A TAXONOMIC STUDY ON THE GENUS PULICARIA IN THE SUDAN

A thesis submitted to the University of Khartoum for The degree of M.Sc. In Biology

By Eiman Jafar Mohammed Abdalla B.Sc. (Hons.) Biology – Chemistry (U.of K.) 2000

Supervisor Dr. A/Gabbar Nasir Gumaa

Faculty of Education Department of Biology

May 2009

DEDICATION

To my parents… who lit my life candles to highlight my way… To my Brothers… To my dearest friends who brought to me all the success and love… To every one who helped me in every thing… To every one who tried to lead me to a better life… I dedicate this work.

EIMAN

I

Acknowledgement

Firstly, thanks to Allah who enabled me to complete this work. I am greatful to my parents for their encouragement & support. I wish to express deep gratitude and sincere appreciation to my supervisor Dr. AbdelGabbar Nasir Gumaa for his ultimate help and guidance. Also I am grateful to anyone who helped me and contributed their time and effort to help me in completing this work. At last, but not the least, I thank Mr. Hassan Monir for typing the manuscript.

II

TABLE OF CONTENTS

Page DEDICATION I ACKNOWLEDGEMENTS II Table of contents III List of tables IV List of Figures VI Abstract VII Arabic Abstract VIII CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO : LITERATURE REVIEW 4 2.1 Botanical studies 4 2.1.1 Classification of the genus Pulicaria 4 2.1.2 Common uses of the genus 4 2.2 Chemical studies 6 2.2.1 Volatile oils 6 2.2.2 Other chemical constituents 8 CHAPTER THREE: THE STUDY AREA 9 3.1 Location 9 3.2 People of Khartoum city & their activities 9 3.3 Geology 11 3.4 Soils 11 3.5 Climate 12 3.5.1 Temperature 12 3.6 Rainfall 12 3.6.1Wind 15 3.6.2 Relative Humidity 15

III CHAPTER FOUR: MATERIAL AND METHODS 19 4.1 Preparation of plant material 19 4.1.1 Field and herbarium methods 19 4.1.2 Drying and grinding of samples 20 4.2 Chemical analysis of plant samples 20 4.2.1 Preparation of the digestion solution 20 4.2.2 Preparation of stock and working solutions 21 4.2.3 determination of Ca and Mg elements 21 4.2.4 Preparation of phosphorus 22 4.2.5 Estimation of crude protein 23 4.2.6 Estimation of ash content 23 4.2.7 Estimation of total sugars 24 CHAPTER FIVE: RESULTS AND DISCUSSION 25 5.1 Results 25 5.1.1 Botanical studies 25 5.1.2 Chemical analysis of plant material 31 5.1.2.1 Chemical analysis of leaves 31 5.1.2.2 Chemical analysis of stems 32 5.2.3 Volatile oils of Pulicaria species 33 5.3 Discussion 35 5.4 Summary, Recommendations and Suggestions 37 5.4.1 Summary 37 5.4.2 Recommendations & Suggestions 38 References 40 Appendix 1 46 Appendix 2 47

IV

LIST OF TABLES

Table Page 1 The monthly wind direction at Khartoum area. 16 2 Mean monthly wind speed (Knots) at Khartoum area. 17 3 The mean relative humidity (%)at Khartoum area. 18 4 Preparation of stock and working solutions used for 21 elemental analysis of plant sample. 5 Distribution of Pulicaria species in the Sudan. 26 6 Geographical distribution of Pulicaria species in Africa 26 and other countries. 7 Results of chemical analysis of leaves of Pulicaria 31 undulata and P.crispa. 8 Results of chemical analysis of stems of Pulicaria 32 undulata and P.crispa. 9 Volatile oils of some Pulicaria spp. 33

V

LIST OF FIGURES

Figure Page 1 The study Area. 10 Mean monthly air temperature (ْ C) at Khartoum area. 13 2 3 Mean annual rainfall (mm) at Khartoum area. 14 4 Pulicaria crispa (Forssk.) Oliv. 27 5 Pulicaria undulata (L.) Mey. 28

VI Abstract

This research is a taxonomic study on the genus Pulicaria (Family Asteraceae) in the Sudan. Results of the botanical study revealed that there were seven species of the genus in the Sudan.Brief botanical descriptions and geographical distribution were given for the family and the species of the genus. Scientific names and synonyms were updated and the species were illustrated by digital camera images.Taxonomic keys were constructed for the species of the genus. Stems and leaves of two species of the genus, namely P.crispa and P. undulata were chemically analyzed. The results of chemical analysis showed that the two species differ in their chemical constituents. Chemical data was statistically analyzed using F-test (Variance ratio test). Results of statistical analysis revealed that there were no highly significant differences (P=0.01) between the two species with regard to ash, carbohydrates, proteins, calcium, magnesium and phosphorus in the leaves and stems of the two species. It was observed in the study that the odour of stems, leaves and flowers of P.undulata was sharper than that of P.crispa. This was attributed to the difference in quality and quantity of the volatile oils of the two species. The volatile oils of P.undulata were richer in oxygenated compounds than those of P.crispa and these compounds are responsible for the sharp characteristic odour of the oils. The study surveyed the various types and uses of the volatile oils of Pulicaria species in the fields of folk medicine, food flavouring and as bacteriocidal and insecticidal agents.

