Euphorbia Trigona and E

Qualitative Phytochemical Screening, Thin Layer Chromatography and Toxicity Tests for the Stems of Two Cactus Genera: Euphorbia trigona and E. abyssinica

Hanadi Alwan Ali Abd Alla Hamid

B.Sc. (Hon.) in Medical Science of Laboratory, College of Medical Science, University of Hodaidah (Yemen), 2003

A Dissertation

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

Biosciences and Biotechnology (Biotechnology) Center of Biosciences and Biotechnology Faculty of Engineering and Technology

June 2015

Qualitative Phytochemical Screening, Thin Layer Chromatography and Toxicity Tests for the Stems of Two Cactus Genera: Euphorbia trigona and E. abyssinica

Hanadi Alwan Ali Abd Alla Hamid

Supervision Committee:

Name Position Signature

Dr. Mutaman Ali Kehail Supervisor ……………..

Dr. Yasir Mohamed Abdelrahim Co-Supervisor ……………..

Date: June, 2015

Qualitative Phytochemical Screening, Thin Layer Chromatography and Toxicity Tests for the Stems of Two Cactus Genera: Euphorbia trigona and E. abyssinica

Hanadi Alwan Ali Abd Alla Hamid

Examination Committee:

Name Position Signature

Dr. Mutaman Ali Kehail Chair Person ……………..

Prof. Elnaem Abdalla Ali External Examiner ……………..

Dr. Abdalla Ibrahim Abdalla Internal Examiner ……………..

Date: 12 June, 2015

Dedication

To my Family

To my Husband

To my sons

To my Sisters and Brothers

To those who stand closer and make it possible

To my Friends and Colleges

Qualitative Phytochemical Screening, Thin Layer Chromatography and Toxicity Tests for the Stems of Two Cactus Genera: Euphorbia trigona and E. abyssinica Hanadi Alwan Ali Abd Alla Hamid

Abstract

There are over 2000 species of Euphorbia (Euphorbiaceae) in the world. The plants are succulent and the aerial parts of some species exude a milky fluid that exerts a number of physiological and a wide range of biological properties, including antioxidant, anti- inflammatory, pesticidal, cytotoxic, antibacterial and multi-drug resistance reversing activities. The objective of this study was to run qualitative phytochemical screening, thin layer chromatography (TLC) and toxicity tests for the stems of two cactus genus: Euphorbia trigona and E. abyssinica. Stems of E. trigona, were collected from within Wad Medani Town, Gezira State, whereas one large stem of E. abyssinica was brought from Erkwit mountain, Red Sea State. All of the laboratory tests were done in the Food Analysis Laboratory, Faculty of Engineering and Technology, University of Gezira. The collected stems of both plants were firstly dissected into outer parts (cortex) and inner part (pith). These parts were dried, grounded and then subjected to phytochemical screening (presence of the main classes), TLC and toxicity tests, using recommended methods. The results of this study showed that, all plant samples contained the same phytochemicals similarly, but with different concentrations for some classes, even in the same plant. The TLC tests of each of E. trigona and E. abyssinica, separated one spot from each of the stem pith and cortex per each extract (polar and apolar), but with different Rf values. ANOVA proved that, the susceptibility of Anopheles and Culex larvae toward Euphorbia stem parts (at 1.2 mg/L) were not similar, and it also proved that, the toxicity of the used plant parts were statistically not similar. The recommendations of this study were to run proper chemical and phytochemical tests (instead of some tests). An antimicrobial and pharmaceutical activity tests should be run, and also to run a proper toxicity test.

إختبارات المسح الكيميائي النباتي النوعي, كروماتوغرافيا الطبقة الرقيقة والسمية لسيقان جنسين من الصبار:

إيوفوربيا ت اريقونا وايوفوربيا أبسينيكا

هنادي علوان علي عبد اهلل حميد

ملخص البحث

يوجد أكثر من ألفي نوع من اإليوفوربيا )عائلة اإليوفوربيا( في العالم. النبات عصيري واألجزاء الخضرية لبعض

األنواع تفرز سوائل لبنية لها العديد من الخواص الفسيولوجية ومدي واسع من الخواص البيولوجية, والتي تشمل مضاد التأكسد,

مضاد اإللتهاب, مبيد لآلفات, سم خلوي, مضاد للبكتريا وذو نشاطات عاكسة للمقاومة العديدة للعقاقير. هدف هذا البحث إلي

إجراء إختبارات مسح كيميائي نباتي نوعي, كروماتوغرافيا الطبقة الرقيقة والسمية لسيقان جنسين من صبار إيوفوربيا ترايقونا

وايوفوربيا إبسيكنيكا. جمعت سيقان نبات إيوفوربيا ت اريقونا من داخل مدينة ود مدني, والية الجزيرة, بينما تم إحضار ساق واحد

