils are used to season all categories of three to six weeks in a mixture of sugar, salt, and spices. In Eastern Europe, the shoots are pickled and eaten as an appetizer. Laba garlic, prepared by soaking garlic in vinegar, is a type of pickled garlic served with dumplings in northern China to celebrate the Chinese New year (Block, 2010). Lightly smoked garlic is becoming increasingly popular in British and European cuisine. It is particularly prized for stuffing poultry and game, and in soups and stews. In both these cases it is important to utilize the undiscarded skin, as much of the smoke flavor is situated there, rather than in the cloves themselves. Immature scapes are tender and edible. They are also known as “garlic spears”, “stems”, or “tops”. Scapes generally have a milder (New York Times, 2008). Garlic leaves are a popular vegetable in many parts of Asia. The leaves are cut, cleaned, and then stir-fried with eggs, meat, or vegetables. Garlic powder has a different taste from fresh garlic. If used as a substitute for fresh garlic, 1/8 teaspoon of garlic powder is equivalent to one clove of garlic (Block, 2010). 2.1.3. Medicinal and Pharmaceutical Uses Global Herbal Supplies (2009), reported the following uses: anthelmintic [an agent that destroys or expels intestinal worms and/or parasites; vermicide; vermifuge]; anti-bacterial [an agent that destroys bacteria; bactericide]; antibiotic [an agent that destroys or stops the growth of micro-organisms] (a powerful natural antibiotic which does not destroys the body’s natural flora); anti-cancerous activities; anticoagulant [an agent that prevents the formation of clots in a liquid, as in blood]; antioxidant [contributing to the oxidation of free radicals which are believed to contribute to premature aging the dementia] (very potent one); antiseptic [an agent for inhibiting the growth of microorganism on living tissue or destroying pathogenic or putrefactive bacteria]; antispasmodic [an agent which relieves or eases muscular spasms, cramps or convulsions]; anti-tumor [inhibits tumor cell formation]; anti-viral [an agent that destroys viruses]; blood thinner, carminative [an agent for easing griping pains, colic and expelling gas from the intestines]; cholagogue [an agent for increasing the flow of bile into the intestines]; diaphoretic [an agent that promotes perspiration]; digestive [aids the digestive system]; diuretic

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[an agent that increases the volume and flow of urine which cleanses the urinary system]; expectorant [an agent that promotes the discharge of mucous and secretions from the respiratory passages]; febrifuge [an agent that reduces or eliminates fevers]; stimulant [an agent excites or quickens the functional activity of the tissues giving more energy] and stomachic [an agent that strengthens, stimulates or tones the stomach]. 2.1.4. Nutritional Contents Garlic is an excellent source of minerals and vitamins that are essential for optimum health. The bulb is one of the richest sources of potassium, iron, calcium, manganese, zinc and selenium. Selenium is a heart-healthy mineral, and is important factor for antioxidant enzymes within the body (USDA, 2006; Table 2.1). 2.1.5. Adverse Effects Garlic is known for causing bad breath (halitosis), as well as causing sweat to Allyl methyl sulfide (AMS) is a volatile liquid which absorbed into the blood during the metabolism of garlic-derived sulfur compounds; from the blood it travels to the lungs and from through the skin pores. Washing the skin with soap is only a partial and imperfect solution to the smell. Studies have shown sipping milk at the same time as consuming garlic can significantly neutralize bad breath. Mixing garlic with milk in the mouth before swallowing reduced the odor better than drinking milk afterward. Plain water, mushrooms and may also reduce the odor; the mix of fat and water found in milk, however, was the most effective (BBC News, 2010). The green, dry “folds” in the center of the garlic clove are especially pungent. The sulfur compound , produces ulacks allicin, but may have some activity due to the presence of S-allylcysteine. Some people suffer from allergies to garlic and other species of Allium. Symptoms can include irritable bowel, diarrhea, mouth and throat ulcerations, nausea, breathing difficulties, and in rare cases, anaphylaxis. Garlic-sensitive patients show positive tests to , allylpropyldisulfide, allylmercaptan and allicin, all of which are present in garlic. Several reports of serious burns resulting from garlic being applied topically for various purposes, including naturopathic uses and acne treatment, indicate care must be taken for these uses, usually testing a small area of skin using a very low concentration of garlic (Baruchin et al., 2001). On the basis of numerous reports of such burns, including burns to children, topical use of raw garlic, as well as insertion of raw garlic into body cavities, is discouraged. In particular,

