A Pharmacognostical study of Eucalyptus cinerea cultivated in Egypt A thesis submitted by Fatema Reda S. El-Sayed For The Degree of Master in Pharmaceutical Sciences (Pharmacognosy) Under the Supervision of Prof. Dr. Fathy M. Soliman Professor of Pharmacognosy Faculty of Pharmacy Cairo University Prof. Dr. Magda M. Fathy Professor of Pharmacognosy Faculty of Pharmacy Cairo University Dr. Maha M. Salama Associate Professor of Pharmacognosy Faculty of Pharmacy Cairo University (2012) بسم اهلل الرحمن الرحيم ِ } إِ َّن اأْلَبأَراَر يَ أشَربُو َن م أن كَأأ ٍس ِ كَا َن مَزا ُج َها كَافُوًرا { سورة اﻹنسان )5( Approval sheet A Pharmacognostical study of Eucalyptus cinerea cultivated in Egypt Approved by: …………….………………………………… …………….………………………………… …………….………………………………… …………….………………………………… …………….………………………………… Date: / / 2012 ACKNOWLEDGEMENT First of all thanks to Allah, by his garce this work was accomplished. I would like to express my heartily appreciation and sincere gratitude for Professor Dr. Fathy Mohamed Soliman, professor of pharmacognosy, faculty of pharmacy, Cairo University for supervising this work, his kind help, valuable guidance and unlimited encouragement during the course of this study. I wish to express my honest and most sincere gratitude and my heartily appreciation to Professor Dr. Mageda Mohamed Fathy, professor of pharmacognosy, faculty of pharmacy, Cairo University, for her guidance and continuous encouragement. I refer my great indebtedness to Dr. Maha Mahmoud Salama, Associate Professor of pharmacognosy, faculty of pharmacy, Cairo University. No words could express my gratitude and my deep appreciation for Dr. Ali El Halawany and Dr. Reham Salah El-Din, Assistant Professors of pharmacognosy, Faculty of Pharmacy, Cairo University, for their precious help throughout the study. I am deeply thankful to all my professors and colleagues in the Pharmacognosy Depatement, Faculty of Pharmacy, Cairo University, who gave me the benefits of their experience and encouragement and all those who helped in making this work possible. Finally, my great indebtedness and indefinite affection are due to the members of my family especially my mother, father, husband and my dear son for their help and continuous encouragement during the course of this study. Introduction Introduction Introduction The plant kingdom includes many medicinally useful plants. For a long time, they were considered as an essential source of many drugs for treatment of different human diseases. Despite of the great advances in synthetic drugs, their adverse side effects directed researchers' eyes to the more safe herbal products. A large number of plants were constantly screened for their possible pharmacological activities, among which are those which belong to family Myrtaceae. The Myrtaceae (Myrtle, eucalypts, clove, or guava family) is a large family of dicotyledonous woody plants placed within the order Myrtales containing over 5,650 species organized in 130 to 150 genera. (1) Recognized as the eighth largest flowering plant family, it comprises several genera of outstanding ecological and economic relevance worldwide. The family occurs mainly in the Southern Hemisphere. It has centers of diversity in the wet tropics, particularly South America, Australia, and tropical Asia with occurrences in Africa and Europe. (1) 1 Introduction Eucalyptus L' Hér. is a large genus of family Myrtaceae. It is comprised of about 900 species and subspecies.(2) Though it is a native of Australia, it is found in almost all parts of the world due to human introduction. It is now cultivated in many tropical, sub- tropical and even sub-temperate countries.(3) Eucalyptus is generally grown for its wood, which is primarily used in making cellulose pulp and secondly for boards and panels.(2) Eucalyptus species are well- known for their essential oils that are extracted through steam or hydrodistillation. These have been widely used in the perfumery and fragrance industries, and in fact, these are among the world's top- traded oils.(4) Essential oil from Eucalyptus species was used as an antiseptic, antipyretic and analgesic since ancient times.(2) It has been reported to possess a wide range of biological activities including antimicrobial, fungicidal, insect-repellant, fumigant, pesticidal and acaricidal activity.