Qualitative Phytochemical Screening, Thin Layer Chromatography and Toxicity Tests for the Stems of Two Cactus Genera: Euphorbia trigona and E. abyssinica Hanadi Alwan Ali Abd Alla Hamid B.Sc. (Hon.) in Medical Science of Laboratory, College of Medical Science, University of Hodaidah (Yemen), 2003 A Dissertation Submitted to the University of Gezira in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science in Biosciences and Biotechnology (Biotechnology) Center of Biosciences and Biotechnology Faculty of Engineering and Technology June 2015 1 Qualitative Phytochemical Screening, Thin Layer Chromatography and Toxicity Tests for the Stems of Two Cactus Genera: Euphorbia trigona and E. abyssinica Hanadi Alwan Ali Abd Alla Hamid Supervision Committee: Name Position Signature Dr. Mutaman Ali Kehail Supervisor …………….. Dr. Yasir Mohamed Abdelrahim Co-Supervisor …………….. Date: June, 2015 2 Qualitative Phytochemical Screening, Thin Layer Chromatography and Toxicity Tests for the Stems of Two Cactus Genera: Euphorbia trigona and E. abyssinica Hanadi Alwan Ali Abd Alla Hamid Examination Committee: Name Position Signature Dr. Mutaman Ali Kehail Chair Person …………….. Prof. Elnaem Abdalla Ali External Examiner …………….. Dr. Abdalla Ibrahim Abdalla Internal Examiner …………….. Date: 12 June, 2015 3 Dedication To my Family To my Husband To my sons To my Sisters and Brothers To those who stand closer and make it possible To my Friends and Colleges 4 Qualitative Phytochemical Screening, Thin Layer Chromatography and Toxicity Tests for the Stems of Two Cactus Genera: Euphorbia trigona and E. abyssinica Hanadi Alwan Ali Abd Alla Hamid Abstract There are over 2000 species of Euphorbia (Euphorbiaceae) in the world. The plants are succulent and the aerial parts of some species exude a milky fluid that exerts a number of physiological and a wide range of biological properties, including antioxidant, anti- inflammatory, pesticidal, cytotoxic, antibacterial and multi-drug resistance reversing activities. The objective of this study was to run qualitative phytochemical screening, thin layer chromatography (TLC) and toxicity tests for the stems of two cactus genus: Euphorbia trigona and E. abyssinica. Stems of E. trigona, were collected from within Wad Medani Town, Gezira State, whereas one large stem of E. abyssinica was brought from Erkwit mountain, Red Sea State. All of the laboratory tests were done in the Food Analysis Laboratory, Faculty of Engineering and Technology, University of Gezira. The collected stems of both plants were firstly dissected into outer parts (cortex) and inner part (pith). These parts were dried, grounded and then subjected to phytochemical screening (presence of the main classes), TLC and toxicity tests, using recommended methods. The results of this study showed that, all plant samples contained the same phytochemicals similarly, but with different concentrations for some classes, even in the same plant. The TLC tests of each of E. trigona and E. abyssinica, separated one spot from each of the stem pith and cortex per each extract (polar and apolar), but with different Rf values. ANOVA proved that, the susceptibility of Anopheles and Culex larvae toward Euphorbia stem parts (at 1.2 mg/L) were not similar, and it also proved that, the toxicity of the used plant parts were statistically not similar. The recommendations of this study were to run proper chemical and phytochemical tests (instead of some tests). An antimicrobial and pharmaceutical activity tests should be run, and also to run a proper toxicity test. 5 إختبارات المسح الكيميائي النباتي النوعي, كروماتواغرفيا الطبقة الرقيقة والسمية لسيقان جنسين من الصبار: إيوفوربيا ت اريقونا وايوفوربيا أبسينيكا هنادي علوان علي عبد اهلل حميد ملخص البحث يوجد أكثر من ألفي نوع من اﻹيوفوربيا )عائلة اﻹيوفوربيا( في العالم. النبات عصيري واﻷجزاء الخضرية لبعض اﻷنواع تفرز سوائل لبنية لها العديد من الخواص الفسيولوجية ومدي واسع من الخواص البيولوجية, والتي تشمل مضاد التأكسد, مضاد اﻹلتهاب, مبيد لﻵفات, سم خلوي, مضاد للبكتريا وذو نشاطات عاكسة للمقاومة العديدة للعقاقير. هدف هذا البحث إلي إجراء إخاتبارت مسح كيميائي نباتي نوعي, كروماتوغرافيا الطبقة الرقيقة والسمية لسيقان جنسين من صبار إيوفوربيا اتريقونا وايوفوربيا إبسيكنيكا. جمعت سيقان نبات إيوفوربيا ت اريقونا من داخل مدينة ود مدني, وﻻية الجزيرة, بينما تم إحضار ساق واحد كبير من إيوفوربيا أبيسينيكا من جبال أركويت, وﻻية البحر اﻷحمر. تمت كل اﻹخاتبارت المعملية في معمل تحليل اﻷغذية, كلية الهندسة والتكنولوجيا, جامعة الجزيرة. تم تشريح السيقان التي تم جمعها أوﻻً لجزء خارجي )قشرة( وجزء داخلي )نخاع(. تم تجفيف هذه اﻷجزاء, سحقها ومن ثم تعريضها للمسح الكيميائي النباتي )وجود المجاميع الرئيسية(, إختبار الطبقة الرقيقة واختبار