VII ﻣﺴﺘﺨﻠـــﺺ اﻟﺒﺤﺚ هﺬا اﻟﺒﺤﺚ ﻋﺒﺎرة ﻋﻦ دراﺳﺔ ﺗﺼﻨﻴﻔﻴﺔ ﻟﺠﻨﺲ Pulicaria (اﻟﻔﺼﻴﻠﺔ اﻟﻤﺮآﺒﺔ ) ﻓﻲ اﻟﺴﻮدان ، آﺸﻔﺖ ﻧﺘﺎﺋﺞ اﻟﺪراﺳﺔ اﻟﻨﺒﺎﺗﻴﺔ ﻋﻦ وﺟﻮد ﺳﺒﻌﺔ اﻧﻮاع ﻣﻦ هﺬا اﻟﺠﻨﺲ ﻓﻲ اﻟﺴﻮدان . ﺗﻢ اﻋﻄﺎء اﻟﻮﺻﻒ اﻟﻨﺒﺎﺗﻲ اﻟﻤﺨﺘﺼﺮ واﻟﺘﻮزﻳﻊ اﻟﺠﻐﺮاﻓﻲ ﻟﻠﻔﺼﻴﻠﺔ واﻧﻮاع اﻟﺠﻨﺲ ﺛﻢ ﺗﺤﺪﻳﺚ اﻻﺳﻤﺎء اﻟﻌﻠﻤﻴﺔ وﻣﺮادﻓﺎﺗﻬﺎ آﻤﺎ ﺗﻢ اﻟﺘﻮﺛﻴﻖ ﻟﻠﻨﺒﺎﺗ ﺎت ﺑﻮاﺳﻄﺔ آﺎﻣﻴﺮا رﻗﻤﻴﺔ. آﻤﺎ ﺗﻢ ﺗﺼﻤﻴﻢ ﻣﻔﺎﺗﻴﺢ ﺗﺼﻨﻴﻒ ﻷﻧﻮاع اﻟﺠﻨﺲ ﻓﻲ اﻟﺴﻮدان. أﺟﺮي ﺗﺤﻠﻴﻞ آﻴﻤﻴﺎﺋﻲ ﻟﺴﻴﻘﺎن واروراق ﻧﻮﻋﻴﻦ ﻣﻦ أﻧﻮاع اﻟﺠﻨﺲ هﻤﺎ: .P crispa و P. undulata . أﻇﻬﺮت ﻧﺘﺎﺋﺞ اﻟﺘﺤﻠﻴﻞ اﻟﻜﻴﻤﻴﺎﺋﻲ أن اﻟﻨﻮﻋﻴﻦ ﻳﺨﺘﻠﻔﺎن ﻓﻲ ﻣﻜﻮﻧﺎﺗﻬﻤﺎ اﻟﻜﻴﻤﻴﺎﺋﻴﺔ ، ﺣﻠﻠﺖ اﻟﺒﻴﺎﻧﺎت اﻟﻜﻴﻤﻴﺎﺋﻴﺔ ﺗﺤﻠﻴﻼً أﺣﺼﺎﺋﻴﺎً ﺑﺎﺳﻨﺨﺪام أﺧﺘﺒﺎر (ف) (أﺧﺘﺒﺎر ﻧﺴﺒﺔ اﻟﺘﺒﺎﻳﻦ ). آﺸﻔﺖ ﻧﺘﺎﺋﺞ اﻟﺘﺤﻠﻴﻞ اﻻﺣﺼﺎﺋﻲ ﻋﻦ ﻋﺪم وﺟﻮد ﻓﺮوﻗﺎت ذات دﻻﻟﺔ إﺣﺼﺎﺋﻴﺔ ﻋﺎﻟﻴﺔ ﺑﻴﻦ اﻟﻨﻮﻋ ﻴﻦ ﻓﻴﻤﺎ ﻳﺘﻌﻠﻖ ﺑﺎﻟﺮﻣﺎد ، اﻟﻜﺮﺑﻮهﻴﺪاﺗﺎت ، اﻟﺒﺮوﺗﻴﻨﺎت ، اﻟﻜﺎﻟﺴﻴﻮم ، اﻟﻤﺎﻏﻨﻴﺴﻴﻮم واﻟﻔﺴﻔﻮر ﻓﻲ ﺳﻴﻘﺎن واوراق اﻟﻨﻮﻋﻴﻦ. ﻟﻮﺣﻆ ﻓﻲ هﺬا اﻟﺒﺤﺚ ان راﺋﺤﺔ ﺳﻴﻘﺎن واوراق ﻧﻮع P. undulata . أآﺜﺮ ﺣﺪة ﻣﻦ ﻧﻮع P. crispa . ﻋﺰي هﺬا ﻟﻮﺟﻮد أﺧﺘﻼﻓﺎت ﻓﻲ ﻧﻮﻋﻴﺔ وآﻤﻴﺔ اﻟﺰﻳﻮت اﻟﻄﻴﺎرة ﻟﻠﻨﻮﻋﻴﻦ . اﻟﺰﻳﻮت اﻟﻄﻴﺎرة ﻟﻨﻮع P. undulata أﻏﻨﻲ ﺑﺎﻟﻤﺮآﺒﺎت اﻟﻤﺆآ ﺴﺪة ﻣﻘﺎرﻧﺔ ﻣﻊ P. crispa وهﺬﻩ اﻟﻤﺮآﺒﺎت هﻲ اﻟﻤﺴﺆوﻟﺔ ﻋﻦ اﻟﺮاﺋﺤﺔ اﻟﺤﺎدة اﻟﻤﻤﻴﺰة اﻟﻤﻮﺟﻮدة ﻓﻲ اﻟﺰﻳﻮت اﻟﻄﻴﺎرة. ﻓﻲ هﺬا اﻟﺒﺤﺚ ﺛﻢ ﻣﺴﺢ أﻧﻮاع واﺳﺘﺨﺪﻣﺎت اﻟﺰﻳﻮت اﻟﻄﻴﺎرة ﻓﻲ ﻣﺠﺎﻻت اﻟﻄﺐ اﻟﺸﻌﺒﻲ ، واآﺴﺎب ﻧﻜﻬﺔ ﻟﻠﻄﻌﺎم وآﻌﻮاﻣﻞ ﻗﺎﺗﻠﺔ ﻟﻠﺒﻜﺘﺮﻳﺎ واﻟﺤﺸﺮات.