كبير من إيوفوربيا أبيسينيكا من جبال أركويت, والية البحر األحمر. تمت كل اإلختبارات المعملية في معمل تحليل األغذية,

كلية الهندسة والتكنولوجيا, جامعة الجزيرة. تم تشريح السيقان التي تم جمعها أوالً لجزء خارجي )قشرة( وجزء داخلي )نخاع(. تم

تجفيف هذه األجزاء, سحقها ومن ثم تعريضها للمسح الكيميائي النباتي )وجود المجاميع الرئيسية(, إختبار الطبقة الرقيقة

واختبار السمية, بإستخدام الطرق الموصي بها. أوضحت نتائج هذه الد ارسة أن كل عينات النباتات تحتوي علي نفس المواد

الكيميائية النباتية بصورة متشابهة, ولكن بتراكيز مختلفة لبعض المجاميع, وحتي في نفس النبات. فصل إختبار الطبقة الرقيقة

لكل من النباتين بقعة واحدة في كل من قشرة ونخاع الساق لكل مستخلص )القطبي والالقطبي(, ولكن مع قيم معامل إعاقة

مختلفة. أثبت إختبار تحليل التباين أن حساسية يرقات األنوفلس والكيولكس تجاه أجزاء ساق نباتات اإليوفوربيا )عند 1.2

ملغ/لتر( ليست متساوية, وايضاً أن سمية أج ازء النباتات المستخدمة ليست متماثلة إحصائياً. توصيات هذه الد ارسة هي إج ارء

إختبا ارت كيميائية وكيميائية نباتية متكاملة )بدالً من بعض اإلختبا ارت(. يجب إج ارء بعض إختبا ارت النشاط المضاد

للميكروبات والنشاط الصيدالني وكذلك إجراء إختبار سمية متكامل.

List of Contents

Subject Page Dedication iii Abstract iv Arabic Abstract v List of Contents vi List of Tables viii CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO: LITERATURE REVIEW 3 2.1. Cactus 3 2.1.1 Morphology 3 2.1.2.Stems 3 2.1.3 Uses 4 2.2. Euphorbia 5 2.2.1. Euphorbia trigona 6 2.2.2. Euphorbia abyssinica 6 2.2.3 Researches on Euphorbia species 8 2.3. Phytochemicals 8 2.4. Chromatography 9 CHAPTER THREE: MATERIALS AND METHODS 3.1 Samples 11 3.2. Methods 11 3.2.1. The phytochemical screening 11 3.2.2.1 For glycosides 11 3.2.2.2 For flavonoids and flavonones 11

3.2.2.3 For saponnins 12 3.2.2.4 For tannins 12 3.2.2.5 For sterols and or triterpenes 12 3.2.2.6 For alkaloids 12 3.3. Thin layer chromatography test 13 3.4. Toxicity test of stem parts powder 13 3.4. Statistical analysis 13 CHAPTER FOUR: RESULTS AND DISCUSSION 4.1. The phytochemical screening in the stem parts 14 4.2 Thin layer chromatography tests for the stem parts of both plants 16 4.3 Toxicity of E. trigona and E. abyssinica stem parts against mosquito larvae 18 CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions 20 5.2. Recommendations 20 References 21

List of Tables

Table Title Page No

The presence and absence of some phytochemicals in stem pith 4.1 15 and cortex of both plants

4.2 TLC for the stem parts of both plants 17

The percentage mortalities of Aopheles and Culex larvae toward

4.3 the powders of E. trigona and E. abyssinica stem parts (at 0.3 g/ 19

250 ml)

Chapter One Introduction

Cacti are commonly grown as houseplants. They are pretty and easy to grow. Some cacti are grown in gardens, especially in dry areas. Cactus can be used as a living fence. The wood of dead cactus is sometimes used for building. People eat the fruit of some kinds of cactus, such as dragon fruit and prickly pear. Ciouvhiul insects also eat prickly pears. These insects produce a red coloring used in food and lipstick (Anderson, 1999). There are over 2000 species of Euphorbia (Euphorbiaceae) in the world. The plants have a unique flower structure and a significant percentage of them are succulent. When broken or cut, the aerial parts of some species exude a milky fluid that exerts a number of physiological effects including skin irritation, tumor promotion, and pro-inflammatory properties. Recently, certain Euphorbia species were found to exhibit a wide range of biological properties, including anti-anaphylactic, antioxidant, neuritogenic, anti-HIV, anti-arthritic, anti-inflammatory, pesticidal, cytotoxic, analgesic, antiplasmodial, antibacterial and multi-drug resistance reversing activities (Faky et al., 2008). The Euphorbia plants are annual or perennial herbs, woody shrubs or trees with a caustic, poisonous milky sap (latex). The roots are fine or thick and fleshy or tuberous. Many species are more or less succulent, thorny or unarmed. The main stem and mostly also the side arms of the succulent species are thick and fleshy, 15–91 cm (6–36 inches) tall. The fruits are three (rarely two) compartment capsules, sometimes fleshy but almost always ripening to a woody container that then splits open (explosively, see explosive dehiscence). The seeds are 4-angled, oval or spherical, and in some species have a caruncle (Carter, 2002). The toxic constituents of Euphorbia species were considered to be a kind of specific diterpenes, globally called phorboids, which comprise tigliane, ingenane and daphnane diterpene derivatives. Terpenes, including diterpenes and triterpenes, have been frequently found in Euphorbia species. Steroids, cerebrosides, glycerols, phenolics and flavonoids were also isolated from plants of the genus, but the compounds most relevant to the toxicity and considerable biological activities in Euphorbia are diterpenes, especially those with abietane, tigliane, and ingenane skeletons (Haba, et al., 2009; Cateni et al., 2003).

