Phytochemical Screening for Leaves,Cortex and Pith of the trigona L.

Ola Ali Abdelmageed Boshara B.Sc. (Hons.) in Pharmacy, Faculty of Pharmacy, University of Gezira , 2004

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

in Biosciences and Biotechnology (Biotechnology) Center of Biosciences and Biotechnology Faculty of Engineering and Technology University of Gezira

May 2014

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Phytochemical Screening for Leaves, Cortex and Pith of the Cactus Euphorbia trigona L.

Ola Ali Abdelmageed Boshara

Supervision Committee

Name Position Signature Dr. Mutaman Ali Kehail Main Supervisor ………………………… Dr. Nizar Sirag Eltayeb Co-Supervisor ………………………… Date: May 2014

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Phytochemical Screening for Leaves,Cortex and Pith of the Cactus Euphorbia trigona L.

Ola Ali Abdelmageed Boshara

Examination Committee

Name Position Signature Dr. Mutaman Ali Kehail Chair Person ……………………..… Dr. Eltayeb Mohamed Teyrab External Examiner ……………………..… Dr. Elnour Elamin Abdelrahman Internal Examiner ……………………..…

Date of Examination: 16 May 2014

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Dedication

To me..

“ Success is the journey, not just the destination.”

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Acknowledgements

I would like to aknowledge Dr. Mutaman Ali Kehail for his great assistance with his knowledge and experience. I also aknowledge Dr. Nizar Sirag for his great assistance in this work. Deepfull thanks for my future husband Ahmad Abdelgaffar for support,encouragement, and helpfull opinions.

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Phytochemical screening for Leaves,Cortex and Pith of the Cactus Euphorbia trigona L. Ola Ali Abdelmageed Boshara

Abstract

The vast majority of people on this planet still rely on their traditional medicine for their everyday health care needs. The present study aimed to run a phytochemical screening for the leaves, stem cortex and pith of Euophorbia trigona L. materials, were collected from within Wad Medani City. The targeted parts were washed while they were in their original plant. The collected parts were transferred directly to the Faculty of Engineering and Technology, University of Gezira, where all of the laboratory tests were done. The experimental tests involved: proximate analysis (percentage moisture, ash and protein contents), the percentage polar and apolar contents and the phytochemical screening for the main components in the three plant parts. The results of this work revealed that, the proximate analysis of Euphorbia trigona parts indicated that, stem pith had significantly, the highest percentages of moisture contents (95.76%), while the moisture in the stem cortex (89.91%) were also more than that of leaves (82.72%). Stem pith was found to possess significantly, the lowest percentages of ash contents (3.05%) than that of the stem cortex (7.70%) and leaves (7.71%). Stem pith showed the lowest percentages of protein contents (0.91%), whereas, the protein content in leaves (2.77%) were the highest. The percentage mean polar content of the leaves (10.47) was more than other parts collectively (6.77 + 2.88%). Meanwhile, the apolar content was relatively high in stem cortex (1.37%) than the other parts (0.81and 1.07%). The preliminary phytochemical screening of E. trigona leaves revealed the presence of saponins, alkaloids, flavonoids, flavonones and flavonols, glycosides and sterols and triterpenoids while tannins were not detected while the stem cortex and stem pith contained all phytoconstituents except tannins and flavonones. It can be concluded that, E. trigona could be a potential source of active principles which may help to discover new chemical classes of drugs that could serve as selective agents for the maintenance of health, and hence, this study recommends to run a further studies to identify the active constituents, as well as to verify the biological activities of this .

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المسح الكيميائي النباتي لمختلف أجزاء نبات صبار اإليوفوربيا ترايقونا

عال علي عبد المجيد بشارة

ملخص الدراسة

يعتمد غالبية سكان هذا الكوكب علي الطب التقليدي في االحتياجات اليومية للمحافظة علي الصحة. هدفت هذه الدراسة

إلجراءمسح كيميائي نباتي ألوراق, قشرة ساق ولب ساق نبات صبار االيوفوربيا ترايقونا. جمعت العينات النباتية من داخل

مدينة ود مدني. غسلت اء االجزالمستهدفة أثناء وجودها في النبات االصل. تم تحويل االجزاء المجمعة الي كلية الهندسة

والتكنولوجيا, جامعة الجزيرة حيث أجريت جميع االختبارات المعملية. شملت االختبارات المعملية: التحليل التقريبي )النسبة

المئوية للرطوبة, الرماد والبروتين(, النسبة المئوية لل مكونات القطبية والالقطبية, إضافة الي المسح الكيميائي النباتي للمكونات

الرئيسية في اجزاء النبات الثالثة. أوضحت النتائج, أن التحليل التقريبي قد دلل علي أن لب الساق يحتوي بصورة معنوية علي

أعلي نسبة رطوبة )95.76%(, بينما كانت الرطوبة في قشرة الساق )89.91%( ايضا اعلي من االوراق )82.72%(. وجد

أن لب الساق يحتوي معنوياً علي أقل نسبة رماد )3.05%( عن قشرة الساق )7.70%( واالوراق )7.71%(. يحتوي لب

الساق علي أقل نسبة بروتين )0.91%(, في حين أن بروتين االوراق )2.77%( هو األعلي. متوسط نسبة المكونات القطبية

في االوراق )10.47( كانت اكثر من ما في بقية االج ازء مجتمعةً )6.77 + 2.88%(. أما بالنسبة للمكونات الالقطبية, فقد

كانت عالية معنوياً في قشرة الساق )1.37%( عن االجزاء االخري )0.81 و 1.07%(. أوضح المسح الكيميائي االولي

لالوراق وجود المواد الصابونية, القلويدية, الفالفونويدية, الفالفونولية والفالفونونية, الجاليكوسيدات واالستروالت والتريبينويدات,

في حين أن التانينات لم يتم الكشف عنها, بينما احتوت قشرة ولب الساق علي كل المكونات الكيميائية النباتية عدا التانينات

والفالفونونات. يمكن وضع الخالصة بأن نبات االيوفوربيا ترايقونا يمكن ان يكون مصدر محتمل لمكونات فعالة يمكن ان

تساعد في اكتشاف اصناف دوائية كيميائية جديدة يمكنها أن تشكل عامل انتقائي للمحافظة علي الصحة, وعليه, فقد اوصت

هذه الدراسة بإجراء المزيد من الدراسات المستقبلية للتعرف علي المكونات الفعالة وكذلك تحديد الفاعلية الحيوية لهذا النبات.

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List of Contents

Subject Page Dedication iv Acknowledgements v Abstract vi Abstract (Arabic) vii Table of contents viii List of Tables x List of Figures and Plates xi Chapter One: Introduction 1 Chapter Two: Literature Review 3 2.1. Cactus 3 2.1.1. Adaptations 3 2.1.2. Morphology 3 2.1.3. Growth habit 4 2.1.4. Stems 5 2.1.5. Areoles 5 2.1.6. Leaves 6 2.1.7. Spines 6 2.1.8. Roots 7 2.1.9. 7 2.1.10. Adaptations for water conservation 8 2.1.11. 9 2.1.12. Uses of cactus 10 2.2. Euphorbia: 14 2.2.1. Description 15 2.2.2. Xerophytes and succulents plants 15 2.2.3. Irritance 17 2.2.4. Uses of Euophorbia 17 2.2.5. Systematics and taxonomy 17 2.2.6. Euphorbia trigona 18 2.2.6.1. Scientific Classification: 18 2.2.6.2. Medicinal benefits: 18

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2.2.7 Researches on Euphorbia species 19 2.3. Phytochemicals 21 2.3.1. Clinical trials and health claim status 21 2.3.2. Food processing 21 2.3.3. Screening and Extraction: 23 2.3.4. Plant material 26 2.3.5. Choice of solvents 26 2.4. Proximate Analysis: 28 2.5. Chromatography: 28 2.5.1. Plate preparation in TLC 29 2.5.2. Procedure: 29 2.5.3. Separation Process and Principle 30 2.5.4. Analysis 31 2.5.5. Applications 31 Chapter Three: Materials and Methods 33 3.1 Samples of plant material: 33 3.2. Preparation of plant material 33 3.3. Approximate analysis 33 3.3.1. Moisture content 33 3.3.2. Ash content 34 3.3.3. Protein content 34 3.4. The polar and apolar contents 35 3.5. Phytochemical screening for the different parts of E. trigona: 36 3.5.1. Qualitative analysis Test for glycosides 36 3.5.2. Qualitative analysis Test for flavonoids 36 3.5.3. Qualitative analysis Test for saponins: 36 3.5.4. Qualitative analysis Test for tannins: 36 3.5.5. Qualitative analysis Test for sterols and or triterpenes: 37 3.5.6. Qualitative analysis Test for alkaloids and /or nitrogenous bases 37 3.7. Statistical analysis 37 Chapter Four: Results and Discussion 38 4.1. The proximate analysis of Euphorbia trigona parts 38 4.2. The polar and apolar contents of Euphorbia trigona parts 44 4.3. The phytochemical screening in Euphorbia trigona parts 47 Chapter Five: Conclusions and Recommendations 49 References 50