VIII CHAPTER ONE INTRODUCTION

Sudan is a vast country with diverse climate and soil types, and as such it favours the cultivation and adaptation of many plants including aromatic plants. Aromatic plants belong to different families such as Asteraceae (Asteraceae), Apiaceae (Umbelliferae), Piperaceae, Apocynaceae and Lamiaceae (Labiatae). Some of the genera of these families are cultivated e.g. Coriandrum (Apiaceae), Mentha and Ocimum (Lamiaceae) while others grow in the wild e.g. Pulicaria. Pulicaria crispa and P.undulata are wild aromatic plants found in the Sudan. Their vernacular Arabic name is "tager" or "rabul". These two species have recently received a lot of attention due to their local and traditional uses for medical and other purposes (Elghazali, et al, 1994). Most of the research in the Sudan was conducted on medicinal plants rather than aromatic plants. Only since the late eighties of the past century, aromatic plants have received attention from researchers (El Egami, 1989). Traditional Taxonomy, which is based on morphological and histological characters, is not always conclusive in the accurate delimitation of taxa. Hence there is a need for other disciplines such as Biochemistry, Molecular Biology, Electrophoresis, Cytogenetics … etc to demonstrate differences and similarities between taxa. Photochemistry, also called Chemotaxonomy, is an attempt to identify and classify plants according to their biochemical composition. This new approach had a remarkable impact on Plant Taxonomy (Wink, 2003).

1

Secondary metabolites (SM) of higher plants have recently received a lot of interest from taxonomists (Willian el al, 1994;Maffei,1996; Santos and Saltino, 2000; Onyilagha et al, 2003; Wink, 2003; Viera et al, 2003; Urdampilleta et al, 2005; Tomczyky, 2006). Plant chemical constituents had led to the revision of many plant taxa, and those families have been grouped successfully on the bases of their secondary metabolites. Volatile oils are plant secondary metabolites which have recently received considerable attention in literature due to their biological properties (Burt, 2004; Bezic et al, 2005; Hambali et al, 2005; Okoh et al, 2008). Tao et al (2008) reported that essential oils can be used in food and flavour industry. Senatore et al (2007) reported that essential oils of plants have proved to be valuable in taxonomic and evolution studies of plants. Essential oils of family Asteraceae are mainly mono-and sequiterpenes (El Egami, 1989). Many authors studied the chemical composition of essential oils of many genera belonging to this family (El Egami, 1989; Al-Yousuf et al, 2001; Fadwa et al, 2005; Urdampilleta et al 2005; Okoh et al, 2008). Urdampilleta et al (2005) reported that monoterpenes are useful in systematic studies and that the occurrence of sesquiterpenes lactones in Asteraceae is of taxonomic interest. Volatile oils also have wide applications in a number of fields including food and beverage flavours, perfume and cosmetic industries and as bacteriocidal agents.

2 Objectives of the study The present study aims to

1- Study the morphology and geographical distribution of the genus Pulicaria in the Sudan, with special reference to Khartoum State. 2- Construct taxonomic keys for Pulicaria spp. based on their morphological and chemical characters. 3- Identify the major chemical macromolecules of P.crispa and P.undulata and run statistical analysis for their chemical components. 4- Study the volatile oils of Pulicaria spp. with special emphasis on their uses in folk medicine, perfume, cosmetic and pesticide industries.

3 CHAPTER TWO LITARATURE REVIEW

2.1. Botanical Studies

2.1.1. Classification of the genus Pulicaria This genus was classified by Cronquist et al (1972) according to the following system: Division Trachaeophyta Class Angiospermopsida Subclass Dicotyledonidae Order Asterales Family Asteraceae Genus Pulicaria

Andrews (1956) reported the presence of seven species of the genus Pulicaria in the Sudan, namely: P. petiolaris, P. undulata, P. crispa, P. attenuata, P. dysenterica var. stenophylla, P. vulgaris and P. grantii.

2.1.2. Common Uses of the Genus

Pulicaria species have been widely used in folk medicine. El Ghazali et al (1994) reported that P.undulata was used traditionally for medicinal purposes in the Sudan, and that the poultices of the whole plant were used against alopecia and as a tea substitute in Egypt. El Egami (1989) reported that the leaves of P.dysenterica possessed an antihistaminic effect. Karryev (1968) mentioned the use of P.undulata as a tonic, a tea substitute, an antispasmoic, a hypreglycaemic and as an ingredient of local perfumes in the Sudan.

4 Aqueous leaf extracts of P.crispa were found to inhibit the production of aflatoxins by Aspergillus flavus. Farmers in the Gezira State considered P.crispa as a noxious weed of cultivation and thus the plant was collected, dried and burnt or used in building shelters. Fadwa et al (2005) reported that the roots of P.odora L. were known for their anti-inflommatory properties. Pulicaria species were also reported to have many medicinal and folkoric uses in Sudan, Egypt, Europe and other countries. Bishay et al (1975) reported that the essential oils of P.undulata were used in the preparation of tonics. Karryev (1986) reported antibacterial properties for the essential oils (3.5%) of P.salveola and added that the oils produced rapid healing of inflammations of rabbit eyes. Mahfouz et al (1973) discovered antihistaminic properties of leaf extracts from P. dysenterica . They added that extracts had heart-stimulating effects in mammals, toads and rabbits. P.crispa was found to have many folkloric medicinal uses in many countries (Batonouny, 1981; El Ghazali et al, 1998; Rizk, 1986). Ali (1997 and 2008) reported that leaves, flowers and stems of P.crispa contained lethal doses against Biomphalaria pfeifferi snails. Fadwa et al (2005) reported that P.odora from Morocco contained thymol oil which was found to exhibit a significant antibacterial activity against seven types of bacteria and added that the activity of the oil was higher than those of standard antibiotics. El Egami (1989) reported insecticidal activity of volatile oils of P.undulata on the larvae and adult stages of beetles. She reported a percentage mortality for the insect and larvae at different concentrations of the volatile oils.