The desired phytochemicals can be obtained through extraction. The purpose of standardized extraction procedures for crude drugs (medicinal plant parts) is to attain the therapeutically desired portions and to eliminate unwanted material by treatment with a selective solvent known as menstrum. The extract thus obtained, after standardization, may be used as medicinal agent as such in the form of tinctures or fluid extracts or further processed to be incorporated in any dosage form such as tablets and capsules. These products contain complex mixture of many medicinal plant metabolites, such as alkaloids, glycosides, terpenoids, flavonoids and lignans. The general techniques of medicinal plant extraction include maceration, infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet), aqueous- alcoholic extraction by fermentation, counter-current extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, and phytonic extraction (with hydrofluorocarbon solvents). For aromatic plants, hydrodistillation techniques (water distillation, steam distillation, water and steam distillation), hydrolytic maceration followed by distillation, expression and effleurage (cold fat extraction) may be employed. Some of the latest extraction methods for aromatic plants include headspace trapping, solid phase micro-extraction, protoplast extraction, microdistillation, thermomicrodistillation and molecular distillation (Handa et al., 2008). Thin-layer chromatography (TLC) is a chromatography technique used to separate non- volatile mixtures. Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the stationary phase (Harry and Christopher, 1989).

Main Objective: The objective of the study was to run phytochemicals screening, thin layer chromatography and toxicity tests for two cactus genera: Euphorbia trigona and E. abyssinica

Chapter Two Literature Review 2.1. Cactus A cactus is a kind of plant. They are xerophytes, specializing in hot, dry climates. Cacti are members of the plant family Cactaceae, in the order Caryophyllales. Cacti are native to the Americas, ranging from Patagonia in the south to parts of western Canada in the north—except for Rhipsalis baccifera, which also grows in Africa and Sri Lanka. Many people like to grow cactus in pots or gardens. Now cacti have spread to many other parts of the world. They are part of an important food chain. Many cacti live in dry places, such as deserts. Most cacti have sharp thorns (stickers) and thick skin. There are many shapes and sizes of cacti. Some are short and round; others are tall and thin. Many cactus flowers are big and beautiful. Some cactus flowers bloom at night and are pollinated by moths and bats. Some cactus fruits are brightly coloured and good to eat. Goats, birds, ants, mice, bats and people eat cactus fruits (Anderson, 2001). 2.1.1. Morphology The 1,500 to 1,800 species of cacti mostly fall into one of two groups of "core cacti": opuntias (subfamily Opuntioideae) and "cactoids" (subfamily Cactoideae). Most members of these two groups are easily recognizable as cacti. They have fleshy succulent stems that are major organs of photosynthesis. They have absent, small, or transient leaves. They have flowers with ovaries that lie below the sepals and petals, often deeply sunken into a fleshy receptacle (the part of the stem from which the flower parts grow). All cacti have areoles—highly specialized short shoots with extremely short internodes that produce spines, normal shoots, and flowers (Edwards and Donoghue, 2006). 2.1.2. Stems The leafless, spiny stem is the characteristic feature of the majority of cacti (and all of those belonging to the largest subfamily, the Cactoideae). The stem is typically succulent, meaning it is adapted to store water. The surface of the stem may be smooth (as in some species of Opuntia) or covered with protuberances of various kinds, which are usually called tubercles. These vary from small "bumps" to prominent, nipple-like shapes in the genus Mammillaria and outgrowths almost like leaves in Ariocarpus species. The stem may also be ribbed or fluted in shape. The prominence of these ribs depends on how much water the stem is storing: when full