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List of Tables

Table No. Title Page 2.1 Proximate and phytochemical composition of Euphorbia heterophylla 16 2.2 Phytochemical analysis of E. trigona extract 20 2.3 Activities of various phytochemicals from plants 22 2.4 Solvents used for active component extraction 25 4.1 The moisture contents (%) in Euphorbia trigona parts 40 4.2 The ash contents (%) in Euphorbia trigona parts 41 4.3 The protein contents (%) in Euphorbia trigona parts 42 4.4 The polar contents (%) in Euphorbia trigona parts 45 4.5 The apolar contents (%) in Euphorbia trigona parts 46 4.6 The presence of some phytochemical in E. trigona parts 48

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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 also eat prickly pears. These insects produce a red coloring used in food and lipstick (Anderson, 1999). 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 deciduous leaves are opposite, alternate or in whorls. In succulent species the leaves are mostly small and short-lived. The stipules are mostly small, partly transformed into spines or glands, or missing. Like all members of the family , all spurges have unisexual flowers. In Euphorbia these are greatly reduced and grouped into pseudanthia called cyathia. The majority of species are monoecious (bearing male and female flowers on the same plant), although some are dioecious with male and female flowers occurring on different plants. It is not unusual for the central cyathia of a cyme to be purely male, and for lateral cyathia to carry both sexes. Sometimes young plants or those growing under unfavorable conditions are male only, and only produce female flowers in the cyathia with maturity or as growing conditions improve. The bracts are often leaf-like, sometimes brightly coloured and attractive, sometimes reduced to tiny scales. 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

11 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).

Main Objective: The objective of the study is to run a phytochemical screening for different parts of the cactus Euphorbia trigona L. Specific objectives: 1- To run a proximate analysis for leave, stem cortex and stem pith of E. trigona 2- To determine the polar and apolar percentages in the three parts of E. trigona 3- To determine the phyochemicals in the parts of E. trigona

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Chapter Two Literature Review 2.1. Cactus cacti is a kind of plants. Its xerophytic, specializing in hot, dry climates. Cacti are members of the plant family Cactaceae, in the order . 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 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. Adaptations An adaptation is anything that helps a living thing survive and make more of its own kind. Cacti have many adaptations for living in places that are sometimes dry for a long time. At other times these places can get lots of rain. Cacti can have many small, thin roots near the top of the soil. These roots take in water quickly after a rain. The same cactus may have one long, thick root called a taproot. The taproot grows deep in the soil. It can reach water when the soil on top is dry. Cacti store water in thick stems. The stems are covered with tough skin, and the skin is covered with wax. The thick waxy skin slows down loss of water. The leaves of cacti are sharp spines (thorns, stickers). Many want the water inside the cactus, but the sharp spines and thick skin protect the cactus (Anderson, 2001). 2.1.2. 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 ). Most members of these two groups are easily recognizable as cacti. They have fleshy succulent stems that are major organs of photosynthesis. Their leaves are either , small, transient or absent . 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 parts grow). All cacti have areoles—

13 highly specialized short shoots with extremely short internodes that produce spines, normal shoots, and flowers. The remaining cacti fall into only two genera, Pereskia and Maihuenia, and are rather different, which means any description of cacti as a whole must frequently make exceptions for them. Pereskia species superficially resemble other tropical forest trees. When mature, they have woody stems that may be covered with bark and long-lasting leaves that provide the main means of photosynthesis. Their flowers may have superior ovaries (i.e., above the points of attachment of the sepals and petals), and areoles that produce further leaves. The two species of Maihuenia have small, globe-shaped bodies with prominent leaves at the top (Edwards and Donoghue, 2006). 2.1.3. Growth habit Cacti show a wide variety of growth habits, which are difficult to divide into clear, simple categories. They can be tree-like (arborescent), meaning they typically have a single more-or-less woody trunk topped by several to many branches. In the genus Pereskia, the branches are covered with leaves, so the species of this genus may not be recognized as cacti. In most other cacti, the branches are more typically cactus-like, bare of leaves and bark, and covered with spines, as in Pachycereus pringlei or the larger opuntias. Some cacti may become tree-sized but without branches, such as larger specimens of Echinocactus platyacanthus. Cacti may also be described as shrubby, with several stems coming from the ground or from branches very low down, such as in thurberi(Anderson,2001). Smaller cacti may be described as columnar. They consist of erect, cylinder-shaped stems, which may or may not branch, without a very clear division into trunk and branches. The boundary between columnar forms and tree-like or shrubby forms is difficult to define. Smaller and younger specimens of Cephalocereus senilis, for example, are columnar, whereas older and larger specimens may become tree-like. In some cases, the "columns" may be horizontal rather than vertical. Thus, Stenocereus eruca has stems growing along the ground, rooting at intervals. Cacti whose stems are even smaller may be described as globular (or globose). They consist of shorter, more ball-shaped stems than columnar cacti. Globular cacti may be solitary, such as Ferocactus latispinus, or their stems may form clusters that can create large mounds. All or some stems in a cluster may share a common root(Anderson,2001).

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Other cacti have a quite different appearance. In tropical regions, some grow as forest climbers and epiphytes. Their stems are typically flattened, almost leaf-like in appearance, with fewer or even no spines. Climbing cacti can be very large; a specimen of Hylocereus was reported as 100 meters (330 ft) long from root to the most distant stem. Epiphytic cacti, such as species of Rhipsalis or Schlumbergera, often hang downwards, forming dense clumps where they grow in trees high above the ground (Anderson, 2001). 2.1.4. 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) The stems of most cacti are some shade of green, often bluish or brownish green. Such stems contain chlorophyll and are able to carry out photosynthesis; they also have stomata (small structures that can open and close to allow passage of gases). Cactus stems are often visibly waxy. Areoles are structures unique to cacti. Although variable, they typically appear as woolly or hairy areas on the stems from which spines emerge. Flowers are also produced from areoles. In the genus Pereskia, believed similar to the ancestor of all cacti, the areoles occur in the axils of leaves (i.e. in the angle between the leaf stalk and the stem). In leafless cacti, areoles are often borne on raised areas on the stem where leaf bases would have been (Anderson, 2001). 2.1.5. Areoles Areoles are highly specialized and very condensed shoots or branches. In a normal shoot, nodes bearing leaves or flowers would be separated by lengths of stem (internodes). In an areole, the nodes are so close together, they form a single structure. The areole may be circular, elongated into an oval shape, or even separated into two parts; the two parts may be visibly connected in some way (e.g. by a groove in the stem) or appear entirely separate (a dimorphic

15 areole). The part nearer the top of the stem then produces flowers, the other part spines. Areoles often have multicellular hairs (trichomes) that give the areole a hairy or woolly appearance, sometimes of a distinct color such as yellow or brown. In most cacti, the areoles produce new spines or flowers only for a few years, and then become inactive. This results in a relatively fixed number of spines, with flowers being produced only from the ends of stems, which are still growing and forming new areoles. In Pereskia, a genus close to the ancestor of cacti, areoles remain active for much longer; this is also the case in Opuntia and Neoraimondia (Anderson, 2001). 2.1.6. Leaves The great majority of cacti have no visible leaves; photosynthesis takes place in the stems (which may be flattened and leaflike in some species). Exceptions occur in three groups of cacti. All the species of Pereskia are superficially like normal trees or shrubs and have numerous leaves. Many cacti in the opuntia group (subfamily Opuntioideae, opuntioids) also have visible leaves, which may be long-lasting (as in Pereskiopsis species) or be produced only during the growing season and then be lost (as in many species of Opuntia). The small genus Maihuenia also relies on leaves for photosynthesis. The structure of the leaves varies somewhat between these groups. Pereskia species have "normal" leaves, with a midrib and a flattened blade (lamina) on either side. Opuntioids and Maihuenia have leaves that appear to consist only of a midrib. Even those cacti without visible photosynthetic leaves do usually have very small leaves, less than 0.5 mm (0.02 in) long in about half of the species studied and almost always less than 1.5 mm (0.06 in) long. The function of such leaves cannot be photosynthesis; a role in the production of plant hormones, such as auxin, and in defining axillary buds has been suggested (Mauseth, 2007). 2.1.7. Spines Botanically, "spines" are distinguished from "thorns": spines are modified leaves, and thorns are modified branches. Cacti produce spines, always from areoles as noted above. Spines are present even in those cacti with leaves, such as Pereskia, Pereskiopsis and Maihuenia, so they clearly evolved before complete leaflessness. Some cacti only have spines when young, possibly only when seedlings. This is particularly true of tree-living cacti, such as Rhipsalis or Schlumbergera, but ground-living cacti, such as Ariocarpus, also lack spines when mature.