6 topical application of raw garlic to young children is not advisable. The side effects of long-term garlic supplementation are largely unknown, and no FDA-approved study has been performed. Possible side effects include gastrointestinal discomfort, sweating, dizziness, allergic reactions, bleeding, and menstrual irregularities (Garty, 1993). Some breastfeeding mothers have found their babies slow to feed and noted a garlic odor coming from their babies after consuming garlic (Hogg, 2002). Garlic may interact with warfarin, antiplatelets, saquinavir, antihypertensives, calcium-channel blockers, quinolone family of antibiotics such as ciprofloxacin, and hypoglycemic drugs, as well as other medications. might be toxic to cats or dogs. The composition of the bulbs is approximately 84.09% water, 13.38% organic matter, and 1.53% inorganic matter, while the leaves are 87.14% water, 11.27% organic matter, and 1.59% inorganic matter (Goldbamboo.com, 2006). The phytohemicals responsible for the sharp flavor of garlic are produced when the plant’s cells are damaged. When a cell is broken by chopping, chewing, or crushing, enzymes, stored in cell vacuoles trigger the breakdown of several sulfur-containing compounds stored in the cell fluids (cytosol). The resultant compounds are responsible for the sharp or hot taste and strong smell of garlic. Some of the compounds are unstable and continue to react over time. Among the members of the onion family, garlic has by far the highest concentrations of initial reaction products, making garlic much more potent than onion, shallot, or . Although many humans enjoy the taste of garlic, these compounds are believed to have evolved as a defensive mechanism, deterring animals such as birds, insects, and worms from eating the plant (Macpherson et al., 2005). A large number of sulfur compounds contribute to the smell and taste of garlic. Allicin has been found to be the compound most responsible for the “hot” sensation of raw garlic. This chemical opens thermo-transient receptor potential channels that are responsible for the burning sense of heat in foods. The process of cooking garlic removes allicin, thus mellowing is spiciness. Allicin, along with its decomposition products diallyl disulfide and , are major contributors to the characteristic odor of garlic, while other allicin- derived compounds, such as vinyldithiins and ajoene show beneficial in vitro biological activity. Because of its strong odor, garlic is sometimes called the “stinking rose”. When eaten in quantity, garlic may be strongly evident in the diner’s sweat and garlic breath the following day. This is because garlic’s strong-smelling sulfur compounds are metabolized, forming allyl methyl sulfide. Allyl methyl sulfide (AMS) cannot be digested and is passed into the blood. It is carried

7 to the lungs and the skin, where it is excreted. Since digestion takes several hours and release of AMS several hours more, the effect of eating garlic may be present for a long time (Block, 2010). The well-known phenomenon of “garlic breath” is the allegedly alleviated by eating fresh . The herb is therefore, included in many garlic recipes, such as pistou, , and the spread used in garlic bread. However, since the odor results mainly from digestive processes placing compounds such as AMS in blood, and AMS is then released through the lungs over the course of many hours; eating parsley provides only a temporary masking. One way of accelerating the release of AMS from the body is the use of a sauna. Because of the AMS in the bloodstream, it is believed by some to act as a mosquito repellent, but no clinically evidence suggests it is actually effective. Abundant sulfur compounds in garlic are also responsible for turning garlic green or blue during pickling and cooking. Under these conditions (i.e. acidity, heat) the sulfur-containing compound alliinase react with common amino acids to make pyrroles, clusters of carbon-nitrogen rings (Shinsuke et al., 2006; Jungeun et al., 2007). These rings can be linked together into polypyrrole molecules. Ring structures absorb particular wavelengths of light and thus appear colored. The two-pyrrole molecule looks red, the three-pyrrole molecule looks blue and the four-pyrrole molecule looks green (like chlorophyll, a tetrapyrrole). Like chlorophyll, the pyrrole pigments are safe to eat (McGee, 2006).