(up to 90% of the mass of a cactus may be water), the ribs may be almost invisible on the swollen stem, whereas when the cactus is short of water and the stems shrink, the ribs may be very visible (Anderson, 2001). 2.1.3. Uses There is still controversy as to the precise dates when humans first entered those areas of the New World where cacti are commonly found, and hence when they might first have used them. An archaeological site in Chile has been dated to around 15,000 years ago, Goebel et al., (2008) suggesting cacti would have been encountered before then. Early evidence of the use of cacti includes cave paintings in the Serra da Capivara in Brazil, and seeds found in ancient middens (waste dumps) in Mexico and Peru, with dates estimated at 12,000–9,000 years ago. Hunter-gatherers likely collected cactus fruits in the wild and brought them back to their camps (Anderson, 2001). The plant now known as Opuntia ficus-indica, or the Indian fig cactus, has long been an important source of food (Griffith, 2004). Both the fruit and pads are eaten (Daniel, 2007). Almost any fleshy cactus fruit is edible. The fruit of the saguaro (Carnegiea gigantea) has long been important to the indigenous peoples of northwestern Mexico and the southwestern United States, including the Sonoran Desert. The bodies of cacti other than opuntias are less often eaten (Anderson, 2001). A number of species of cacti have been shown to contain psychoactive agents, chemical compounds that can cause changes in mood, perception and cognition through their effects on the brain (Zimmerman and Parfitt, 1982; Seedi et al., 2005; Bussmann and Sharon, 2006). Cacti may also be planted outdoors in regions with suitable climates. Concern for water conservation in arid regions has led to the promotion of gardens requiring less watering (xeriscaping). Cacti are one group of drought-resistant plants recommended for dry landscape gardening (Harlow and Coate, 2004). Cacti have many other uses. They are used for human food and as fodder for animals, usually after burning off their spines. In addition to their use as psychoactive agents, some cacti are employed in herbal medicine. The practice of using various species of Opuntia in this way has spread from the Americas, where they naturally occur, to other regions where they grow, such as India.

Cacti are used as construction materials. Living cactus fences are employed as barricades. The woody parts of cacti, such as Cereus repandus and Echinopsis atacamensis, are used in buildings and in furniture. The frames of wattle and daub houses built by the Seri people of Mexico may use parts of Carnegiea gigantea. The very fine spines and hairs (trichomes) of some cacti were used as a source of fiber for filling pillows and in weaving (Shetty et al., 2011). 2.2. Euphorbia: Euphorbia is a genus of flowering plants belonging to the family Euphorbiaceae. Consisting of 2008 species, Euphorbia is the fourth largest genus of flowering plants. The family is primarily found in the tropical and subtropical regions of Africa and the Americas, but also in temperate zones worldwide. Succulent species originate mostly from Africa, the Americas and Madagascar. There exists a wide range of insular species: on the Hawaiian Islands, where spurges are collectively known as "akoko", and on the Canary Islands as "tabaibas" (Stebbins and Hoogland, 1976; Carter, 2002). The botanical name Euphorbia derives from Euphorbus, the Greek physician of king Juba II of Numidia (52–50 BC – 23 AD), who married the daughter of Anthony and Cleopatra. He wrote that one of the cactus-like Euphorbias was a powerful laxative. In 12 B.C., Juba named this plant after his physician Euphorbus in response to Augustus Caesar dedicating a statue to Antonius Musa, his own personal physician. Botanist and taxonomist Carl Linnaeus assigned the name Euphorbia to the entire genus in the physician's honor (Nancy, 1986; Linnaeus, 1753). According to recent studies of DNA sequence data (Victor and Mark, 2002; Victor, 2003; Peter et al., 2006), most of the smaller "satellite genera" around the huge genus Euphorbia nest deep within the latter. Consequently these taxa, namely the never generally accepted genus Chamaesyce as well as the smaller genera Cubanthus, Elaeophorbia, Endadenium, Monadenium, Synadenium and Pedilanthus were transferred to Euphorbia (Víctor et al., 2007). The entire subtribe Euphorbiinae now consists solely of the genus Euphorbia. Some issues has been reported; the plants description and uses (Carter, 2002), the proximate and the nutrient contents (Omale and Emmanuel (2010), the skin irritating and caustic effects (Tom et al., 2000) and classification (Victor and Mark, 2002; Victor, 2003; Peter et al., 2006).