5 2.2. Chemical Studies Few chemical studies were carried on the genus Pulicaria in the Sudan (El Egami, 1989; Ali, 1997 and 2008). These studies analyzed volatile oils, tannins, alkaloids, glycosides and other chemical constituents.

2.2.1. Volatile oils The characteristic odour of Pulicaria species is due to the presence of terpenoid volatile oils. Plant families with volatile oils include Asteraceae, Lamiaceae, Rosaceae, Rutaceae and Apiaceae (Harborne, 1973). Volatile oils are widespread and may occur in almost all plant organs such as leaves, roots, stems, rhizomes, flowers or fruits (Claus, 1965). El Egami (1989) investigated the volatile oils of P.crispa using gas- liquid-chromatography (GLC). She was able to identify the following volatile oils: limonene, linalool, carvolanactone, nerol and eugenol. Adam (1989) studied the essential oils of Pulicaria crispa using Ion Trap Mass Spectroscopy in Illinois, USA. Weyerstahl et al (1999) studied the essential oils of P.gnaphlodes from Iran. They found that the leaves contained the highest amount of volatile oils as compared to the flowers and stems. Al-Yousuf et al (2001) studied the composition of essential oils of P.glutinosa Jaub. in the United Arab Emirates (UAE) and revealed the presence of B-sitosterol, B-amyrin, a neutral triterpene and choline. Williams and Fleming (1980) reported that the major volatile oils of P.undulata were Ketonic in nature and thus are associated with the carbonyl group while those of P.crispa are typical of compounds containing hydroxyl group.

6 Bishay et al (1975) worked on P.undulata from Egypt, using gas- liquid-chromatography (GLC). They detected the presence of 19 types of volatile oils. Assaf (1981) isolated five flavonoids from P.undulata which were identified as follows: rhamnocitrin, 3-7 dimethoxyquercetin, rhamtin (7-methoxyqueretin), dihydrokaempferol and isoquercetin. He also reveled the presence of B-amyrin, B-sitosterol and the fatty acids of linolenic, palmitic and stearic. Metwally et al (1986) studied P.undulata from Egypt, using column chromatography (CC) and thin layer chramotography (TLC). They detected the thymol derivatives, lupenyl acetate and taraxosteryl acetate. Pares et al (1981) used paper chromatography (PC) to study P.dysenterica. They reported the presence of volatile oils such methyl ethers, scutellarin, querectangetin and 6-hydroxykaempferol in the leaves. They added that the flowers yielded 3,4,7 trimethyl ethers (a flavone) as well as kaempferol -3- glucoside. Constantinescu et al (1966) studied the volatile oils of underground and aerial parts of P.vulgaris and reported the presence of flavones and saponins in the leaves and flowers of the plant. Fadwa et al (2005) studied P.odora in Morocco using gas chromatography (GC). They identified 27 components of which the volatile oil thymol (47.8%) and thymol isobutyrate (30%) were the main constituents.

7 2.2.2 Other Chemical Constituents It was revealed that P.uliginosa contained resinous and tannic substances, ascorbic acid, essential oils and other biologically active micronutrients such as Mn, Cu, Ni, Zn, and Fe. Rizk (1986) studied the chemical composition of Pulicaria species and reported the presence of tannins and alkaloids. Investigations of Rizk and Ismail (1981) on P.crispa in Qatar revealed the presence of coumarins, tannins, alkaloids and a number of volatile oils. Karryev (1966) studied P.undulata and determined the contents of ash, nitrogen, water-soluble compounds, alkaloids, glycosides, flavonoids, coumarins, tannins, fats, organic acids and ascorbic acid. Volatile alkaloids, pectins and saponins were not detected.

8 CHAPTER THREE THE STUDY AREA

3.1 Location

The study area falls within the premises of the Faculty of Education, University of Khartoum. The Faculty lies in the northern part of Omdurman City which lies in the northern part of Khartoum. Khartoum is the Sudan's capital, lying between altitudes 15º 10' and 16º 30' (Abusin and Davies, 1991) and has an area of about 20.970Km² (Abusin and Davies,1991). In the Faculty of Education, there is a large water reservoir which, when full, allows surplus water to exude and hence irrigates the vicinity of the reservoir. The water from the reservoir, coupled with rainfall, has created a permanent plant community in the vicinity of the reservoir (fig. 1).

3.2 People of Khartoum city and their activities

Khartoum, like many other capitals in the world, is inhabited mostly by Sudanese nationals in addition to immigrants and foreigners from all over the world. These people work in the public as well as the private sector. Foreign investment in the Sudan has resulted in a substantial influx of people to Khartoum city.

9

(Fig.1): The Study Area

10 3.3 Geology The geology of Khartoum area was divided by Abusin and Davies (1991) into six main divisions, namely: 1. Wind-blown sand and superficial gravel. 2. Sediments of the clay plains. 3. Tertiary volcanic rocks. 4. Nubian sandstone formation. 5. Post-organic igneous complexes. 6. Basement complex metamorphosed and igneous rocks.

3.4 Soils The soils of Khartoum differ in fertility and productivity. Abusin and Davies (1991) reported four types of soils as follows: 1. Clay plains. 2. Nile terraces. 3. Peneplained surfaces. 4. Goz Abu Dulu. Al-Gasim (1996) reported that the soils of Khartoum are composed of 52% sand, 36% clays and 12% gravels.