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The spines of cacti are often useful in identification, since they vary greatly between species in number, color, size, shape and hardness, as well as in whether all the spines produced by an areole are similar or whether they are of distinct kinds. Most spines are straight or at most slightly curved, and are described as hair-like, bristle-like, needle-like or awl-like, depending on their length and thickness. Some cacti have flattened spines (e.g. Schlerocactus papyracanthus). Other cacti have hooked spines. Sometimes, one or more central spines are hooked, while outer spines are straight (e.g., Mammillaria rekoi). In addition to normal-length spines, members of the subfamily Opuntioideae have relatively short spines, called glochids that are barbed along their length and easily shed. These enter the skin and are difficult to remove, causing long-lasting irritation (Anderson, 2001). 2.1.8. Roots Most ground-living cacti have only fine roots, which spread out around the base of the plant for varying distances, close to the surface. Some cacti have taproots; in genera such as Copiapoa, these are considerably larger and of a greater volume than the body. Taproots may aid in stabilizing the larger columnar cacti. Climbing, creeping and epiphytic cacti may have only adventitious roots, produced along the stems where these come into contact with a rooting medium (Anderson, 2001). 2.1.9. Flowers Like their spines, cactus flowers are variable. Typically, the ovary is surrounded by material derived from stem or receptacle tissue, forming a structure called a pericarpel. Tissue derived from the petals and sepals continues the pericarpel, forming a composite tube—the whole may be called a floral tube, although strictly speaking only the part furthest from the base is floral in origin. The outside of the tubular structure often has areoles that produce wool and spines. Typically, the tube also has small scale-like bracts, which gradually change into sepal- like and then petal-like structures, so the sepals and petals cannot be clearly differentiated (and hence are often called "tepals"). Some cacti produce floral tubes without wool or spines (e.g. Gymnocalycium) or completely devoid of any external structures (e.g. Mammillaria) unlike the flowers of other cacti, Pereskia flowers may be borne in clusters. Cactus flowers usually have many stamens, but only a single style, which may branch at the end into more than one stigma. The stamens usually arise from all over the inner surface of the upper part of the floral tube, although in some cacti, the stamens are produced in one or more

17 distinct "series" in more specific areas of the inside of the floral tube. The flower as a whole is usually radially symmetrical (actinomorphic), but may be bilaterally symmetrical (zygomorphic) in some species. Flower colors range from white through yellow and red to magenta (Anderson, 2001). 2.1.10. Adaptations for water conservation All cacti have some adaptations to promote efficient water use. Most cacti—opuntias and cactoids—specialize in surviving in hot and dry environments (i.e. they are xerophytes), but the first ancestors of modern cacti were already adapted to periods of intermittent drought. A small number of cactus species in the tribes Hylocereeae and Rhipsalideae have become adapted to life as climbers or epiphytes, often in tropical forests, where water conservation is less important.

Photosynthesis requires plants to take in carbon dioxide gas (CO2). As they do so, they lose water through transpiration. Like other types of succulents, cacti reduce this water loss by the way in which they carry out photosynthesis. "Normal" leafy plants use the C3 mechanism: during daylight hours, CO2 is continually drawn out of the air present in spaces inside leaves and converted first into a compound containing three carbon atoms (3-phosphoglycerate) and then into products such as carbohydrates. The access of air to internal spaces within a plant is controlled by stomata, which are able to open and close. The need for a continuous supply of

CO2 during photosynthesis means the stomata must be open, so water vapor is continuously being lost. Plants using the C3 mechanism lose as much as 97% of the water taken up through their roots in this way. A further problem is that as temperatures rise, the enzyme that captures

CO2 starts to capture more and more oxygen instead, reducing the efficiency of photosynthesis by up to 25% (Raven and Edwards, 2001). Crassulacean acid metabolism (CAM) is a mechanism adopted by cacti and other succulents to avoid the problems of the C3 mechanism. In full CAM, the stomata open only at night, when temperatures and water loss are lowest. CO2 enters the plant and is captured in the form of organic acids stored inside cells (in vacuoles). The stomata remain closed throughout the day, and photosynthesis uses only this stored CO2. CAM uses water much more efficiently at the price of limiting the amount of carbon fixed from the atmosphere and thus available for growth (Sharkey, 1988; Keeley and Rundel, 2003). CAM-cycling is a less efficient system whereby stomata open in the day, just as in plants using the C3 mechanism. At night, or when the plant is short of water, the stomata close and the CAM mechanism is used to store CO2 produced by

18 respiration for use later in photosynthesis. CAM-cycling is present in Pereskia species (Edwards and Donoghue, 2006). By studying the ratio of 14C to 13C incorporated into a plant—its isotopic signature—it is possible to deduce how much CO2 is taken up at night and how much in the daytime. Using this approach, most of the Pereskia species investigated exhibit some degree of CAM-cycling, suggesting this ability was present in the ancestor of all cacti (Edwards and Donoghue, 2006).

Pereskia leaves are claimed to only have the C3 mechanism with CAM restricted to stems (Keeley and Rundel, 2003). More recent studies show that "it is highly unlikely that significant carbon assimilation occurs in the stem"; Pereskia species are described as having "C3 with inducible CAM." Leafless cacti carry out all their photosynthesis in the stem, using full CAM. It is not clear whether stem- based CAM evolved once only in the core cacti, or separately in the opuntias and cactoids (Edwards and Donoghue, 2006); CAM is known to have evolved convergently many times (Sharkey, 1988). To carry out photosynthesis, cactus stems have undergone many adaptations. Early in their evolutionary history, the ancestors of modern cacti (other than one group of Pereskia species) developed stomata on their stems and began to delay developing bark. However, this alone was not sufficient; cacti with only these adaptations appear to do very little photosynthesis in their stems. Stems needed to develop structures similar to those normally found only in leaves. Immediately below the outer epidermis, a hypodermal layer developed made up of cells with thickened walls, offering mechanical support. Air spaces were needed between the cells to allow carbon dioxide to diffuse inwards. The center of the stem, the cortex, developed "chlorenchyma" – a plant tissue made up of relatively unspecialized cells containing chloroplasts, arranged into a "spongy layer" and a "palisade layer" where most of the photosynthesis occurs (Edwards and Donoghue, 2006). 2.1.11. Taxonomy Naming and classifying cacti has been both difficult and controversial since the first cacti were discovered for science. The difficulties began with Carl Linnaeus. In 1737, he placed the cacti he knew into two genera, Cactus and Pereskia. However, when he published Species Plantarum in 1753—the starting point for modern botanical nomenclature—he relegated them all to one genus, Cactus. The word "cactus" is derived through Latin from the Ancient Greek

19

κάκτος (kaktos), a name used by Theophrastus for a spiny plant, which may have been the cardoon (Cynara cardunculus) (Johnson and Smith, 1972; Sonnante et al., 2007). Later botanists, such as Philip Miller in 1754, divided cacti into several genera, which, in 1789, Antoine Laurent de Jussieu placed in his newly created family Cactaceae. By the early 20th century, botanists came to feel Linnaeus's name Cactus had become so confused as to its meaning (was it the genus or the family?) that it should not be used as a genus name. The 1905 Vienna botanical congress rejected the name Cactus and instead declared Mammillaria was the type genus of the family Cactaceae. It did, however, conserve the name Cactaceae, leading to the unusual situation in which the family Cactaceae no longer contains the genus after which it was named(Anderson,2001). The difficulties continued, partly because giving plants scientific names relies on "type specimens". Ultimately, if botanists want to know whether a particular plant is an example of, say, Mammillaria mammillaris, they should be able to compare it with the type specimen to which this name is permanently attached. Type specimens are normally prepared by compression and drying, after which they are stored in herbaria to act as definitive references. However, cacti are very difficult to preserve in this way; they have evolved to resist drying and their bodies do not easily compress A further difficulty is that many cacti were given names by growers and horticulturalists rather than botanists; as a result, the provisions of the International Code of Nomenclature for algae, fungi, and plants (which governs the names of cacti, as well as other plants) were often ignored. Curt Backeberg, in particular, is said to have named or renamed 1,200 species without one of his names ever being attached to a specimen, which, according to David Hunt, ensured he "left a trail of nomenclatural chaos that will probably vex cactus taxonomists for centuries (Anderson, 2001). 2.1.12. Uses of Cactus 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.