2.2.1. Euphorbia trigona E. trigona is a cactus commonly known as the African milk tree. It originates from Africa and is a popular houseplant in other parts of the world. E. trigona is a spiny shrub that contains a milky sap in its leaves. It grows to 6 feet tall, and some cultivars have red leaves. E. trigona is highly tolerant of drought, as are other species of cactus, and propagates easily from cuttings. E. trigona (also known as "High Chaparall" and "African Milk Tree") is a perennial plant that originally comes from West Africa. It has an upright stem that is branched into three or four sides. The stem itself is dark green with V-shaped light green patterns. The about 5mm long thorns are placed in pairs of two on the stem's ridges. The drop shaped leafs grows from between the two thorns on each ridge. The plant's flowers are white or a combination of white and light yellow. The flowers appear on spring and summer, but potted versions of the plant may not grow flowers at all. The sap (or latex) from the plant can is venomous and can cause skin irritations (EHOW, 2014). The Basque Institute for Agricultural Research and Development has identified, isolated and characterized anti-tumor proteins present in the latex of the plant E. trigona. The purified proteins can inhibit the growth of several tumor cell lines. This property shows that the latex proteins could be considered in clinical trials for cancer treatment due to its anti-tumor activity. Latex comprises various substances including proteins that play a very active role in defending the plant, like proteases, chitinases, oxidases and lectins. These latter proteins constitute a very valuable tool for studying membrane structure and for detecting malignant transformations, among other types of research. Identifying plants which have proteins of interest in biomedicine is one of scientific objectives. In the latex of E. trigona, the scientisit found three proteins belonging to the Ribosome Inactivating Protein (RIP) family. The purified proteins were able to inhibit eukaryotic ribosomes in cell-free systems, and also showed cytotoxic activity (the ability to inhibit cell growth) when tested with different tumor cell lines. In addition to antitumor activity, a possible antifungal activity of the lectins latex. This potential antifungal activity opens an interesting line of research in finding new uses for latex of E. trigona against various diseases (ScienceDaily, 2012). 2.2.2. Euphorbia abyssinica Desert candle (Euphorbia abyssinica J.F. Gmel.) is a succulent tree of dry deciduous and evergreen montane forest, woodland and shrub savanna. It occurs widely throughout dryland

Africa, where it is appreciated as a live fence because it is easily propagated from untreated mature branch cuttings. Euphorbia abyssinica, was noticed to be dominated in Erkwit plateau, Red Sea Mountain, Sudan (Zahran, 2010). The ability of large branches to regenerate with ease in dry soil may be related to the natural plant growth regulator hormone indole acetic acid (IAA) contained in the latex of the plant. The latex samples from northern Ethiopia that were chemically analyzed contained on average 0.06 mg/l latex, as well as the IAA metabolites indole lactate (ILA) and indole ethanol (IEt). One sample also contained IAA conjugated to amino- acids and to glucose (Aerts et al., 2008). Phytochemical investigation of the latex of Euphorbia abyssinica afforded a new hydroxy unsaturated fatty acid, 8(R)-hydroxy-dec-3(E)-en-oic acid, in addition to the four known compounds lupeol, β-sitosterol, oleanolic acid and β-sitosterol-3-O- glucoside. The in vitro antibacterial and antifungal activities of the isolated compounds, as well as the total methanol extract, were studied against different micro-organisms; compound 1 displayed reasonable antifungal activities towards the tested fungi (Fiky et al., 2008). The development of alternative sources for energy and chemicals, particularly the use of plant biomass as a renewable source for fuel or chemical feedstocks from euphorbia plants, has received much recent attention (Kalita, 2008). Treatment of skin diseases remains a matter of concern due to recurrence of the diseases and the difficulties to cure by available drugs. The latex of Euphorbia abyssinica is traditionally claimed for treatment of skin diseases; however its effectiveness is not proven scientifically. This study was conducted to evaluate the effectiveness of the latex of E. abyssinica against common skin diseases and to identify secondary metabolites in the plant. Ointment containing 25 % (v/v) latex of the plant was prepared with honey. Ivermectin, Tetracycline and clotrimazole were used as positive controls against demodex, dermatophillosis and ringworm respectively. Effectiveness of the ointment was tested on cattle naturally infected by dermatophillosis, demodex and ringworm. A cure rate of 100% was obtained against demodex and ringworm infections. However the ointment had cure rate of 25% against dermatophillosis. Honey did not cure any of the diseases (cure rate 0 %). Polyphenols, Saponines, Phytosteroides Cardiac glycosides, Tannins and Alkaloids have been detected in the phytochemical analysis of the plant. This study confirmed that the plant E. abyssinica contains antibacterial and antiparasitic constituents' hints that potential drugs can be isolated from it (Berhan et al., 2014).