(4) Eucalyptus leaves were used as a traditional household remedy in Australia. The leaves and oil were used specially for respiratory conditions such as bronchitis and croup, and the dried leaves were smoked like tobacco for asthma. It is also used for feverish conditions (malaria, typhoid, cholera, etc.) and skin problems such as burns, ulcers and wounds. Aqueous extracts were used for aching joints, 2 Introduction bacterial dysentery, ringworms and tuberculosis. The wood is also used for timber production in Spain (5). A diverse range of biological activities have been demonstrated by the many kinds of non-volatile secondary metabolites isolated from Eucalyptus species. Examples of their pharmacological actions include anti-oxidant, antimalarial, antibacterial, antiviral, HIV-RTase inhibition, aldose reductase inhibition and cancer chemopreventive activities. Such broad pharmacological profiles provided a stimulus to further research and it is likely that more new compounds will be isolated from an increasing number of Eucalyptus species in the future. It is likely that in the years ahead Eucalyptus metabolites other than the volatile constituents will form part of the armory of drugs available to the physician for the treatment or prevention of human diseases.(6) Eucalyptus cinerea F. Muell. ex Benth. is a small to medium- sized tree. It is distributed from the central western slopes of New South Wales in the Wattle Flat area south to Beechworth in northern Victoria. It is notable for the thick rough bark and the mature crown of largely grey or glaucous sub-adult leaves.(7) 3 Introduction Eucalyptus cinerea F. Muell. ex Benth. is commonly known as Silver Dollar Gum, Argyle Apple and Mealy Stingybark. This species of Eucalyptus is widely used in fresh or dried arrangements and makes a beautiful landscape specimen. It is planted single or in groups. It has distinctive blue-green aromatic foliage.(7) Reviewing the current literature few reports were found dealing with the chemical and botanical studies of Eucalyptus cinerea F. Muell. ex Benth. Therefore, this work was performed to investigate its active constituents to find out their probable medicinal effects as well as studying the macro- and micromorphological features to help identification of the plant in entire and powdered forms. The present study includes: Part I: Botanical study: 1. Macromorphological study of the plant organs. 2. Micromorphological study of the plant organs. Part II: Phytochemical study: 1. Preliminary phytochemical screening of the plant organs. 4 Introduction 2. Investigation of the essential oil content of the leaves and stems. 3. Investigation of the lipid content. 4. Investigation of the ethyl acetate fraction of the leaves. 5. Investigation of the chloroform:methanol (80:20) extract of the leaves. 6. Investigation of the total tannin content. Part III: Biological study: 1. Pharmacological study: i) Determination of the safety of the plant. ii) Anti-oxidant activity. iii) Anti-inflammatory activity. 2. Cytotoxic activity. 3. Antimicrobial activity. 4. Molluscicidal, larvicidal and pupicidal activities. 5 Review of Literature Review of literature Review of literature Eucalyptus, an Australian native, belongs to family Myrtaceae and represented by up to 900 species of tall, evergreen and magnificent trees cultivated world over for its oil, gum, pulp, timber and medicinal value. (2, 4) Extensive survey of literature revealed that Eucalyptus species is an important source of many pharmacologically and medicinally important chemicals, such as Essential oils, terpenoids, polyphenolic compounds including flavonoids, tannis and acylphloroglucinol compounds. Various Eucalyptus species were also studied for their various pharmacological activities like analgesic, antifungal, anti-inflammatory, antibacterial, antidiabetic, antioxidant, Antiviral, Antitumor, antihistaminic, anticancer, cytochrome P-450 inhibition and hepatoprotective properties.(9) I. Essential oil Among the various wood and non-wood products, essential oil found in Eucalyptus foliage is the most important one. It finds extensive use in food, perfumery and pharmaceutical industries. In addition, the oil possesses a wide spectrum of biological activities viz., anti-microbial, fungicidal, insecticidal/insect repellent, herbicidal, acaricidal and nematicidal. Eucalyptus oils could be used as environmentally benign pest control against bacteria, fungi, insects, nematodes, weeds and mites. The use of Eucalyptus oil as a natural pesticide is of immense significance in view of the environmental and toxicological implications of the 6 Review of literature indiscriminate use of synthetic pesticides and overcoming or reducing the problem of increasing pest resistance.(4) Singh et al., (1983) (10) studied seventeen Eucalyptus species for their oil content. Results revealed that E. citriodora Hook is a good source of citronellal and citronellol which are used in perfumery industry. Hassan (1985) (11) studied the GC/MS analysis of E. camaldulensis F. Muell., E. diversifolia Bonpl., E. ficifolia F. Muell. and E. lehmanni Preiss., and found that the four essential oils were different in composition. Franich (1985) (12) analyzed the essential oil