Eucalyptus Cinerea Cultivated in Egypt

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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 اإلنسان )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

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)

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,

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)

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.

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.

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

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 of Eucalyptus nitens juvenile foliage harvested from coppiced trees. Alpha-pinene, limonene, 1,8-cineol, cis-ocimene and α- terpineol were the principal components. Minor constituents were monoterpene alcohols, ketones, and aldehydes and 3- methylbutanal.

Simmons and Parsons (1987) (13) investigated seasonal variation in the volatile leaf oils of individual trees of Eucalyptus ovata and Eucalyptus camphora. They concluded that variation is best explained by a leaf ageing effect rather than a seasonal effect.

Li et al., (1994) (14) examined the chemical composition of the leaf oils of Eucalyptus nitens and E. denticulata trees grown in a provenance trial in Tasmania. The leaf oil of E. nitens was characterized by high levels of 1,8-cineole and α- pinene, while that of E. denticulata by high levels of p-cymene

Review of literature and γ-terpinene. It was found that the oil yield from E. nitens juvenile leaves was significantly higher than that from the corresponding adult leaves for all source localities examined.

Li et al., (1995) (15) analysed the volatile oils from juvenile and adult leaves collected from natural populations of five Eucalyptus species from the series Obliquae (E. obliqua, E. delegatensis, E. regnans, E. sieberi and E. pauciflora) and seven species from the series Piperitae (E. risdonii, E. tenuiramis, E. pulchella, E. amygdalina, E. nitida, E. radiata and E. coccifera) of the informal subgenus Monocalyptus. Oil yields (1.3–4.9% dry wt.) from adult leaves of all species were markedly higher than from corresponding juvenile leaves (0.1–3.1% dry wt.). Volatile leaf oils of most species are rich in α- and β-phellandrene, cis- and trans-menth-2-en-1-ol, while some were rich in 1, 8-cineole or α, β and γ-eudesmol. Multivariate analyses indicated that the Tasmanian Eucalyptus species from series Obliquae are well differentiated and exhibited little intraspecific variation in percentage oil composition. In contrast, species from the series Piperitae showed great intraspecific variation and are poorly differentiated which is consistent with morphological studies.

Foudil-Cherif et al., (2000) (16) studied the leaf essential oils of nine Algerian Eucalyptus species. The essential oils were analyzed by GC and GC/MS. The techniques allowed the detection of more than 40 components in each essential oil.

Alitonou et al., (2004) (17) The chemical composition of the leaf oil of Eucalyptus tereticornis Sm. was determined by

Review of literature

HR-GC and GC/MS. Twenty-three compounds, representing 91.0% of the oil were identified. The major components were: p-cymene (31.4%), β-phellandrene (9.77%), spathulenol (8.13%), γ-terpinene (7.03%) and α-phellandrene (6.78%).

Tapondjou et al., (2005) (18) analysed the essential oils distilled from Eucalyptus saligna and Cupressus sempervirens leaves by GC/MS.

Sartorelli et al., (2007) (19) analyzed the leaf essential oils of Eucalyptus robusta and Eucalyptus saligna, the latter in two phenologic stages by GC/MS. The major constituent in E. robusta oil was α-pinene (73.0% of the total amount). On the other hand, the oil composition of the E. saligna was dependent of the phenologic stage. In the vegetative phase, the major constituents were p-cymene (54.2%) and γ-terpinene (43.8%), while during flowering α-pinene became the major constituent followed by p-cymene (22.5%).

Ashour (2007) (20) analyzed the essential oils distilled from different organs of Eucalyptus sideroxylon and Eucalyptus torquata cultivated in Egypt by means of GC/MS.

Elgizawy (2008) (21) studied the effect of seasonal variation on the composition of the essential oil of the leaves of Eucalyptus camaldulensis var. camaldulensis and Eucalyptus citriodora Hook using GC/MS technique.

Cheng et al., (2009) (22) analyzed the chemical composition of the leaf essential oils of Eucalyptus

Review of literature camaldulensis and Eucalyptus urophylla using GC/MS technique. Babu et al., (2009) (23) evaluated the first order kinetic and Langmuir adsorption models to simulate hydrodistillation of E. cinerea oil. The Langmuir model parameters that simulate the hydrodistillation process were determined. GC/MS analysis revealed that the oil produced from fresh foliage contained higher 1,8-cineole content (84.4%) than the dried foliage (77.6%). The other major constituents were limonene and α-terpineol. Rasooli et al., (2009) (24) analyzed the essential oils of Mentha spicata and Eucalyptus camaldulensis by GC and GC/MS.

Palacios et al., (2009) (25) analyzed twelve essential oils obtained by hydrodistillation of edible fruits and herbs by (GC/MS). According to GC/MS analysis, 1,8-cineole (56.86%) was the major constituent of E. cinerea essential oil.