11 3.5 Climate

3.5.1 Temperature Summer is the longest season in Khartoum, with high air- temperature. April, May, June and July are the hottest months. The maximum mean temperature reported ranges between 13.4- 28.7Cº. Winter is the coldest season, with December, January and February being the coldest months of the year (Fig. 2).

3.6 Rainfall The rainy season usually lasts June through October. The highest mean monthly rainfall was reported in August (1995). The highest means of annual rainfalls (1994, 1995) were 231.6 mm and 194.4 mm respectively (Fig. 3.).

12

13

14 3.6.1 Winds The main prevailing winds are the southwesterlies (SW) (Table. 1). The mean monthly wind speeds have been presented in Table (2).

3.6.2 Relative Humidity

The relative humidity in Khartoum area is high during the months of July, August and September (Table. 3).

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18 Chapter Four Materials and Methods

4.1 Preparation of plant material 4.1.1 Field and herbarium methods

The present work is a phytochemical study of the genus Pulicaria of the family Asteraceae in Khartoum area. The plant material was collected from the study area during the period May 2007 to May 2008. Field work included the examination of vegetative, floral and fruit characters of two main species of the genus, namely: Pulicaria crispa (Forssk) Oliv and P.undulata (L.) Mey which prevail in the study area. Aerial parts having leaves, flowers, fruits and stems were clipped from each species. The collected material was deposited at the herbarium of the Department of Biology, Faculty of Education, University of Khartoum for authentication. A preliminary identification of the collected plant material was made using the keys of Andrews (1956) and this identification was later checked using the works of Oliver (1962), Hutchinson and Dalziel (1988) and ElAwad (1995). Scientific names and synonymy have been updated by the works of Babiker (2001)and ElGhazali et al (2007) A number of employees were interviewed on historical background of the genus in the study area, its vernacular names and the commonest medicinal and folkloric uses. Brief family and species descriptions have been given and identification keys based on morphology and chemistry of the two main species have been constructed.

19 4.1.2 Drying and grinding of samples

The collected plant samples were air-dried (25-45Cº) for several days until the samples were sufficiently dry to be ground. The dried material was ground to produce a homogeneous sample and the ground material was then placed in plastic bags and kept in a refrigerator for chemical analysis.

4.2 Chemical Analysis of plant samples 4.2.1 Preparation of the digestion solution

Elemental analysis of plant material requires the preparation of a digestion mixture that destroys the organic matter and hence facilitates the release of elements for chemical analysis. The digestion solution was composed of 60% perchloric acid (HCLO4), concentrated nitric acid

(conc. HNO3) and concentrated sulphuric acid

(conc.H2SO4).

It was prepared as follows: 1ml of 60%HCLO4 was added to 5ml of conc.HNO3 and 0.5ml of conc. H2SO4. The mixture was cooled and the digestions were done as follows:2 ml of the digestion mixture were added to 0.25g of the dry ground plant material sample in a digestion tube. The plant material was first digested at 60º C. The temperature was then raised gradually to 100ºC and the digestion continued until a colorless solution was obtained. The digestion mixture was cooled and completed to 10ml.

20 4.2.2 Preparation of stock and working solutions

Stock solution (primary standard) and working solution (secondary standard) were prepared as in Table (4) below. These solutions were used for the determination of the elements calcium (Ca) and magnesium (Mg).

Table (4) Preparation of stock and working solutions used for elemental analysis of plant samples.

Element Stock solution Working solution

Ca 2.4973 of dry CaCO3 were dissolved in 0.0 – 100ml/g 200 ml of distilled water containing 5ml

of conc. HNO3. The solution was heated

to expel CO2, cooled and diluted to one liter

Mg 1.0136g of MgSO4 .7H2O were dissolved in distilled water containing 1 ml conc.

H2SO4 and the solution was diluted to one liter.

4.2.3 Determination of Ca and Mg elements

The elements Ca and Mg were determined by using an atomic absorption spectrophotometer (AAS) as follows: The spectrophotometer was switched on and the individual element wavelengths, air and gas flows were determined. The cathode lamp was stabilized, a calibration curve was drawn from the standards in turn and the stability calibration was checked. Samples were aspirated under the same conditions as the standards. The spray chamber and burner were flushed frequently with

21 deionized water. Computerized results were obtained and blanks were determined in the same way.

4.2.4 Determination of Phosphorus (P)

This was determined by the molybdenum blue method using a visible spectrophotometer. Phosphorus standards were prepared by dissolving 0.4393g of dry KH2PO4 in distilled water and the solution was diluted to one liter. Ammonium molybate –Sulphuric acid reagent was prepared as follows: 25g of (NH4) MO7. 4H2O were dissolved in 200 ml and the solution was warmed. 280 ml conc.H2SO4 was added to 400 ml of water to prepare the sulphuric acid mixture and the solution was diluted to one liter and stored in the dark. Stannous chloride reagent was prepared by dissolving 0.5g and

SnCl2.2H2O in 25ml of HCL.15 ml of the P standard were pipetted into 50 ml volumetric flash to give a standard range of 0.0 – 0.3 mg P which represents the standard working solution. 10 ml of the sample solution were pipetted into 50 ml volumetric flash and the solution was diluted to two thirds of the flask.2 ml of the ammonium molybdate-sulphuric acid reagent were mixed with 2 ml of stannous chloride. The mixture was diluted to volume and left for 30 minutes. The absorbance was measured at 200 nm. A calibration curve was drawn from the standards and was then used to determine P in the sample aliquots.

22 4.2.5 Estimation of crude protein Crude protein was estimated according to AOAC (1984), using a micro-Kjeldahl method. The basic idea behind this method is that all nitrogen in the sample is contained in proteins. The percentage of nitrogen in the sample was calculated according to the following formula: N% = T × n × 0.01 × D/sample weight × 100 Sample weight Where: T = titration volume.