20

Hunter-gatherers likely collected cactus fruits in the wild and brought them back to their camps (Anderson, 2001). It is not known when cacti were first cultivated. Opuntias (prickly pears) were used for a variety of purposes by the Aztecs, whose empire, lasting from the 14th to the 16th century, had a complex system of horticulture. Their capital from the 15th century was Tenochtitlan (now Mexico City); one explanation for the origin of the name is that it includes the Nahuatl word nōchtli, referring to the fruit of an opuntia (Andrews, 2003). The coat of arms of Mexico shows an eagle perched on a cactus while holding a snake, an image at the center of the myth of the founding of Tenochtitlan (Aveni et al., 1988). The Aztecs symbolically linked the ripe red fruits of an opuntia to human hearts; just as the fruit quenches thirst, so offering human hearts to the sun god ensured the sun would keep moving. Europeans first encountered cacti when they arrived in the New World late in the 15th century. Their first landfalls were in the West Indies, where relatively few cactus genera are found; one of the commonest is the genus Melocactus. Thus, melocacti were possibly among the first cacti seen by Europeans. Melocactus species were present in English collections of cacti before the end of the 16th century by 1570 according to one source (Barroqueiro, 2012), where they were called Echinomelocactus, later shortened to Melocactus by Joseph Pitton de Tourneville in the early 18th century (Rowley, 1997). Cacti, both purely ornamental species and those with edible fruit, continued to arrive in Europe, so Linnaeus was able to name 22 species by 1753. One of these, his Cactus opuntia (now part of Opuntia ficus-indica), was described as "fructu majore ... nunc in Hispania et Lusitania" (with larger fruit ... now in Spain and Portugal), indicative of its early use in Europe (Linnaeus, 1753). The plant now known as Opuntia ficus-indica, or the Indian fig cactus, has long been an important source of food. The original species is thought to have come from central Mexico, although this is now obscure because the indigenous people of southern North America developed and distributed a range of horticultural varieties (cultivars), including forms of the species and hybrids with other opuntias (Griffith, 2004). Both the fruit and pads are eaten, the former often under the Spanish name tuna, the latter under the name nopal. Cultivated forms are often significantly less spiny or even spineless. The nopal industry in Mexico was said to be worth US$150 million in 2007 (Daniel, 2007). The Indian fig cactus was probably already present in the Caribbean when the Spanish arrived, and was soon after brought to Europe. It

21 spread rapidly in the Mediterranean area, both naturally and by being introduced—so much so, early botanists assumed it was native to the area. Outside the Americas, the Indian fig cactus is an important commercial crop in Sicily, Algeria and other North African countries. Fruits of other opuntias are also eaten, generally under the same name, tuna. Flower buds, particularly of Cylindropuntia species, are also consumed (Anderson, 2001). Almost any fleshy cactus fruit is edible. The word pitaya or pitahaya (usually considered to have been taken into Spanish from Haitian creole) can be applied to a range of "scaly fruit", particularly those of columnar cacti. 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. It can be preserved by boiling to produce syrup and by drying. The syrup can also be fermented to produce an alcoholic drink. Fruits of Stenocereus species have also been important food sources in similar parts of North America; Stenocereus queretaroensis is cultivated for its fruit. In more tropical southern areas, the climber Hylocereus undatus provides pitahaya orejona, now widely grown in Asia under the name dragon fruit. Other cacti providing edible fruit include species of Echinocereus, Ferocactus, Mammillaria, Myrtillocactus, Pachycereus, Peniocereus and Selenicereus. The bodies of cacti other than opuntias are less often eaten, although Anderson (2001) reported that Neowerdermannia vorwerkii is prepared and eaten like potatoes in upland Bolivia (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. Two species have a long history of use by the indigenous peoples of the Americas: peyote, Lophophora williamsii, in North America, and the San Pedro cactus, pachanoi, in . Both contain mescaline. L. williamsii is native to northern Mexico and southern Texas. Individual stems are about 2–6 cm (0.8–2.4 in) high with a diameter of 4– 11 cm (1.6–4.3 in), and may be found in clumps up to 1 m (3 ft) wide (Zimmerman et al.,1982). A large part of the stem is usually below ground. Mescaline is concentrated in the photosynthetic portion of the stem above ground. The center of the stem, which contains the growing point (the apical meristem), is sunken. Experienced collectors of peyote remove a thin slice from the top of the plant, leaving the growing point intact, thus allowing the plant to regenerate. Evidence indicates peyote was in use more than 5,500 years ago; dried peyote buttons presumed to be from a site on the Rio Grande, Texas, were radiocarbon dated to around 3780–3660 BC. Peyote is

22 perceived as a means of accessing the spirit world. Attempts by the Roman Catholic church to suppress its use after the Spanish conquest were largely unsuccessful, and by the middle of the 20th century, peyote was more widely used than ever by indigenous peoples as far north as Canada. It is now used formally by the Native American Church (Seedi. et al., 2005). is native to Ecuador and Peru. It is very different in appearance from L. williamsii. It has tall stems, up to 6 m (20 ft) high, with a diameter of 6–15 cm (2.4– 5.9 in), which branch from the base, giving the whole plant a shrubby or tree-like appearance. Archaeological evidence of the use of this cactus appears to date back to 2,000–2,300 years ago, with carvings and ceramic objects showing columnar cacti. Although church authorities under the Spanish attempted to suppress its use, this failed, as shown by the Christian element in the common name "San Pedro cactus"—Saint Peter cactus. Anderson(2002) attributes the name to the belief that just as St Peter holds the keys to heaven, the effects of the cactus allow users "to reach heaven while still on earth." It continues to be used for its psychoactive effects, both for spiritual and for healing purposes, often combined with other psychoactive agents, such as

Datura ferox and tobacco (Bussmann and Sharon, 2006). Cacti were cultivated as ornamental plants from the time they were first brought from the New World. By the early 1800s, enthusiasts in Europe had large collections (often including other succulents alongside cacti). Rare plants were sold for very high prices. Suppliers of cacti and other succulents employed collectors to obtain plants from the wild, in addition to growing their own. In the late 1800s, collectors turned to orchids, and cacti became less popular, although never disappearing from cultivation. Cacti are often grown in greenhouses, particularly in regions unsuited to the cultivation of cacti outdoors, such the northern parts of Europe and North America. Here, they may be kept in pots or grown in the ground. Cacti are also grown as houseplants, many being tolerant of the often dry atmosphere. Cacti in pots may be placed outside in the summer to ornament gardens or patios, and then kept under cover during the winter. Less drought-resistant epiphytes, such as epiphyllum hybrids, Schlumbergera (the Thanksgiving or Christmas cactus) and Hatiora (the Easter cactus), are widely cultivated as houseplants.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). For example in California, the East Bay Municipal Utility District sponsored the

23 publication of a book on plants and landscapes for summer-dry climates. 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 . Cochineal is a red dye produced by a scale that lives on species of Opuntia. Long used by the peoples of Central and North America, demand fell rapidly when European manufacturers began to produce synthetic dyes in the middle of the 19th century. Commercial production has now increased following a rise in demand for natural dyes(Shetty et al.,2011). Cacti are used as construction materials. Living cactus fences are employed as barricades. The woody parts of cacti, such as 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; it also has one of the largest ranges of chromosome counts, along with Rumex and Senecio. Members of the family and genus are commonly referred to as spurges. Euphorbia antiquorum is the type species for the genus Euphorbia; it was described by Carl Linnaeus in 1753 in Species Plantarum. 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 common name "spurge" derives from the Middle English/Old French espurge ("to purge"), due to the use of the plant's sap as a purgative. 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 was a powerful laxative. In 12 B.C., Juba named this plant after his physician Euphorbus in

24 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). 2.2.1. Description The 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 deciduous leaves are opposite, alternate or in whorls. In succulent species the leaves are mostly small and short-lived. The stipules are mostly small, partly transformed into spines or glands, or missing. Like all members of the family Euphorbiaceae, all spurges have unisexual flowers. In Euphorbia these are greatly reduced and grouped into pseudanthia called cyathia. The majority of species are monoecious (bearing male and female flowers on the same plant), although some are dioecious with male and female flowers occurring on different plants. It is not unusual for the central cyathia of a cyme to be purely male, and for lateral cyathia to carry both sexes. Sometimes young plants or those growing under unfavorable conditions are male only, and only produce female flowers in the cyathia with maturity or as growing conditions improve. The bracts are often leaf-like, sometimes brightly coloured and attractive, sometimes reduced to tiny scales. 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). Omale and Emmanuel (2010) determined the proximate and nutrient composition of Euphorbia and also some of the phytochemicals, both in the dry and fresh stem samples (table, 2.1) using the most recommended methods. 2.2.2. Xerophytes and succulents In the genus Euphorbia, succulence has often evolved divergently and to differing degrees. Sometimes it is difficult to decide, and it is a question of interpretation, whether or not a species is really succulent or "only" xerophytic. In some cases, especially with geophytes, plants closely related to the succulents are normal herbs. About 850 species are succulent in the strictest sense. If one includes slightly succulent and xerophytic species, this figure rises to about 1000, representing about 45% of all Euphorbia species (Carter, 2002).