السمية, بإستخدام الطرق الموصي بها. أوضحت نتائج هذه الد ارسة أن كل عينات النباتات تحتوي علي نفس المواد الكيميائية النباتية بصورة متشابهة, ولكن باتركيز مختلفة لبعض المجاميع, وحتي في نفس النبات. فصل إختبار الطبقة الرقيقة لكل من النباتين بقعة واحدة في كل من قشرة ونخاع الساق لكل مستخلص )القطبي والﻻقطبي(, ولكن مع قيم معامل إعاقة مختلفة. أثبت إختبار تحليل التباين أن حساسية يرقات اﻷنوفلس والكيولكس تجاه أجزاء ساق نباتات اﻹيوفوربيا )عند 1.2 ملغ/لتر( ليست متساوية, وايضاً أن سمية أج ازء النباتات المستخدمة ليست متماثلة إحصائياً. توصيات هذه الد ارسة هي إج ارء إختبا ارت كيميائية وكيميائية نباتية متكاملة )بدﻻً من بعض اﻹختبا ارت(. يجب إج ارء بعض إختبا ارت النشاط المضاد للميكروبات والنشاط الصيدﻻني وكذلك إجراء إختبار سمية متكامل. 6 List of Contents Subject Page Dedication iii Abstract iv Arabic Abstract v List of Contents vi List of Tables viii CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO: LITERATURE REVIEW 3 2.1. Cactus 3 2.1.1 Morphology 3 2.1.2.Stems 3 2.1.3 Uses 4 2.2. Euphorbia 5 2.2.1. Euphorbia trigona 6 2.2.2. Euphorbia abyssinica 6 2.2.3 Researches on Euphorbia species 8 2.3. Phytochemicals 8 2.4. Chromatography 9 CHAPTER THREE: MATERIALS AND METHODS 3.1 Samples 11 3.2. Methods 11 3.2.1. The phytochemical screening 11 3.2.2.1 For glycosides 11 3.2.2.2 For flavonoids and flavonones 11 7 3.2.2.3 For saponnins 12 3.2.2.4 For tannins 12 3.2.2.5 For sterols and or triterpenes 12 3.2.2.6 For alkaloids 12 3.3. Thin layer chromatography test 13 3.4. Toxicity test of stem parts powder 13 3.4. Statistical analysis 13 CHAPTER FOUR: RESULTS AND DISCUSSION 4.1. The phytochemical screening in the stem parts 14 4.2 Thin layer chromatography tests for the stem parts of both plants 16 4.3 Toxicity of E. trigona and E. abyssinica stem parts against mosquito larvae 18 CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions 20 5.2. Recommendations 20 References 21 8 List of Tables Table Title Page No The presence and absence of some phytochemicals in stem pith 4.1 15 and cortex of both plants 4.2 TLC for the stem parts of both plants 17 The percentage mortalities of Aopheles and Culex larvae toward 4.3 the powders of E. trigona and E. abyssinica stem parts (at 0.3 g/ 19 250 ml) 9 Chapter One Introduction Cacti are commonly grown as houseplants. They are pretty and easy to grow. Some cacti are grown in gardens, especially in dry areas. Cactus can be used as a living fence. The wood of dead cactus is sometimes used for building. People eat the fruit of some kinds of cactus, such as dragon fruit and prickly pear. Ciouvhiul insects also eat prickly pears. These insects produce a red coloring used in food and lipstick (Anderson, 1999). There are over 2000 species of Euphorbia (Euphorbiaceae) in the world. The plants have a unique flower structure and a significant percentage of them are succulent. When broken or cut, the aerial parts of some species exude a milky fluid that exerts a number of physiological effects including skin irritation, tumor promotion, and pro-inflammatory properties. Recently, certain Euphorbia species were found to exhibit a wide range of biological properties, including anti-anaphylactic, antioxidant, neuritogenic, anti-HIV, anti-arthritic, anti-inflammatory, pesticidal, cytotoxic, analgesic, antiplasmodial, antibacterial and multi-drug resistance reversing activities (Faky et al., 2008). The Euphorbia plants are annual or perennial herbs, woody shrubs or trees with a caustic, poisonous milky sap (latex). The roots are fine or thick and fleshy or tuberous. Many species are more or less succulent, thorny or unarmed. The main stem and mostly also the side arms of the succulent species are thick and fleshy, 15–91 cm (6–36 inches) tall. The fruits are three (rarely two) compartment capsules, sometimes fleshy but almost always ripening to a woody container that then splits open (explosively, see explosive dehiscence). The seeds are 4-angled, oval or spherical, and in some species have a caruncle (Carter, 2002). The toxic constituents of Euphorbia species were considered to be a kind of specific diterpenes, globally called phorboids, which comprise tigliane, ingenane and daphnane diterpene derivatives. Terpenes, including diterpenes and triterpenes, have been frequently found in Euphorbia species. Steroids, cerebrosides, glycerols, phenolics and flavonoids were also isolated from plants of the genus, but the compounds most relevant to the toxicity and considerable biological activities in Euphorbia are diterpenes, especially those with abietane, tigliane, and ingenane skeletons (Haba, et al., 2009; Cateni et al., 2003). 10 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