N = normality of H2SO4 equivalent to NH3. D = dilution factor. N = nitrogen. Since nitrogen forms16% of the crude protein of the sample (=6.25), the percentage of crude protein is calculated by the following equation: Crude protein % = N% × 6.25

4.2.6 Estimation of ash content

The term ash refers to the residue left after combustion of oven- dried samples. it is a measure of the total mineral content in a particular sample.

The ashing procedure was carried out as follows A crucible was preheated in a muffle furnace to about 5500C, cooled in desiccators and weighed. About 1 g of plant sample was transferred to the crucible and the crucible and plant sample were weighed. The crucible and plant sample were then dried at 1050C, weighed, placed in a cold muffle furnace and the

23 temperature was allowed to rise to 5500C. The crucible and the contents were heated for two hours, allowed to cool and then transferred to a desiccator and weighed. The ash percent is calculated as follows:

ASH% = weight of crucible and ash – weight of crucible ×100 Oven-dry weight of sample

4.2.7 Estimation of total sugars Determination of total sugars was carried out on each sample according to AOAC (1984) methods.

24 Chapter Five Results and Discussion

5.1 Results 5.1.1 Botanical studies The genus Pulicaria belongs to family Asteraceae (Compositae) and here is a brief botanical description of this family:

Members are mostly herbs or shrubs. Leaves simple, opposite or alternate. Inflorescences capitate; flower heads solitary or in corymbs; florets of two types: peripheral ray florets and central disc florets; sepals absent and represented by setae or pappus; petals united; stamens (disc florets) 5, syngenesious; ovary inferior, unilocular; placentation basal; Fruit achenes.

The family includes about 100 genera and 2300 species (Hutchinson, 1959). Andrews (1956) reported the following species of the genus Pulicaria in the Sudan: Pulicaria petiolaris Jaub. and Spach., P. grantii Oliv.and Hiern. , P. attenulata Hutch. and Burtt, P. dysenterica var. stenophylla Boiss, and P. vulgaris Gaertn. El Ghazali (1983) reported that only three species of Pulicaria were found in Khartoum State, namely: P. crispa, P. undulata and P. grantii. The geographical distribution of these three species indicates that P.undulata has the widest ecological amplitude. Species distribution in the Sudan according to Andrews (1956) has been shown in Table (4) below.

25 Table (5) Distribution of Pulicaria species in the Sudan. Species Locality Pulicaria petiolaris Red sea district. Fung District. P. undulata Northern and central Sudan. P. crispa Northern and central Sudan. P. attenuta Darfur: Jebel Meidob. P. dysenterica var. stenophylla Darfur: Jebel Marra, 6500-9000 ft. P. vulgaris Kordafan P. grantii Kassala: Gash Delta. Khartoum

In Africa, the genus was reported in Egypt, Ethiopia, Cameroon, Niger, Mali, Senegal and Ghana. Other places include Lebanon, Palestine and India (El Egaimi, 1989). (Table 5).

Table (6) Geographical distribution of Pulicaria species in Africa and other countries:

Species Africa countries Other countries P. undulta Egypt, Mali, Ghana, Niger, Palestine North Nigeria P. crispa Mali, Senegal, Ghana, India, Palestine, Niger, Cameroon, Egypt, Lebanon Ethiopia

26

27

28 Key to the species

A. Leaves Lanceolate, linear or linear-lanceolate: B. Leaf base cuneate; flower heads in corymbs; inner pappus of 4 bristles. ……………….. P. petiolaris. B.B Leaf base not as above; flower heads solitary; inner pappus of 10 or mre bristles: C. Leaf base auriculate,………………P. dysenterica var. stenophylla. C.C. Leaf base not auriculate. …………………. P. grantii A.A Leaves oblong or oblanceolate or variable: D. Leaf base auriculate or amplexicaul; flower heads solitary: E. Leaves variable in shape; achenes glabrous; inner pappus of a few Bristles. …………….. P.crispa. E.E Leaves oblong or oblanceolate; achenes setulose; inner pappus of 10 or more bristles: F. Stems densely white-woolly ; leaf-bases auriculate-amplexicaull inner pappus of 14-15 bristles. …………… P. undulata. F.F Stems puberulous; leaf –bases semi-amplexicaul; inner pappus of 10-12 britles. ……………….. P.vulgaris. D.D Leaf-bases attenuate; flower heads in corymbs. P. attenuata.

Pulicaria cripa and P. undulata are the main species of the genus Pulcaria which are widespread throughout the Sudan. Pulicaria crispa (Forsk.) Oliv., Trans.Linn. Soc., Bot. 29 (1873); Aster crispa Forsk., Fl. Aegypt-Arab: 150 (1775); Frankoeuria crispa (Forsk.) Cass, Dict. Sci. Nat. 38: 374 (1825); Tackholm, St.F.E. ed. 2:563, Pl. 198/B (1974). Tagar; rabul (Ar.).

29

Whitish tomentose or occassionally nearly glabrous much branched herbs. Leaves simple, subsessile, denticulate, obovate-oblong to linear lanceolate, 0.5 – 1.5 ×0.5 - .6 in tomentose to nearly solitary, terminal, 0.3-0.6 in across; flowers yellow, aromatic . Achenes glabrous; pappus uniseriate, with a few short bristles.

Habitat: Waste grounds and water catchment areas. Distribution: Widerspread. P.undulata (L.) Mey, Ferzeichn. Pl.: 79 (1831); Inula undulata L., Syst. Nat. ed. 12: 558(1767), P. undulata L. var. alveolosa (Batt. and Trab.) Maire-Berhaut., Fl. Ser. 1:176 (1831); P. incisa (Lam.) DC., Prodr. 5:479 (1836). Tagar, rabul (Ar.). Perenmial herbs. Leaves oblong or oblanceolate to linear. Flower heads solitary terminal; flowers yellow, aromatic. Achenes setulose, slightly ribbed. Habitat: Water catchment areas on silty soils. Distribution: Widespread.