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Table (2.1) The nutritional values in 100 g of garlic For 100 g (3.5oz) garlic bulb Principle Nutrient Value Percentage Energy 149 Kcal 7.5% Carbohydrates 33.06 g 25% Protein 6.36 g 11% Total Fat 0.5 g 2% Cholesterol 0 mg 0% Dietary Fiber 2.1 g 5.5% Vitamins Folates 3µg 1% Nicin 0.700 mg 4% Pantothenic acid 0.596 mg 12% Pyridoxine 1.235 mg 95% Riboflavin 0.110 mg 8% Thiamin 0.200 mg 17% Vitamin A 9 IU ˂1% Vitamin C 31.2mg 52% Vitamin E 0.08 mg 0.5% Vitamin K 1.7 µg 1.5% Electrolytes Sodium 153 mg 10% Potassium 401 mg 8.5% Minerals Calcium 181 mg 18% Copper 0.299 mg 33% Iron 1.70 mg 21% Magnesium 25 mg 6% Manganese 1.672 mg 73% Phosphorus 153 mg 22% Selenium 14.2 µg 26% Zinc 1.160 mg 10.5%

Source: USDA (2006)

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2.1.6 Sesame oil

Sesame oil is an edible vegetable oil derived from sesame seeds. Besides being used as cooking oil in South India, it is often used as a flavor enhancer in Korean, Chinese, Japanese, Middle Eastern and Southeast Asian cuisine. It has adistinctive aroma and taste. Sesame oil is one of the more stable natural oils, but can still benefit from refrigeration and limited exposure to light and high temperatures during extraction, processing and storage. Sesame oil is polyunsaturated semi-drying oil. In industry, sesame oil may be used as a solvent in injected drugs or intravenous drip solutions, cosmetic carrier oil, coating stored grains to prevent weevil attack. The oil also has synergy with some insecticides (Morries, 2002). The oil content of sesame seed varies from 48 to 55%. The main fatty acids of Sesame oil are oleic and linoleic, roughly with the same percentage (slightly above 40% each). Saturated acids account for approximately 14 percent and mainly consist of palmitic and stearic acid. The main major characteristics of sesame oil are due to its high content of unsaponifiable matter which contains subastances such as sesamin and sesamoline which are not found in other oils give distinctive colour reactions, whereby sesame oil is readily detected in other oils. These compounds have beneficial effects on serum lipid levels and liver function and give sesame seed oil amarked antioxidant activity. The lignans are also responsible for the great stability of sesame seed oil to oxidation. Sesame oil had a following characteristics: iodine value (103-116), saponification value (188-195), refractive index 1.47 at 25C0 and specific gravity 0.92 at 15C0 (Bernarini, 1984).

2.1.7 Sunflower oil Sunflower oil is the non-volatile oil compressed from sunflower (Helianthus annuus) seeds. Sunflower commonly used in food as frying oil, and in cosmetic formulations as an emollient. Sunflower oil is liquid at room temperature. The refined oil is clear and slightly amber-colored with a slightly fatty odor. Sunflower oil is mainly a triglycerides (fats), typically derived from the fatty acids and linoleic acid which is doubly unsaturated (polyunsaturated omega-6; 59%) and oleic acid (monounsaturated omega-9; 30%) (Alfred, 2002). It is interesting to note the influence of climatic conditions on the linoleic acid content. In cold countries, especially in Russia, the linoliec acid content is at peak and it may even reach more than 70 percent of the total fatty acids. As a result, in this case, the oleic acid content is low. When

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Sunflower seeds are grown in a relatively warm climate, such as that of India, the linoleic acid content decrease considerably, with an increase in oleic acid content. In the country of origin the oil had a linoleic acid content of 60-68 percent, whereas in India it had a linoleic and oleic acid content of 40 and 49 percent, respectively. Sunflower oil has a following characteristics: iodine value (125-136), sapoification value (188-194), refractive index 1.47 at 25C0, specific gravity 0.92 at 15C0 (Bernarini, 1984).

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CHAPTER THREE MATERIALS AND METHODS 3.1 Samples The garlic (Allium sativum L.) bulbs (about 1000 g) were obtained from the local Market of Wad Medani City, Gezira State. The transparent covering of garlic bulb was removed manually and the cover free bulbs were room dried for about two weeks Mortar and then pestle were used to ground the dry garlic bulbs into powder and then stored in a covered plastic container for extraction uses. 3.2Methods Cold extraction of sample by adding hexane as solvent to powder of garlic bulbs in beaker (500ml) then shaking and stayed for two days, after that filterd the mixure and put the sample in two container, about 2-3ml mixed one by sesame oil and another by sunflower oil covered by allmonium foil with pores for week, in order to evaporate the solvent and obtained mixure oil.