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Table (2.1): Proximate and phytochemical composition of Euphorbia heterophylla

Parameter Dry sample Fresh sample

Phytochemicals Flavonoids + ++

Alkaloids + ++

Saponins + ++

Terpenoids + +

Steroids + ++

Tannins + +

Proximate Moisture % 13.88 + 1.67 88.47 + 2.68

Ash % 1.37 + 0.08 0.7 + 0.01

Protein % 5.85 + 0.96 1.38 + 0.22

Fat % 1.25 + 0.09 1.10 + 0.05

Fiber % 3.00 + 0.18 0.92 + 0.09

Carbohydrates 70.68 + 2.34 7.59 + 2.68

Source: Omale and Emmanuel (2010).

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2.2.3. Irritance The milky sap of spurges (called "latex") evolved as a deterrent to herbivores. It is white and colorless when dry, except in E. abdelkuri, where it is yellow when dry. The pressurized sap seeps from the slightest wound and congeals after a few minutes in air. The skin irritating and caustic effects are largely caused by varying amounts of diterpenes. Triterpenes such as betulin and corresponding esters are other major components of the latex. In contact with mucous membranes (eyes, nose and mouth), the latex can produce extremely painful inflammation. Therefore, spurges should be handled with caution and kept away from children and pets. Latex on skin should be washed off immediately and thoroughly. Congealed latex is insoluble in water, but can be removed with an emulsifier like milk or soap. A physician should be consulted if inflammation occurs, as severe eye damage including permanent blindness may result from exposure to the sap. When large succulent spurges in a greenhouse are cut, vapours can cause irritation to the eyes and throat several metres away. Precautions, including sufficient ventilation, are required (Tom et al., 2000). 2.2.4. Uses of Euphorbia Several spurges are grown as garden plants, among them Poinsettia (E. pulcherrima) and the succulent E. trigona. E. pekinensis is used in traditional Chinese medicine, where it is regarded as one of the 50 fundamental herbs. Several Euphorbia species are used as food plants by the larvae of some (butterflies and moths), like the Spurge Hawk-moths ( euphorbiae and Hyles tithymali), as well as the Giant Leopard . The diterpenoid ingenol is the core structure of a topical drug recently commercialized to treat actinic keratosis, a precancerous skin condition. It is produced by the Euphorbia plants (Carter, 2002). 2.2.5. Systematics and taxonomy According to recent studies of DNA sequence data (Victor and Mark, 2002; Victor, 2003; Peter et al., 2006),found that 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. The genus Euphorbia is one of the largest and most complex genera of flowering plants and several botanists have made unsuccessful attempts to subdivide the genus into numerous

27 smaller genera. According to the recent phylogenetic studies, Euphorbia can be divided into 4 subgenera, each containing several, not yet sufficiently studied sections and groups. Of these, Esula is the most basal. Chamaesyce and Euphorbia are probably sister taxa but very closely related to Rhizanthium (Victor and Mark, 2002; Victor, 2003; Peter et al., 2006). 2.2.6. 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 is venomous and can cause skin irritations (Ehow, 2014). 2.2.6.1. Scientific Classification: Kingdom: Plantae Phylum: Angiosperms Class: Family: Euphorbiaceae Tribe: Euphorbieae Genus: Euphorbia Species: trigona, (Carter, 2002) 2.2.6.2. Medicinal benefits:- The Basque Institute for Agricultural Research and Development Neiker-Tecnalia 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 of this plant, which is very prolific and easily acclimated, could be considered in clinical trials for cancer treatment due to its anti-tumor activity. Phytochemicals

28 present in E. trigona were presented in Table (2.2. 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. This potential antifungal activity opens an interesting line of research in finding new uses for latex of E. trigona against various diseases (ScienceDaily, 2012). 2.2.7 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, 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(Kedei et al.,2004). 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).

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Table (2.2): Phytochemical analysis of E. trigona extract

Constituents Petroleum Chloroform Ethyl acetate Ethanol

ether extract Extract Extract Extract

Sterols + - - +

Alkaloids + + + +

Tannins + - + +

Flavonoids + - - -

Saponins - - - +

Source: Nashikkar et al., (2012)

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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 (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 (Table, 2.3), and cellular repair. Highly colored vegetables and fruits tend to be highest in these chemicals, but tea, chocolate, 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). 2.3.1. Clinical trials and health claim status Lycopene from tomatoes has been tested in human studies for cardiovascular diseases and prostate cancer. These studies, however, did not attain sufficient scientific agreement to conclude an effect on any disease. Very limited and preliminary scientific research suggests that eating one-half to one cup of tomatoes and/or tomato sauce a week may reduce the risk of prostate cancer. The United States Food and Drug Administration concluded that there is little scientific evidence supporting this claim." Phytochemical-based dietary supplements can also be purchased. According to the American Cancer Society, "Available scientific evidence does not support claims that taking phytochemical supplements is as good for long-term health as consuming the fruits, vegetables, beans, and grains from which they are taken (FDA, 2004). 2.3.2. Food processing Phytochemicals in freshly harvested plant foods may be destroyed or removed by modern processing techniques, including cooking (Bongoni et al., 2013). For this reason, industrially processed foods likely contain fewer phytochemicals and may thus be less beneficial than unprocessed foods. Absence or deficiency of phytochemicals in processed foods may contribute to increased risk of preventable diseases (Liu, 2004; Rao and Rao, 2007).

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Table (2.3): Activities of various phytochemicals from plants

Phytochemicals Activities

Phenols and Polyphenols Antimicrobial, Anthelmintic, Antidiarrhoeal

Flavones Antimicrobial

Flavonoids Antimicrobial, Antidiarrhoeal

Tannins Antimicrobial, Anthelmintic, Antidiarrhoeal

Terpenoids Antimicrobial, Antidiarrhoeal

Alkaloids Antimicrobial, Anthelmintic

Glycosides Antidiarrhoeal

Saponins Antidiarrhoea

Source: Kumar et al., 2010

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A converse example may exist in which lycopene, a phytochemical present in tomatoes, is either unchanged in content (Agarwal et al., 2001) or made more concentrated by processing to juice or paste, maintaining good levels for bioavailability (Dewanto et al., 2002). 2.3.3. Screening and Extraction: Plants are source of large amount of drugs comprising to different groups such as antispasmodics, emetics, anti-cancer, antimicrobials etc. A large number of the plants are claimed to possess the antibiotic properties in the traditional system and are also used extensively by the tribal people worldwide. It is now believed that nature has given the cure of every disease in one way or another. Plants have been known to relieve various diseases in Ayurveda. Therefore, the researchers today are emphasizing on evaluation and characterization of various plants and plant constituents against a number of diseases based on their traditional claims of the plants given in Ayurveda. Extraction of the bioactive plant constituents has always been a challenging task for the researchers(Ncube et al.,2008). Plant-derived substances have recently become of great interest owing to their versatile applications. Medicinal plants are the richest bio-resource of drugs of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs (Ncube et al., 2008). Extraction (as the term is pharmaceutically used) is the separation of medicinally active portions of plant (and ) tissues using selective solvents through standard procedures. The products so obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or (after removing the solvent) in dry powder form, and are intended for oral or external use. These include classes of preparations known as decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts or powdered extracts. Such preparations have been popularly called galenicals, named after Galen, the second century Greek physician. Extraction methods used pharmaceutically involves the separation of medicinally active portions of plant tissues from the inactive/inert components by using selective solvents. During extraction, solvents diffuse into the solid plant material and solubilize compounds with similar polarity (Ncube et al., 2008). 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