30 5.1.2 Chemical Analysis of plant Material 5.1.2.1 Chemical Analysis of leaves Dried leaf samples of Pulicaria crispa and P.undulata were analyzed for the following components: ash %, protein %, carbohydrate %, ca% and weight of Mg and P mg/100g ( Table 7).

Table (7) Results of chemical Analysis of leaves of Pulicaria undulata and P. crispa.

Chemical component P.undulata P.crispa Ash% 15.02% 13.38% Proteins % 6.1% 6.8% Carbohydrate % 1.5% 1.8% Ca% 12.3% 10.2% Mg mg/100g 18.6% 16.3% P mg/100g 28.0% 22.0%

It can be seen from the above table that ash%, ca% and the weight of Mg in mg/100g. are higher in the leaves of P.undulata as compared to the leaves of P. crispa . Moreover, the weight of P is considerably higher in P. undulata than in P. crispa. However, the two species are more or less similar in their percentages of proteins and carbohydrates. Results of statistical analysis, using F-test, showed no significant differences between the two species in the chemical components of their leaves (p=0.05) (Appendix 1).

31 5.1.2.2 Chemical Analysis of stems Dried samples of the stems of Pulicaria undulata and P.crispa were analyzed for ash%. protein %, carbohydrate %, weight of Ca, Mg and P. in mg /100g. (Table 8).

Table (8) Results of chemical Analysis of stems of Pulicaria undulata and P.crispa

Chemical component P.undulata P.crispa Ash% 10.3 8.7 Protein % 3.3 5.2 Carbohydrate% 1.2 2.8 Ca mg/100g 9.2 7.6 Mg mg/100g 12.1 10.6 P. mg/100g 18.2 14.3

From the above table, it can be observed that the stems of the two species differ in all chemical components. Pulicaria undulata has relatively higher values of P. ash% and the weight of Ca, Mg and P. As in the leaves, the weight of P in P.undulata is considerably higher than that of P.crispa, however, P.crispa has higher values of % protein and carbohydtares as compared to P.undulata. Results of statistical analysis, using F-test, showed no significant difference between the two species in the chemical components of their stems (P=0.05) (Appendix 2).

32 5.2.3 Volatile oils of Pulicaria species:

Attempts have been made to extract; volatile oils from the two species of Pulcaria, namely P.crispa and P.undulata. However, the volume of the extracted material was too small to be analyzed. For the volatile oils of the above two species mentioned in this section is extracted from El Egami (1989) and other sources of literature as can be seen in Table (9).

Table (9) Volatile oils of some Pulicaria spp.

. Species Main volatile oils Reference P.crispa Limonene, Linalool El Egami carvolanactone, nerol, eugenol (1989) P.undulata Rhamnocitrin, 3-7 dimethoxy Metwally et al quercetin (7- methoxy quercetin), (1981) dihydro-kaempferol, B-amyrim, B-sitosterol, Thymol derivatwes lupenyl acetate taraxosteryl acetate P.dysenterica Methyl ethers, scutellarin, Pares et al quercetangetin, 6-hydrooxy (1981) Kaempferol, 3,4,7 trimethyl ether, Kaempferol 3- glucoside P. vulagris Flavones, saponins Constantinescu et al (1966) P.odora Thymol, thymol isobutyrate Fadwa et al (2005)

33

Taxonomic key for Pulicaria spp. based on their volatile oils.

A. Main volatile oils are monoterpenes or sesquiterpenes: B. Volatile oils are mainly limonene, limalool, carvolanactone, nerol and eugenol. ………………….. P.crispa B.B Volatile oils are rhamnocitrim, 3-7 dimethoxyquercetin, rhametin, Kaempferol and B- sitosterol. ------P.undulata. A.A Main volatile oils are methylethers, flavones, saponins, thymol or thymol derivatives: C. Volatile oils are 3,4,7 trimethyl ether, quercetangetin and B-hydroxy Kaempferol. ------P.dysenterica. C.C Volatile oils are flavones and Saponins -----P.vulgaris C.C.C Volatile oils are thymol and thymol derivatives. ---- P.odora.

34

5.3 Discussion This study reported seven Pulicaria species in the Sudan, a finding which agrees with Andrews (1956). However, the study identified only two species of Pulicaria in Khartoum State, namely P.crispa and P.undulata. This finding partially agrees with El Ghazali et al (1988) who reported three species of Pulicaria in Khartoum State, namely Pulicaria crispa, P.undulata and P. grantii. Like many other species in the Sudan, the geographical distribution of Pulicaria species depends on climatic and edaphic factors (Andrews, 1956). Likewise, the habitat also plays an important role in the distribution of the genus Pulicaria. Consequently, the disappearance of P.grantii from Khartoum State might be due to change in climatic, edaphic and habitat conditions,excessive use and poor adaptation. Pulicaria is a weed of cultivation and thus prefers moist habitats. It is also widespread in moist places and water catchment areas. Thus, the genus Pulicaria prevails on river banks in Khartoum and Gezira States. The results of chemical analysis revealed that the species of Pulicaria crispa and P. undulata differed in their chemical constituents of the different plants organs. Statistical analysis using F-test (P=0.05) showed that there were no significant differences between the two species with regard to ash %, carbohydrate, proteins, P, Ca and Mg. There were also no significant differences between these chemical components in the leaves and stems of each of the two species. It is clear from Table (7) and (8) that the stems of the two species differ in all chemical components, but the differences are not statistically significant. Pulicaria undulata stems had relatively higher values of ash % and the weight of Ca, Mg and P as compared to the stems of P.crispa. Similarly the leaves of