3.2.1. Physical Tests: 3.2.1.1. Determination of Refractive Index Refractive index was measured by using refractometer in the Food Analysis Laboratory, Faculty of Engineering and Technology, University of Gezira. 3.2.1.2 .Determination of density According to John (2003), 10 ml of the oil were measured in a pre- weighed measuring cylinder. The weight of the cylinder and oil were measured, the weight of the oil was then obtained by subtracting the weight of the cylinder from the weight of the oil and cylinder.

Density of oil =W1 -W0/V0 Where:

W1= weight of empty measuring cylinder + oil

W0= weight of measuring cylinder

V0= volume of oil used

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3.2.2.1. Determination of saponification Value According to Akpan (2006), about 2 g of the garlic oil were added to flask with 30 ml of ethanolic KOH and was then attached to acondenser for 30 minutes to ensure the sample was fully dissolved. After sample had cooled 1 ml of phenolphthalein was added and titrated with 0.5M HCL until a pink endpoint has reached. Saponification value was calculated as: Saponification value = (S-B) ×M×56.1/Sample weight Where: S= sample titer B= blank titer value M= molarity of the HCL 56.1= molecular weight of KOH 3.2.2.2. Determination of Peroxide Value According to Nielsen (2003), 2.0 g of oil extracted were added to 22ml of a solution mixture of 12ml chlorofom and 10 ml acetic acid. 0.5 ml of saturated potassium iodide was added to the flask. The flask was corked and allowed to stay with occasional shaking for 1 minute. 30 ml of distilled water were then added to the mixture and titrated against 0.1M of sodium thiosulphate until yellow color is almost gone. 0.5 ml of starch indicator was quickly added and titration continued until blue color just disappeared. A blank titration was also carried out at the same condition. Peroxide value= (S-B) × N × 1000/W Where: Peroxide value= Meq peroxide per 100 g of sample S= volume of titrant (cm3) for sample B= volume of titrant (cm3) for blank

N= molarity of sodium thiosulphate solution (mEq/cm3)1000= conversion of units (g/kg) W= Weight of oil sample 3.2.2.3. Determination of Iodine Value According to Akpan (2006), 0.4 g of the garlic oil sample was weighed into a conical flask and 20 ml of carbon tetrachloride were added to dissolve the oil. Then 25 ml of Wij’s

13 reagent were added to the flask using a safety pipette in fume chamber. Stopper was then inserted and the content of the flask was vigorously swirled. The flask was then placed in the dark for two hours and 30 minutes. At the end of this period, 20 ml of 10% aqueous potassium iodide and 125 ml of water were added using a measuring cylinder. The content was titrated with 0.1M sodium- thiosulphate solutions until the yellow color almost disappeared. Few drops of 1% starch indicator were added and the titration continued by adding thiosulphate drop wise until blue coloration disappeared after vigorous shaking. The same procedure was used for blank test and other samples. The iodine value (I.V) is given by the expression:

Iodine value=12.69C (V1-V2)/M Where: C= Concentration

V1= Volume of sodium thiosulphate used for blank

V2= Volume of sodium thiosulphate used for determination M= Weight of the sample. 3.2.2.4. Determination of Free Fatty Acid According to Chopra and Kanwar (1991), 2.0 g of oil were measured into 250 ml Erlenmeyer flask, 100 ml of ethanol were added and followed by 2 ml of phenolphthalein indicator. The mixture was shaken and titrated against 0.1M NaOH with continuously shaking until the endpoint is reached, which is indicated by a slight pink color that persists for 30 seconds, the free fatty acid is expressed as: %FFA= V×N×282×100/W Where: %FFA= Percent free fatty acid (g/100g) V= Volume of NaOH (ml) N= Molarity of NaOH 282= Molecular weight of oleic acid W= Weight of oil sample 3.2.2.5. Determination of Acid Value According to AOAC (1998), 25 ml of 5% ethanol were boiled on a water bath the heating is to ensure the removal of dissolved gases. 2.5 g of garlic oil were added to 25 ml of hot ethanol and the mixture was heated to boil. Then few drops of 1% phenolphthalein indicator were added

14 and titrated against 0.1M KOH with constantly shaking until a permanent pink color was obtained, the acid value is expressed as: Acid value=56.1×M×V/W Where: M= Concentration; V= Titer value 56.1= Molecular weight of KOH; W= Weight of oil sample 3.3. Thin Layer Chromatography Test Identification of the individual components of the crude polar (methanol) or a polar (petroleum ether) extracts by thin layer chromatography was run according to Vogel et al., (2003). The garlic oil extract was applied as a spot, using a micro-syringe on a TLC plate coated with silica gel (0.5 mm thickness), the plate was developed in a tank containing the solvent mixture (hexane: acetone; 2:1) for about 45 minutes. After solvent drying at room temperature, the provision was made by using iodine sprays (as detecting reagent) and visualized under UV light.