33 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). The basic parameters influencing the quality of an extract are : 1- Plant part used as starting material 2- Solvent used for extraction (Table, 2.4) 3- Extraction procedure Effect of extracted plant phytochemicals depends on 1- The nature of the plant material 2- Its origin 3- Degree of processing 4- Moisture content 5- Particle size The variations in different extraction methods depend upon: 1- Type of extraction 2- Time of extraction 3- Temperature 4- Nature of solvent 5- Solvent concentration 6- Polarity (Ncube et al., 2008)

34

Table (2.4): Solvents used for active component extraction

Water Ethanol Methanol Chloroform Ether Acetone

Anthocyanins Tannins Anthocyanins Terpinoids Alkaloids Phenol

Starch Polyphenols Terpinoids Flavonoids Teroinoids Flavonols

Tannins Polyacetylenes Saponins Coumarins

Saponins Flavonols Tannins Fattyacids

Terponoids Terinoids Xanthoxylenes

Polypeptides Sterols Totarol

Lectins Alkaloids Quassinoids

Source: Cowan (1999)

35

2.3.4. Plant material Plants are potent biochemists and have been components of phytomedicine since times immemorial; man is able to obtain wondrous assortment of industrial chemicals. Plant based natural constituents can be derived from any part of the plant like bark, leaves, flowers, roots, fruits, seeds (i.e. any part of the plant may contain active components). The systematic screening of plant species with the purpose of discovering new bioactive compounds is a routine activity in many laboratories. Plants are collected either randomly or by following local healers in geographical areas where the plants are found (Parekh et al., 2006). Fresh or dried plant materials can be used as a source for the extraction of secondary plant components. Many authors had reported about plant extract preparation from the fresh plant tissues. The logic behind this came from the ethno medicinal use of fresh plant materials among the traditional and tribal people. But as many plants are used in the dry form (or as an aqueous extract) by traditional healers and due to differences in water content within different plant tissues, plants are usually air dried to a constant weight before extraction. Other researchers dry the plants in the oven at about 40°C for 72 hours. In most of the reported works, underground parts (roots, tuber, rhizome, bulb etc.) of a plant were used extensively compared with other above ground parts in search for bioactive compounds possessing antimicrobial properties (Ncube et al., 2008; Das et al., 2010). 2.3.5. Choice of solvents Successful determination of biologically active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. Properties of a good solvent in plant extractions includes, low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption of the extract, preservative action, inability to cause the extract to complex or dissociate. The factors affecting the choice of solvent are quantity of phytochemicals to be extracted, rate of extraction, diversity of different compounds extracted, diversity of inhibitory compounds extracted, ease of subsequent handling of the extracts, toxicity of the solvent in the bioassay process, potential health hazard of the extractants (Eloff, 1998). The choice of solvent is influenced by what is intended with the extract. Since the end product will contain traces of residual solvent, the solvent should be non-toxic and should not interfere with the bioassay. The choice will also depend on the targeted compounds to be extracted (Ncube et al., 2008; Das et al., 2010).

36

The various solvents that are used in the extraction procedures are: 1. Water: Water is universal solvent, used to extract plant products with antimicrobial activity. Though traditional healers use primarily water but plant extracts from organic solvents have been found to give more consistent antimicrobial activity compared to water extract. Also water soluble flavonoids (anthocyanins) have no antimicrobial significance and water soluble phenolics only important as antioxidant compound (Das et al., 2010). 2- Acetone: Acetone dissolves many hydrophilic and lipophilic components , is miscible with water, is volatile and has a low toxicity to the bioassay used, it is a very useful extractant, especially for antimicrobial studies where more phenolic compounds are required to be extracted. In a study by Ncube et al(2008) they found that extraction of tannins and other phenolics was better in aqueous acetone than in aqueous methanol. Both acetone and methanol were found to extract saponins which have antimicrobial activity. 3- Alcohol: The higher activity of the ethanolic extracts as compared to the aqueous extract can be attributed to the presence of higher amounts of polyphenols as compared to aqueous extracts. It means that they are more efficient in cell walls and seeds degradation which have unpolar character and cause polyphenols to be released from cells. More useful explanation for the decrease in activity of aqueous extract can be ascribed to the enzyme polyphenol oxidase, which degrade polyphenols in water extracts, whereas in methanol and ethanol they are inactive. Water is a better medium for the occurrence of the micro-organisms as compared to ethanol (Lapornik et al., 2005). By adding water to the pure ethanol for preparing ethanol 70% the polarity of solvent was increased (Bimakr, 2010). Additionally, ethanol was found easier to penetrate the cellular membrane to extract the intracellular ingredients from the plant material (Wang, 2010). Since nearly all of the identified components from plants active against microorganisms are aromatic or saturated organic compounds, they are most often obtained through initial ethanol or methanol extraction (Cowan, 1999). Methanol is more polar than ethanol but due to its cytotoxic nature, it is not suitable for extraction in certain kind of studies as it may lead to incorrect results. 4- Chloroform: Terpenoid lactones have been obtained by successive extractions of dried barks with hexane, chloroform and methanol with activity concentrating in chloroform fraction. Occasionally tannins and terpenoids can be found in the aqueous phase, but they are obtained by treatment with less polar solvents (Cowan, 1999).

37

5- Ether: Ether is commonly used selectively for the extraction of coumarins and fatty acids (Cowan, 1999). 6- Dichloromethanol: It is used for carrying out the extraction procedures. It is specially used for the selective extraction of terpenoids only (Cowan, 1999). 2.4. Proximate Analysis: Proximate Analysis is a partitioning of compounds in a feed into six categories (AOCS, 2000) based on the chemical properties of the compounds. The six categories are: 1- Moisture 2- Ash 3- Crude protein 4- Crude lipid 5- Crude fiber 6- Nitrogen-free extracts (digestible carbohydrates) It is important to remember that, proximate analysis is not a nutrient analysis, rather it is a partitioning of both nutrients and non-nutirents into categories based on common chemical properties(Vogel et al.,2003). 2.5. 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(Vogel et al.,2003). 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(Vogel et al.,2003).

38

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"(Vogel et al.,2003). 2.5.1. Plate preparation 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 a thick slurry on an unreactive 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). The process of technique is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products(Vogel et al.,2003). 2.5.2. Procedure of TLC: A small spot of solution containing the sample is applied to a plate, about 1.5 centimeters from the bottom edge. The solvent is allowed to completely evaporate off, otherwise a very poor or no separation will be achieved. If a non-volatile solvent was used to apply the sample, the plate needs to be dried in a vacuum chamber. A small amount of an appropriate solvent (eluent) is poured into a glass beaker or any other suitable transparent container (separation chamber) to a depth of less than 1 centimeter. A strip of filter paper (aka "wick") is put into the chamber so that

39 its bottom touches the solvent and the paper lies on the chamber wall and reaches almost to the top of the container. The container is closed with a cover glass or any other lid and is left for a few minutes to let the solvent vapors ascend the filter paper and saturate the air in the chamber. (Failure to saturate the chamber will result in poor separation and non-reproducible results). The TLC plate is then placed in the chamber so that the spot(s) of the sample do not touch the surface of the eluent in the chamber, and the lid is closed. The solvent moves up the plate by capillary action, meets the sample mixture and carries it up the plate (elutes the sample). The plate should be removed from the chamber before the solvent front reaches the top of the stationary phase (continuation of the elution will give a misleading result) and dried (Vogel et al., 2003). 2.5.3. Separation Process and Principle Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be adjusted. Also, the separation achieved with a TLC plate can be used to estimate the separation of a flash chromatography column (Fair and Kormos, 2008).