35 P.undulata had considerably higher values of P elment than the leaves of P.crispa. However, the `leaves of P.crispa had higher percentages of proteins and carbohydrates as compared to the leaves of P.undulata. These findings agree with El Egami (1989) and Hafiz (1985). It was observed in this study that the odour of the leaves and flowers of Pulicaria undulata was more intense than that of P.crispa. This observation is supported by El Egami (1989) who also noticed that the odour of volatile oils extracted from P.undulata was sharper than of P.crispa. It was also supported by Fadwa et al (2005) who reported that the volatile oils of Pulicaria odora were rich in oxygenated compounds, a finding which might explain the characteristic fragrant odour of the oil. The current study reviewed the various uses of volatile oils in the Sudan and worldwide. Volatile oils had wide applications in folk medicine, as therapeutic and bacteriocidal agents (Fadwa et al, 2005;Burt, 2004; Pellecuer et al,1980;Panizzi et al ,1993, Bezic et al, 2003; Sivropolou et al, 1996). Their bacteriocidal properties were attributed to their high thymol content (47.8%) which was found to have a significant antibiotic activity. Other uses are food flavours, tea substitute in addition to molluscicdal, insecticidal and larvaecidal activities (El Egami,1989; Ali, 1997and 2008; Fadwa et al, 2005). In the Sudan, Pulicaria species have now been in wide use as a starting material in the synthesis of chemicals of new fragrance for the cosmetics industry.

36 5.4 Conclusions and Recommendation 5.4.1 Conclusions ♦ This work is a botanical chemical study on the genus Pulicaria which belongs to the family Asteraceae (Compositae). ♦ Seven species of the genus were identified in the Sudan, namely: P.crispa, P.undulata, P.grantii, P.petiolaris, P.attenuta, P.dysenterica var. stenophylla and P.vulgaris. ♦ The distribution of species in the Sudan, Africa and other countries has been indicated. ♦ Only two species pf Pulicaria were identified in Khartoum State, namely:P.crispa and P.undulata. These two species are widespread in Khartoum and Gezira States as weeds of cultivation and domains of moist habitats and water catchments areas. ♦ A brief family description has been given together with despcrition and digtal camera image illustrations for P.undulata and P.crispa. Scientific names and synonyms have been updated. Herbarium specimens have been prepared and deposited at the herbarium of the Department of Biology, Faculty of Education, University of Khartoum. ♦ Pulcraia species are aromatic herbs with characteristic odour due to their high content of volatile oils. They also contain other chemical constituents including tannins alkaloids and saponins. ♦ The major volatile oils of P.crispa were limonene, linalool, carvotanactone, nerol and eugenol. ♦ The major volatile oils of P.undulata were rhamnocitrin, 3-7 dimethoxquercetin, rhametin (7-methoxyquercetin), dihydroxykaempferol, B-amyrin, B-sitosterol and thymol derivatives.

37 ♦ The major volatile oils of P.dysenterica were methyl ethers, scutellarin, quercetangetin and 6-hydroxykaempferol. ♦ The major volatile oils of P.vulgaris were flavones. ♦ The major volatile oils of P.odora were thymol and thymol isobutyrate. ♦ Volatile oils of Pulicaria species are diverse in type and amount among the different organs of even the same species. ♦ Remarkable attention has now been given to the volatile oils due to their wide application in the fields of folk medicine as therapeutic and antibacterial agents. Volatile oils are used as food flavours, tea substitute and have recently been found to have molluscicidal and insecticidal properties.

5.4.2 Recommendations The author recommends the following: ♦ Adequate attention, interest and care are required for the good of medicinal plants with special reference for Pulicaria spp. This is because these species are widespread in the Sudan. ♦ Funding and research facilities should be availed for researchers in order to develop extraction of volatile acids of Pulicaria spp. since the volatile oils of these species are of high quality and have wide applications in folk medicine, perfume and cosmetic industry, in addition to their bacteriocidal and insecticidal properties. ♦ Pharmocologists and biochemists are urged to get more involved in the study of the medicinal and bacteriocidal properties of the volatile oils of Pulicaria spp. This is because the antibacterial activity of these oils exceeds that of the antibiotics currently used in the Sudan.

38 ♦ The author suggests the conduction of two or more research projects on Puliacria spp. in order to cover research areas that have not been dealt with in this work, such as the study of alkaloids, resins and tannins and their various uses in the Sudan.

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45 Appendix 1 Results of statistical analysis of chemical component of the stems of P. undulata and P. crispa using F-test (variance ratio test).

2 Species Σ X Σ X2 S F

P. undulata 54.3 680.71 18.20 1.11 N.S. P. crispa 49.2 485.18 16.35

N.S. = Variance not significant at P=0.01.

46 Appendix 2 Results of statistical analysis of chemical component of the leaves of P. undulata and P. crispa using F-test (variance ratio test).

2 Species Σ X Σ X2 S F

P. undulata 81.25 1746.31 127.74 2.55 N.S. P. crispa 70.48 1082.23 50.86

N.S. = Variance not significant at P=0.01.

47

Appendix 1 Results of statistical analysis of chemical component of the stems of P. undulata and P. crispa using F-test (variance ratio test).

2 Species Σ X Σ X2 S F

P. undulata 54.3 680.71 18.20 1.11 N.S. P. crispa 49.2 485.18 16.35

N.S. = Variance not significant at P=0.01.

Appendix 2 Results of statistical analysis of chemical component of the leaves of P. undulata and P. crispa using F-test (variance ratio test).

2 Species Σ X Σ X2 S F

P. undulata 81.25 1746.31 127.74 2.55 N.S. P. crispa 70.48 1082.23 50.86

N.S. = Variance not significant at P= 0.01.