The Rf of the separated spots were measured.

Rf = the distance driven by component (cm)/ the distance driven by solvent 3.4. Statistical Analysis Microsoft office, Excel program, 2007, was used to present and analyze the obtained data. Simple descriptive statistics and ANOVA two factors were also used to clear the differences observed in the values of the physical and chemical parameters of the local prepared garlic oil mixed with sesame oil and another which was mixed with sunflower oil.

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CHAPTER FOUR RESULTS AND DISCUSSION

4.1 Physical Characteristics of Garlic Bulb-Oil Fixed with Sesame and Sunflower oils Table (4.1) showed some physical characteristics of the garlic bulb-oil fixed with sesame oil and with sunflower oil samples. The color was yellow for both oils samples. The refractive index (at 27Co) was 1.467 for the sample fixed with sesame oil, while it was 1.466 for the sample fixed with sunflower oil. The refractive index was found to be 1.55 - 1.585 according to Gafar et al., (2012), which were not matched (relatively more) with the study results. The density (at 27 Co) was 1.0 for the sample fixed with sesame oil, while it was 1.2 for the sample which fixed with sunflower oil. The density of garlic oil was 1.073 g/ml (at 25Co) according to Gafar et al., (2012). The sample which fixed with sesame oil has a density closer to that, but the sample which was fixed with sunflower oil seemed to be relatively higher in its density. The statistical analysis revealed a non significant difference between the two fixed samples of garlic bulb-oil (f= 0.98; f-crit= 161.45), i.e. both samples are similar in their physical characteristics (according to the obtained data), although the sample that fixed with sesame oil showed relative better density compared to Gafar et al., (2012) ranges.

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Table (4.1) Some Physical Characteristics of Garlic Bulb-Oil fixed by Sunflower and Sesame oils

Characteristic Sample fixed with sesame oil Sample fixed with sunflower oil

Color Yellow Yellow

Refractive index 1.467 1.466

Density 1.00 1.20

SUMMARY Count Sum Average Variance Refractive index 2 2.93 1.47 5E-07 Density 2 2.2 1.1 0.02

ANOVA Source SS Df MS F P-value F crit Rows 0.13 1 0.13 13.30 0.17038 161.45 Columns 0.01 1 0.01 0.98 0.50318 161.45 Error 0.01 1 0.01

Total 0.15 3

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4.2 Chemical Characteristics of Garlic Bulb-Oil Fixed with Sesame and Sunflowers oils Table (4.2) showed the chemical characteristics (peroxide, saponification, Iodine, acid values and free fatty acids) of garlic bulb-oil fixed with sesame and sunflower oils. The results showed that, the peroxide value in term of (mmol /kg) was 2.00 in the sample fixed with sesame oil, while it was 5.00 in the sample fixed with sunflower oil. Gafar et al., (2012) found that, the peroxide value was 2.5 ± 0.5mmol/kg), i.e. the peroxide value ranged between 2-3. The results obtained in this study confirmed that, the sample fixed with sesame oil was closer to Gafar et al., (2012) result than the sample fixed with sunflower oil. The iodine value in term of (g/100g) of sample fixed with sesame oil was 65 and it was 82 in the sample fixed with sunflower oil. The value obtained in Gafar et al., (2012) study was 12.69 ± 0.05 (g/100 g). Both the samples were far more than this range, and this finding reflected that, the samples used in this experiment were far unsaturated. The saponification value was 162 in sample which fixed with sesame oil, while it was 179 in sample fixed with sunflower oil. It was 192+10 (mg KOH/g), according to study of Gafar et al., (2012). The values reflected the amount of KOH (in mg) required to make soap from one gram of each oil, which was low in the both samples, specially, sesame fixed sample. Table (4.2) also showed that, the acid value and the free fatty acids were 3.38 and 1.69% in the sample fixed with sesame oil, while they were 2.25 and 1.12% in the sample which fixed with sunflower oil, respectively. The acid value and the free fatty acids obtained in the study of Gafar et al., (2012) were found 4.18 ± 0.01 and 2.1 ± 0.05, respectively. This finding showed that, both samples were not matched with the results of Gafar et al., (2012), but sesame fixed sample was relatively the best one. Anova revealed that, there was no significant differences between the two fixed samples of garlic bulb essential oil in their chemical characteristics (f= 2.94; f-crit= 7.71), i.e. both samples are similar in their chemical characteristics, although that, sesame fixed sample showed a relative best characteristics in comparison to Gafar et al., (2012) ranges.