Development of a TLC plate, a purple spot separates into a red and blue spot. Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds that differ in polarity, the more polar compound has a stronger interaction with the silica and is, therefore, more capable to dispel the mobile phase from the binding places. As a consequence, the less polar compound moves higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. It is commonly said that "strong" solvents (eluents) push the analyzed compounds up the plate, whereas "weak" eluents barely move them. The order of strength/weakness depends on the

40 coating (stationary phase) of the TLC plate. For silica gel coated TLC plates, the eluent strength increases in the following order: Perfluoroalkane (weakest), Hexane, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile, Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest). For C18 coated plates the order is reverse. This means that, if a mixture of ethyl acetate and hexane as the mobile phase is used, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will normally not result in reversed order of running of the compounds on the TLC plate. An eluotropic series can be used as a guide in selecting a mobile phase. If a reversed order of running of the compounds is desired, an apolar stationary phase should be used, such as C18- functionalized silica (Jork et al., 1990; Jork et al., 1994). 2.5.4. Analysis As the chemicals being separated may be colorless, several methods exist to visualize the spots: fluorescent analytes like quinine may be detected under blacklight (366 nm), often a small amount of a fluorescent compound, usually manganese-activated zinc silicate, is added to the adsorbent that allows the visualization of spots under UV-C light (254 nm). The adsorbent layer will thus fluoresce light-green by itself, but spots of analyte quench this fluorescence. Iodine vapors are a general unspecific color reagent. Specific color reagents into which the TLC plate is dipped or which are sprayed onto the plate exist (Potassium permanganate – oxidation or Bromine). In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis, for example mass spectrometry, a technique known as Far-

Eastern blotting. Once visible, the Rf value, or retardation factor, of each spot can be determined by dividing the distance the product traveled by the distance the solvent front traveled using the initial spotting site as reference. These values depend on the solvent used and the type of TLC plate and are not physical constants (Jonathan et al., 2007; Geiss, 1987). 2.5.5. Applications In organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a capillary tube are placed on the plate: a spot of starting material, a spot from the reaction mixture, and a cross-spot with both. A small (3 by 7 cm) TLC plate takes a couple of minutes to run. The analysis is qualitative, and it will show if the starting material has disappeared, i.e. the

41 reaction is complete, if any product has appeared, and how many products are generated (although this might be underestimated due to co-elution). Unfortunately, TLCs from low- temperature reactions may give misleading results, because the sample is warmed to room temperature in the capillary, which can alter the reaction—the warmed sample analyzed by TLC is not the same as what is in the low-temperature flask. One such reaction is the DIBALH reduction of ester to aldehyde (Jonathan et al., 2007).

42

Chapter Three Materials and Methods

3.1 Samples of plant material: Stems and leaves of the cactus plant (Euophorbia trigona), that were used as a natural product materials in this study, were collected from Wad Medani City. The targeted parts were washed while they were in their original plant, so as to avoid loss of some components. The collected parts were transferred directly to the Faculty of Engineering and Technology, University of Gezira, during April 2014, where all of the laboratory tests were done. 3.2. Preparation of plant material The collected parts of E. trigona plants were firstly divided into leave and stem parts. The stem were cut and divided into outer parts (cortex) and inner part (pith). The three 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 were subjected to some approximate analysis (moisture, ash, protein and polar and apolar contents) tests, while the other part were subjected to phytochemical screening (presence of some main components such as alkaloids, glycosides, .. etc), using recommended methods. 3.3. Approximate analysis Tests of Approximate analysis were based on triplicates. 3.3.1. Moisture contents Accurate weight (3 g) of each of the dried and grounded plant material was placed in a special dish and put in a vacuum oven at 105 oC for approximately 1:30 hours. The dishes were then removed from the oven, covered, cooled in desiccator, and weighed (AOAC, 1990).

The percentage moisture can be calculated as follows:

100 (P-a)

percent moisture = % P

43

P = weight in g of sample (= 3g) a = weight in g of dried sample

3.3.2. Ash contents

A weight of 3 g of each of the dried and grounded plant material was placed in a crucible and put in muffle furnace at 550 + 5 oC for approximately four hours. The dishes were then removed from the oven, covered, cooled in desiccator, and weighed (AOCS, 2000). The ash fraction contains all the mineral elements occur in these parts. It was observed that, the high temperature may cause the volatilization of certain elements (particularly K, Na, Cl, and P) and may also cause the mineral matter to melt and fuse (Okwu and Morah, 2004). % ash (in g)

% ASH (dry)= x 100

(100 - % moisture)

3.3.3. Protein content It was discovered that, all proteins contains about the same amount of nitrogen (16%), which is relatively easy to calculate crude protein according to nitrogen on the basis: 100/16 = 6.25, therefore: Nitrogen x 6.25 = Crude Protein (AOAC, 1990) About 2 g of each of the dry grounded plant material placed in digestion flask. About

15g Na2SO4, 1 g CuSO4, one or two selenized boiling granules and 25 mL of conc H2SO4 were added sequentially to the flask. Digestion was continued until the solution in the flask was almost colourless or light green. When cooled 200 mL water was added. Additional boiling granules were added when necessary to prevent bumping. 100 mL 0.1 N HCl was pipetted into a 500 mL erlenmeyer flask, and 1 mL Conway's indicator was added and the flask was placed under the condenser ensuring that the condenser tip was immersed in the acid solution. Standardized HCl was used in distillation for nitrogen content of the sample. The Kjeldahl flask containing the digested sample was tilted and 100 mL of 50% NaOH solution was added slowly down the side of the Kjeldahl flask so that it forms a layer underneath the digestion mixture. Immediately connect the flask to the distilling bulb of the distillation apparatus. Rotate flask to thoroughly mix contents. The mixture was heated until all ammonia has passed over into the standard acid.

44

The tip of condenser was washed and the excess standard HCl was titrated in distillate with NaOH standard solution (AOAC, 1990).

(A - B) x 1.4007

% Nitrogen (wet) = x 100

weight (g) of sample where: A = vol. (mL) std. HCl x normality of std. HCl B = vol. (mL) std. NaOH x normality of std. NaOH Calculate nitrogen content on dry basis (when moisture content is known) as follows: % Nitrogen (wet)

% Nitrogen (dry)= x 100

(100 - % moisture)

The percentage protein (wet or dry basis) was calculated as follows: % PROTEIN = % nitrogen x 6.25

3.4. The polar and apolar contents About 2 g of each of the dried plant materials were subjected to soxhelt extraction by using hexane solvent so as to calculate the apolar contents. After 4-5 hours the samples were removed from the soxhlet, dried and then weighed. Other 2 g samples were subjected also to a soxhlet extraction using ethanol solvent (so as to calculate the polar contents) for about 4-5 hours. The samples were then removed from the soxhlet, dried and weighed. The fraction difference in weight of each sample (before and after extract) in accordance to the original weight (2 g) was used to calculate the percentage polar and apolar contents (Sofowora, 1993).

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3.5. Phytochemical screening for the different parts of E. trigona: 3.5.1. Qualitative analysis test for glycosides About 3.0 g of the dried powder of each part of the E. trigona plant, 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 Harbone (1973); Sofowora (1993); 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. 3.5.2. Qualitative analysis test for flavonoids About 2.0 g of the dried powder of each part of the E. trigona plant, 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. 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). 3.5.3. Qualitative analysis test for saponins: About 2 g of the dried powder of each part of the Euophorbia plant was 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). 3.5.4. Qualitative analysis test for tannins: The dried powder of each part of Euophorbia plant (5 g), was 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).

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3.5.5. Qualitative analysis test for sterols and or triterpenes: The dried powder of each part of Euophorbia plant (1 g) 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). 3.5.6. Qualitative analysis test for alkaloids and /or nitrogenous bases The dried powder of each part of the Euophorbia plants (5 g) was extracted with ethanol and filtered. Aliquots from the ethanolic extract (10 ml) 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).

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

47

Chapter Four Results and Discussion

4.1. The proximate analysis of Euphorbia trigona parts The percentage moisture contents (on the fresh bases) of E. trigona leaves, stem cortex and stem pith were presented in Table (4.1) and Figure (4.1). Stem pith showed the highest percentages of moisture contents (95.56, 95.84 and 95.87%) with a mean of 95.76%, while the moisture in the stem cortex were 90.50, 89.74 and 89.50%, with a mean of 89.91%, whereas, the moisture in leaves were 83.74, 83.72 and 80.71%, with a mean of 82.72%. The observed difference here was significant (f= 114.77; f-crit=5.143). It was clear that, the moisture content of the leaves was less than that (88.47%) calculated by Omale and Emmanuel (2010). Also, the internal part of the stem reserve large quantity of moisture than the outer part and the leave. Table (4.2) and Figure (4.1), showed the percentage ash contents (on the fresh bases) in E. trigona leaves, stem cortex and stexxh `m pith. Stem pith possessed the lowest percentages of ash contents (3.52, 2.37 and 3.25%) with a mean of 3.05%, while the ash content in the stem cortex were 7.69, 8.20 and 7.20%, with a mean of 7.70%, whereas, the ash content in leaves were 8.04, 7.73 and 7.36%, with a mean of 7.71%. The observed difference here was significant (f= 89.41; f-crit=5.143). It was clear that, the ash content was relatively high than that (0.7%) obtained by Omale and Emmanuel (2010), and this may be due to the dusty weather during the preparation of samples or it can be attributed to differences in the geographical parameters. Table (4.3) and Figure (4.1), showed the percentage protein contents (on the fresh bases) in E. trigona leaves, stem cortex and stem pith. Stem pith possessed the lowest percentages 123 protein contents (0.91, 0.89 and 0.93%) with a mean of 0.91%, while the protein content in the stem cortex were 1.44, 1.51 and 1.55%, with a mean of 1.50%, whereas, the protein content in leaves were 2.31, 3.11 and 2.89%, with a mean of 2.77%. The observed difference here was significant (f= 89.41; f-crit=5.143).