2.2.3 Researches on Euphorbia species Researches on parts in various Euphorbia species include the roots, seeds, latex, lactiferous tubes, stem wood, stem barks, leaves, and whole plants. Many studies have suggested that these plants have not only therapeutic relevance but that they also display toxicity (Hohmann and Molnár, 2004). Some constituents of Euphorbia species may be promising lead compounds for Certain Euphorbia species have been reported to possess antitumor activity and have been recommended for use as anticancer remedies (Ahmad et al., 1988; Duarte et al., 2006). Their antitumor activity was mainly attributed to the presence of abietane diterpene derivatives, most of which contain lactone structures reported to possess potent antineoplastic activity toards various cancer cell lines (Yan et al., 2008; Luo and Wang, 2006). Moreover, some Euphorbia species have been also used as medicinal plants for the treatment of skin diseases, gonorrhea, migraines, intestinal parasites, and warts and for mediating pain perception. Many researchers have shown that Euphorbia species also possess antiproliferative activity, cytotoxicity, antimicrobial activity, antipyretic-analgesic activity, inhibition of HIV-1 viral infection, inhibitory activity on the mammalian mitochondrial respiratory chain, etc. As mentioned, there are also some reports of toxicity in Euphorbia species. Their toxic substances originate from the milky sap, which is a deterrent to insects and herbivores. Besides, they may possess extreme proinflammatory and tumor promoting toxicities. Severe pain and inflammation can result from contact with the eyes, nose, mouth and even skin, which may be due to the activation of protein kinase C enzyme (Kedei et al., 2004). 2.3. Phytochemicals Phytochemicals are chemical compounds that occur naturally in plants (phyto means "plant" in Greek). Some are responsible for color and other organoleptic properties, such as the deep purple of blueberries and the smell of garlic. The term is generally used to refer to those chemicals that may have biological significance, for example antioxidants, but are not established as essential nutrients. Scientists estimate that there may be as many as 10,000 different phytochemicals having the potential to affect diseases such as cancer, stroke or metabolic syndrome (FDA, 2000).

Compounds in plants (apart from vitamins, minerals, and macronutrients) that have a beneficial effect to the human body are termed phytonutrients. There are over 10,000 of them, and they have effects such as antioxidant, boosting the immune system, anti-inflammatory, antiviral, antibacterial, and cellular repair. Highly colored vegetables and fruits tend to be highest in these chemicals, but tea, chocolate, nuts, flax seeds, and olive oil are all excellent sources as well. Various families of plants tend towards certain families of phytonutrients, for example, orange foods tend to have the caretenoid group (Brown and Arthur, 2001; Papp et al., 2007).

2.4. Chromatography: Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for more advanced use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved (Vogel et al., 2003). Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Specific examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents (Reich and Schibli, 2007).

A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantitative analysis. This method is referred to as HPTLC, or "high-performance TLC". TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as thick slurry on an un-reactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110°C. The thickness of the absorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC (Vogel et al., 2003).

Chapter Three Materials and Methods

3.1 Samples Stems of the cactus plant (Euophorbia trigona), were collected from within Wad Medani Town, Gezira State, whereas one large stem of Euphorbia abyssinica, plant was brought from Erkwit Mountain, Red Sea State. The collected parts were transferred directly to the Faculty of Engineering and Technology, University of Gezira, during October 2014, where all of the laboratory tests were done. The collected stems of both plants were firstly dissected into outer parts (cortex) and inner part (pith). These parts were dried separately, at room temperature away from direct sunlight, and then grounded to fine particles by using mortar and pestle. One part of the prepared plant materials was subjected to the toxicity tests (against mosquito larvae), while the other part were subjected to phytochemical screening (presence of the main classes) and thin layer chromatography, using recommended methods. 3.2. Methods 3.2.1. The phytochemical screening 3.2.1.1. For glycosides About 3.0 g of the dried powder of each stem part were boiled with an aliquot of distilled water (100) ml and filtered. Aliquots (2 ml each) of the filtrate were tested for glycosides as described by Sofowora (1993) and Trease and Evans (1989). The filtrate was dissolved in 2 ml of glacial acetic acid. To this solution two drops of ferric chloride solution were added and mixed. The mixture was transferred to a narrow test tube. 2 ml conc H2SO4 was added carefully on the side of the tube using a pipette to form upper layer gradually acquired a bluish green colour which darkened on standing. 3.2.1.2. For flavonoids and flavonones About 2.0 g of the dried powder of each stem part was macerated in 50 ml (1%) of hydrochloric acid over night, filtered and the filtrate was subjected to the following tests a) About 10 ml from each filtrate was rendered alkaline with sodium hydroxide 10% w/v; the yellow colour indicated the presence of flavonoids.