Gilles et al., (2010) (26) determined the chemical composition of the essential oils of three Australian Eucalyptus species, namely E. olida, E. staigeriana and E. dives by GC/MS. A total of 24 compounds were identified from the essential oil of E. dives, with the major compounds being piperitone (40.5%), α-phellandrene (17.4%), p-cymene (8.5%) and terpin-4-ol (4.7%). For E. staigeriana, 29 compounds were identified with 1,8-cineole (34.8%), neral (10.8%), geranial (10.8%), α-phellandrene (8.8%) and methyl geranate (5.2%) being the dominant ones. In contrast, a single

Review of literature compound, (E)-methyl cinnamate, accounted for 99.4% of the essential oils of E. oilda, although 20 compounds were identified.

Maciel et al., (2010) (27) studied the chemical composition of essential oils of three Eucalyptus species. The major constituents of the oils were Z-citral and α-citral (E. staigeriana), citronellal (E. citriodora) and 1,8-cineole (E. globulus).

Toloza et al., (2010) (28) reported the chemical composition of essential oils of Eucalyptus dunnii, Eucalyptus gunni, and another five Eucalyptus species.

Medhi et al., (2010) (29) analyzed the chemical compositions of the essential oil of Eucalyptus camaldulensis leaves using (GC/MS). The main constituents in the leaf essential oil were 1,8-cineole (69.46%), γ–Terpinene (15.10%), α–Pinene (5.47%) and Globulol (2%). Tyagi and Malik (2011) (30) determined the chemical composition of the esential oil of E. globulus in liquid and the vapour phases by GC, GC/MS and solid phase micro extraction-gas chromatography mass spectrometry (SPME GC-MS), respectively. The dominant compounds in E. globulus oil were 1,8-cineole (45.4%), limonene (17.8%), p- cymene (9.5%), γ-terpinene (8.8%), α-pinene (4.2%) and α- terpineol (3.4%), while in the vapour phase, 1,8-cineole (34.6%), limonene (29.9%), p-cymene (10.5%), γ-terpinene (7.4%), α-pinene (4.0%) and α-phellandrene (2.4%) were the major compounds identified.

Review of literature

Elaissi et al., (2011) (31) analysed the essential oils of twenty Eucalyptus species harvested from Tunisia by GC and GC/MS for the study of the chemical variability. The main identified compounds were 1,8-cineole followed by α-pinene, p-cymene, borneol, cryptone, spathulenol, viridiflorol and limonene. The chemical principal components analysis identified 10 chemotypes.

Ogunwande et al., (2011) (32) analyzed the essential oils obtained by separate hydrodistillation of three different plants cultivated in Nigeria ,including Eucalyptus toreliana L, by means of GC and GC/MS. The oil contained 1,8-cineole (39.4%) and α-terpinyl acetate (10.7%) as the major components. The oil also features high levels of sabinene (5.9%), caryophyllene-oxide (4.7%) and α-pinene (4.2%).

Ben Jemâa et al., (2012) (33) investigated seasonal variation in the chemical composition of essential oils isolated from leaves of five Eucalyptus species (Namely: E. camaldulensis, E. astringens, E. leucoxylon, E. lehmannii and E. rudis) Gas chromatography and GC/MS analyses showed that chemical composition varied with Eucalyptus species and seasons. The five essential oils contained 1,8-cineole, α- pinene, and α-terpineol as major compounds. beta-pinene and p-cymene were only present in E. rudis essential oil. In addition, o-cymene was specific only to E. camaldulensis and E. rudis essential oils.

Review of literature

II. Sterols and triterpenes

Horn and lamberton (1964) (34) identified 11, 12- dehydro-ursolic acid lactone acetate as a triterpene in E. globulus Labill., E. cinerea F. Muell. ex Benth., E. risdoni Hook and E. urnigera Hook. leaf waxes.

Dayal (1982) (35) isolated betulinic acid [1], ursolic acid [3] and β-sitosterol from the acetone extract of the roots of E. citriodora Hook.

Hongcheng and Fujimoto (1993) (36) isolated two new triterpene esters, namely tereticornate A [15] and B from the dried leaves of Eucalyptus tereticornis.

Siddiqui et al., (1997) (37) isolated ten pentacyclic triterpenoids including two new constituents, eucalyptic acid and eucalyptolic acid and eight known compounds (ursolic acid lactone, betulinic acid, oleanolic acid [5], ursolic acid, 3β- O-cis-p-coumaroyl-alphitolic acid, alphitolic acid, 3-β-O- trans-p-coumaroyl-alphitolic acid, and 3-β-O-trans-p- coumaroyl-maslinic acid) from the fresh and uncrushed leaves of Eucalyptus camaldulensis var. obtusa. Their structures were elucidated by detailed 1D and 2D NMR studies.

Siddiqui et al., (2000) (38) investigated the constituents of the fresh leaves of Eucalyptus camaldulensis var. obtusa and isolated the new triterpenoid amirinic acid [7] and four known triterpenoids: ursolic acid lactone, betulinic, oleanolic and ursolic acids.