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Table (4.2) Some Chemical Characteristics of Garlic Bulb-Oil fixed by Sunflower and Sesame oils

Characteristic Sample fixed with sesame oil Sample fixed with sunflower oil

Peroxide value 2 5

(mmol/kg)

Iodine value 65 82

(g/100g)

Saponification value 162 179

(mgKOH/g)

Acid value (%) 3.38 2.25

Free fatty acid (%) 1.69 1.12

SUMMARY Count Sum Average Variance Peroxide value 2 7 3.5 4.5 Iodine value 2 147 73.5 144.5 Saponification value 2 341 170.5 144.5 Acid value 2 5.63 2.82 0.64 Free fatty acid 2 2.81 1.41 0.16

ANOVA Source SS Df MS F P-value F crit Rows 43644.11 4 10911.03 257.2 4.49E-05 6.39 Columns 124.61 1 124.61 2.94 0.161709 7.71 Error 169.69 4 42.42

Total 43938.41 9

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4.3 TLC Test of the Fixed Garlic Bulb-Oil Samples

Table (4.3) showed the fractionation of garlic bulb-oil fixed with sesame oil and

sunflower oil in the TLC plate. Both samples separated one spot with Rf value of (0.60) for garlic bulb-oil fixed with sesame oil and (0.80) for garlic bulb-oil fixed with sunflower in visual light.

According to Gafar et al, (2012) study, five spots with Rf values of (0.81, 0.63, 0.62, 0.38 and 0.14) were observed. There was clear difference in TLC results, which can be attributed to the variety of garlic plant, soil factors, fixer oil used, solvent system used, quality of chemicals used, accuracy of UV and visual monitoring. Also, some or all of the active substances in the individual sesame oil, sunflower oil or garlic essential oil, may be activated or inhibited each other in the mixtures, though original or new spots may be separated or disappears.

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Table (4.3) TLC Test of Garlic Bulb-Oil fixed by Sunflower and Sesame oil using solvent system (hexane: acetone; 2:1)

Spot No. Sample fixed with Sesame oil Sample fixed with Sunflower oil 1 0.6 0.8

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CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions 1- The color was yellow for both samples oils. 2- The refractive index (at 27Co) was 1.467 for the sample fixed with sesame oil, while it was 1.466 for the sample which fixed with sunflower oil. 3- The density (at 27 Co) was 1.0 for the sample fixed with sesame oil, while it was 1.2 for the sample which fixed with sunflower oil. 4- The peroxide value in term of (mmol /kg) was 2.00 in the sample fixed with sesame oil, while it was 5.00 in the sample fixed with sunflower oil. 5- The iodine value in term of (g/100 g) of sample fixed with sesame oil was 65 and it was 82 in the sample fixed with sunflower oil. 6- The saponification value (mg KOH/g) was 162 in sample which fixed with sesame oil, while it was 179 in sample fixed with sunflower oil. 7- The acid value and free fatty acids were 3.38 and 1.69% in the sample fixed with sesame oil, while they were 2.25 and 1.12% in the sample which fixed with sunflower oil, respectively. 8- Anova revealed that, both samples are similar in their physical and chemical characteristics, although that, sesame fixed sample showed a relative best characteristics in comparison to the standards. 9- The fractionation (TLC) showed only one spot was separated from each fixed sample.

5.2 Recommendations 1- Garlic essential oil should not be imported, and it can be prepared locally, for a multipurpose, that saves a hard currency for the Sudan. 2- The Sesame and Sunflower oils can be used to fix Garlic essential oil, with preference to sesame oil. 3- Further quantitative, nutritional and pharmaceutical studies should be conducted to make benefit of garlic essential oil.

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