20 b) Shinoda’s test: 5 ml of each filtrate was mixed with concentrated HCl 1 ml and magnesium ions were added. The formation of red colour indicated the presence of flavonoids, flavonones and or flavonols (Harbone, 1973). 3.2.1.3. For saponins: About 2 g of the dried powder of each stem part were extracted with 20 ml ethanol (50%) and filtered. Aliquot of the alcoholic extracts (10 ml) were evaporated to dryness under reduced pressure. The residue was dissolved in distilled water (2 ml) and filtered. The filtrate was vigorously shaken; if a voluminous forth was developed and persisted for almost one hour, this indicated the presence of saponins (Harbone, 1973). 3.2.1.4. For tannins: Five grams of the dried powder sample were extracted with ethanol (50%) and filtered. Ferric chloride reagent (5%w\v in methanol) was added. The appearance of green colour which changed to a bluish black colour or precipitate indicated the presence of tannins (Harbone, 1973). 3.2.1.5. For sterols and or triterpenes: One gram of the dried powder of each stem part was extracted with petroleum ether (10 ml) and filtered. The filtrate was evaporated to dryness and the residue was dissolved in chloroform (10 ml). Aliquots of chloroform extract (3 ml) were mixed with concentrated acetic acid anhydride (3 ml), and a few drops of sulphuric acid were added. The formation of a reddish violet ring at the junction of the two layers indicated the presence of unsaturated sterols and\or triterpenes (Harbone, 1973). 3.2.1.6. For alkaloids Five grams of the dried powder of each stem part were extracted with ethanol and filtered. 10 ml of the ethanolic extract were mixed with hydrochloric acid (10 ml; 10%v/v) and filtered. The filtrate was rendered alkaline with ammonium hydroxide and extracted with successive portions of chloroform. The combined chloroform-extract was evaporated to dryness. The residue was dissolved in hydrochloric acid (2 ml; 10%v/v) and tested with Mayer’s reagent, and Dragendorff’s reagent, respectively. The formation of a precipitate was indicated the presence of alkaloids and \or nitrogenous bases (Harbone, 1973).

3.3. Thin layer chromatography test Identification of the individual components of each stem part of the crude polar (methanol) or apolar (petroleum ether) extracts by thin layer chromatography according to Vogel et al., (2003). The solution was applied as a band, 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 (chloroform : 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 value = the distance driven by component (cm) the distance driven by solvent

3.4. Toxicity test of stem parts powder Twenty of mosquitos' larvae (either Anopheles or Culex) were put in a beaker containing 250 ml water and 0.3 g of the dried powder of stem pith or cortex, which were thrown gently and individually on those beakers. Each test was triplicate. After 24 hours, the dead larvae were counted as percentage mortalities according to WHO (1980). A control batch was also designed so as to correct the tested mortalities. Whenever the mortality in the control batch reached 20%, the test will be canceled. The toxicity of each stem part from both plants were evaluated against mosquito larvae.

3.5. Statistical analysis Microsoft office, Excel program, 2007, was used analyze the obtained data. Simple descriptive statistics and ANOVA single factor were also used to clear the differences observed in the values of the stem parts of Euphorbia plants.

Chapter Four Results and Discussion

4.3. The phytochemical screening in the stem parts The presence or absence of some phytochemicals in E. trigona and E. abyssinica, stem cortex and stem pith were presented in table (4.1). Concerning E. trigona plant, saponins, alkaloids, flavonoids and flavonones, glycosides, sterols and triterpenoids were detected in stem pith, while tannins was not detected. The phytochemical composition in the stem cortex was similar completely to those of the stem pith of the same plant. The flavonoids and flavonones of the stem pith were noticed to be more concentrated in comparison to that of stem cortex. Concerning E. abyssinica plant, saponins, alkaloids, flavonoids and flavonones, glycosides, sterols and triterpenoids (as same as E. trigona) were detected in stem pith, while tannins was not detected. The phytochemical composition in the stem cortex was similar completely to those of the stem pith of the same plant and of the stem pith and cortex of the other plant. The alkaloids and the glycocides of the stem cortex were noticed to be more concentrated in comparison to that of stem pith. It was clear that, the screening for the presence of the main classes in the stem parts of both plants, detected the presence and absence of phytochemicals in all samples similarly, but with different concentrations for some classes, even in the same plant. Nashikkar et al., (2012), detected the presence of sterols, alkaloids, tannins (which was not detected in this study), flavonoids and saponins in E. trigona using different extracts. The enrichment of E. trigona and E. abyssinica stems with their detected phytochemicals makes them a source of medicine for several diseases according to Kumar et al., (2010).

Table (4.1): The presence and absence of some phytochemicals in stem pith and cortex of both plants

E. trigona E. abyssinica Item Pith Cortex Pith Cortex

Tannins _ _ _ _ Saponins + + + + Alkaloids + + + ++ Flavonoids and ++ + + + flavonones Glycosides + + + ++ Sterols + + + + Triterpenes + + + +

++ indicated the presence of the class (in relatively high concentration), + indicated the presence of the class, – indicated the absence of the class.

4.2 Thin layer chromatography tests for the stem parts of both plants The results obtained from the TLC separation tests for the polar and apolar extracts of the stem pith and stem cortex of E. trigona and E. abyssinica were presented in Table (4.2). The TLC tests of the polar and apolar extracts of stem pith and cortex of E. trigona (using solvent mixture of chloroform: acetone; 2:1) proved the present of one spot for each stem parts per each extract, but with different Rf values, and hence, each part of the stem contained two different spots (active ingredients) one in the polar component and the other in the apolar component. The TLC of E. abyssinica (using solvent mixture of chloroform: acetone; 2:1) proved the present of one spot for each stem parts per each extract (as same as E. trigona), but also with different Rf values, and hence, each part of the stem contained two different active ingredients; one in the polar component and the other within the apolar component. A similar test conducted by Anjaneyulu et al., (1985) found that, the TLC for E. trigona resulted in a single spot only.