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It was clear that, the mean protein content in stem part (0.91 + 1.50 / 2 = 1.21%) was relatively similar to that obtained by Omale and Emmanuel (2010); which was 1.38 + 0.22, while the protein content of leaves was relatively double to that quantity. The proteins of E. trigona played a very active role in defending the plant. The purified proteins were able to inhibit eukaryotic ribosomes in cell-free systems, and also showed cytotoxic activity and possible antifungal activity (ScienceDaily, 2012).

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Table (4.1): The moisture contents (%) in Euphorbia trigona parts

Rep. Leaves Stem cortex Stem pith

1 83.74 90.50 95.56

2 83.72 89.74 95.84

3 80.71 89.50 95.87

SUMMARY Groups Count Sum Average Variance Leaves 3 248.17 82.72 3.04 Stem cortex 3 269.74 89.91 0.27 Stem pith 3 287.27 95.76 0.03

1 Source of Variation SS Df MS F P-value F crit Between Groups 255.70 2 127.85 114.77 1.65E-05 5.14 Within Groups 6.68 6 1.114

Total 262.39 8

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Table (4.2): The ash contents (%) in Euphorbia trigona parts

Rep. Leaves Stem cortex Stem pith

1 8.04 7.69 3.52

2 7.73 8.20 2.37

3 7.36 7.20 3.25

SUMMARY Groups Count Sum Average Variance

Leaves 3 23.13 7.71 0.12

Stem cortex 3 23.09 7.70 0.25

Stem pith 3 9.14 3.05 0.36

ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 43.376 2 21.68 89.41 3.42E-05 5.14

Within Groups 1.453 6 0.24

Total 44.82 8

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Table (4.3): The protein contents (%) in Euphorbia trigona parts

Rep. Leaves Stem cortex Stem pith

1 2.31 1.44 0.91

2 3.11 1.51 0.89

3 2.89 1.55 0.90

SUMMARY Groups Count Sum Average Variance

Leaves 3 8.31 2.77 0.17

Stem cortex 3 4.5 1.5 0.003

Stem pith 3 2.7 0.91 0.0001

ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 5.47 2 2.73 47.15 0.0002 5.14

Within Groups 0.35 6 0.06

Total 5.82 8

52

100

90

80

70

60 leaves

50 cortex pith 40

30

20

10

0 moisture % ash % protein %

Figure (4.1): The % moisture, ash and protein contents in the leave, stem cortex and stem pith of E. trigona

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4.2. The polar and apolar contents of Euphorbia trigona parts The polar content (on the fresh bases) of E. trigona leaves, stem cortex and stem pith were presented in Table (4.4). Stem pith showed the lowest polar contents (2.91, 2.72 and 2.83%) with a mean of 2.88%, while the % polar content in the stem cortex were 7.03, 6.51 and 6.78%, with a mean of 6.77%, whereas, the % polar content in leaves were 9.76, 10.77 and 10.89%, with a mean of 10.47%. The observed difference here was significant (f= 285.33; f- crit=5.14). It was clear that, the % mean polar content of the leaves (10.47) was more than other parts collectively (6.77 + 2.88%). Table (4.5), showed the percentage apolar contents (on the fresh bases) in E. trigona leaves, stem cortex and stem pith. Stem pith possessed, also, the lowest percentages of apolar content (0.88, 0.76 and 0.80%) with a mean of 0.81%, while the apolar content in the stem cortex were 1.13, 1.53 and 1.45%, with a mean of 1.37%, whereas, the apolar content in leaves were 1.03, 0.98 and 1.21%, with a mean of 1.07%. The observed difference here was also significant (f= 11.05; f-crit=5.14). It was clear that, the apolar content was relatively high in stem cortex (the mean was 1.37%) than the other parts. Cowan (1999), revealed that, ethanol can be used to extract 7 components (tannins, polyphenols, polyacetylenes, flavonols, terpenoids and lectins), while chloroform (as apolar solvent) can be used to extract terpenoids and flavonoids only. This fact explains why percentage polar content was more than that of apolar contents in all plant parts. Successful determination of biologically active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. Properties of a good solvent in plant extractions includes, low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption of the extract, preservative action, inability to cause the extract to complex or dissociate. The factors affecting the choice of solvent are quantity of phytochemicals to be extracted, rate of extraction, diversity of different compounds extracted, diversity of inhibitory compounds extracted, ease of subsequent handling of the extracts, toxicity of the solvent in the bioassay process, potential health hazard of the extractants (Eloff, 1998). The choice of solvent is influenced by what is intended with the extract. Since the end product will contain traces of residual solvent, the solvent should be non-toxic and should not interfere with the bioassay. The choice will also depend on the targeted compounds to be extracted (Ncube et al., 2008; Das et al., 2010).

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Table (4.4): The polar contents (%) in Euphorbia trigona parts

Rep. Leaves Stem cortex Stem pith

1 9.76 7.03 2.91

2 10.77 6.51 2.72

3 10.89 6.78 2.83

SUMMARY Groups Count Sum Average Variance

Leaves 3 31.42 10.47 0.38

Stem cortex 3 20.32 6.77 0.07

Stem pith 3 8.46 2.82 0.01

ANOVA Source of Variation SS Df MS F P-value F crit

Between Groups 87.89 2 43.95 285.38 1.13E-06 5.14

Within Groups 0.92 6 0.15

Total 88.82 8

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Table (4.5): The apolar contents (%) in Euphorbia trigona parts

Rep. Leaves Stem cortex Stem pith

1 1.03 1.13 0.88

2 0.98 1.53 0.76

3 1.21 1.45 0.80

SUMMARY Groups Count Sum Average Variance

Leaves 3 3.22 1.07 0.01

Stem cortex 3 4.11 1.37 0.04

Stem pith 3 2.44 0.81 0.004

ANOVA Source of Variation SS Df MS F P-value F crit

Between Groups 0.46 2 0.23 11.05 0.01 5.14

Within Groups 0.13 6 0.02

Total 0.59 8

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4.3. The phytochemical screening in Euphorbia trigona parts The presence (+) or absence (-) of some main phytochemicals in E. trigona leave, stem cortex and stem pith were presented in table (4.6). respective to E. trigona leaves, saponins, alkaloids, flavonoids, flavonones and flavonols, glycosides and sterols and triterpenoids were detected, while tannins were not detected. The same was noticed for stem cortex and stem pith, except in absence of flavonones and flavonols. The flavonoids of leaves and stem cortex were more in comparison to that of stem pith. 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 with the detected phytochemicals makes it a source of medicine for several diseases as was showed in Table (2.3; page 21), according to Kumar et al.,

(2010).

It can be concluded that E. trigona could be a potential source of active principles which may help to discover new chemical classes of drugs that could serve as selective agents for the maintenance of health. Further studies should be conducted to identify the active constituents present therein as well as to verify the biological activities of this plants.

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Table (4.6): The presence of some phytochemical in E. trigona parts

Item Leaves Stem cortex Stem pith

Tannins _ _ _ Saponins + + + Alkaloids + + + Flavonoids ++ ++ + Flavonones and + _ _ Flavonols Glycosides + + + Sterols and + + + Triterpenes

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Chapter Five

Conclusions and Recommendations

5.1 Conclusions

1- The proximate analysis of Euphorbia trigona parts indicated that, stem pith had significantly, the highest percentages of moisture contents, while the moisture in the stem cortex were also more than that of leaves.

2- Stem pith was found to possess the lowest percentages of ash contents than that of the stem cortex and leaves.

3- Stem pith showed the lowest percentages of protein contents, whereas, the protein content in leaves were the highest.

4- The % mean polar content of the leaves (10.47) was more than other parts collectively (6.77 + 2.88%). Meanwhile, the apolar content was relatively high in stem cortex than the other parts.

5- The preliminary phytochemical screening of E. trigona leaf extract revealed the presence of saponins, alkaloids, flavonoids, flavonones and flavonols, glycosides and sterols and triterpenoids while tannins were not detected while the stem cortex and stem pith contained all phytoconstituents except tannins and Flavonones.

5.2 Recommendations 1- E. trigona could be a potential source of many active principles. 2- Further studies should be conducted to identify the active constituents. 3- The biological activities of this plant should be verified.

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