Table (4.2) TLC (Rf value) for the stem parts of both plants

E. trigona plant

Spot Pith Pith Cortex Cortex

No. (petroleum ether) (ethanol) (petroleum ether) (ethanol)

1 0.77 0.60 0.68 0.51 2 - - - -

E. abyssinica plant

Spot Pith Pith Cortex Cortex

No. (petroleum ether) (ethanol) (petroleum ether) (ethanol)

1 0.34 0.55 0.49 0.54 2 - - - -

4.3 Toxicity of E. trigona and E. abyssinica stem parts against mosquito larvae The toxicity of 0.3 g/250 ml water (1200 mg/L) powder of E. trigona (which were brought from Wad Medani) and E. abyssinica (which were brought from Wad Erkwit areas) stem parts, toward Anopheles and Culex larvae, were presented in Table (4.3). Concerning E. abyssinica samples, the stem pith produced 31.67% mortality in Anopheles and 21.67% mortality in Culex larvae, whereas, the stem cortex produced 41.67% mortality in Anopheles and 33.33% mortality in Culex. It seemed that, Anopheles larvae were more susceptible toward the stem pith and cortex powders than Culex larvae, and also, stem cortex was more toxic than stem pith of the same plant. Concerning E. trigona samples, the stem pith produced 46.67% mortality in Anopheles and 38.33% mortality in Culex larvae, whereas, the stem cortex produced 41.33% mortality in Anopheles and 28.33% mortality in Culex. It seemed that, Anopheles larvae were more susceptible toward the stem pith and cortex powders than Culex larvae, and also, stem pith was more toxic than stem cortex of the same plant. The analysis revealed that, Euphorbia plants could produced a mean of about 40.33% mortality in Anopheles larvae and 30.42% mortality in Culex larvae. Also, the pith part, of E. trigona from Wad Medani area produced relatively high mortality (42.50%) than cortex part (34.83%), while that, the cortex part of E. abyssinica produced relatively high mortality (37.5%) than the pith part (26.67%). ANOVA proved that, the susceptibility of Anopheles and Culex larvae toward Euphorbia different parts were not similar (f= 81.52; f-crit= 10.13, hence, the difference is significant), and it also proved that, the toxicity of the used plant part were statistically not similar (f=36.27; f- ctir=9.28, hence, the difference is significant). The reason for the variation in the toxicity of different Euphorbia stem parts could be attributed to their chemical and phytochemical constituents in addition to the internal defense mechanisms of each mosquito larvae. The TLC tests could also confirm the reason. Only one spot was separated from the polar and another one spot from the apolar extract of each stem part from each plant.

Table (4.3) The percentage mortalities of Aopheles and Culex larvae toward the powders of E. trigona and E. abyssinica stem parts (at 0.3 g/ 250 ml)

Larvae E. abyssinica E. trigona

Pith Cortex Pith Cortex

Anopheles 31.67 41.67 46.67 41.33 Culex 21.67 33.33 38.33 28.33 The control mortality was zero

Variance Average Sum Count SUMMARY 39.33 40.34 161.34 4 Anopheles 50.66 30.42 121.66 4 Culex

40.5 0 26. 67 53.34 2 Pith – abyssinica

34.78 37.50 75 2 Cortex- abyssinica

34.78 42.50 85 2 Pith –trigona

84.50 34.83 69.66 2 Cortex -trigona

ANOVA F crit P-value F MS df SS Source 10.13 0.003 81.52 196.81 1 196.81 Rows 9.28 0.007 36.27 87.57 3 262.71 Columns 2.41 3 7.24 Error

7 466 .77 Total

Chapter Five

Conclusions and Recommendations

5.1 Conclusions 1- All plant samples contained the same phytochemicals similarly, but with different concentrations for some classes, even between the pith and cortex of the same plant. 2- The TLC tests of E. trigona separated one spot from each of the stem pith and cortex per each extract, but with different Rf values. The same was noticed in E. abyssinica, but also with different Rf values. 3- ANOVA proved that, the susceptibility of Anopheles and Culex larvae toward Euphorbia different parts were not similar, and it also proved that, the toxicity of the used plant parts were statistically not similar.

5.2 Recommendations 1- Proper chemical and phytochemical tests (instead of some tests) should be run 2- Antimicrobial and pharmaceutical activity tests should be run

3- Proper toxicity test (involve the obtain of LD50 and LD95) should be run

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