Sarah Samir Mohamed Gharib Abd-ullah Pharmacognosy Department, Faculty of Pharmacy Misr University for Science and Technology A Pharmacognostical Study of Artemisia annua L. (family Asteraceae) Cultivated in Egypt Under the supervision of Prof. Dr. Hesham I. El-Askary Professor of Pharmacognosy, Faculty of Pharmacy Cairo University Prof. Dr. Meselhy R. M. Zayed Professor of Pharmacognosy, Faculty of Pharmacy Cairo University Prof. Dr. Hamida M. A. El-Gohari Professor of Pharmacognosy, Faculty of Pharmacy Cairo University (Misr University for science and technology) Abstract Artemisia annua L. has been used in China for more than 2000 years for treating many disorders including malaria, the main source for the artemisinin. Botanical identification and DNA finger print of the leaves were carried out, to confirm identity of the drug. The present work includes phytochemical screening which revealed the presence of flavonoids, sterols and/or triterpenes, carbohydrates, tannins and volatile oil. Determination of LD50 indicated the safety of alcoholic extract of leaves (4 gm/kg b.wt.). The ethyl acetate fraction showed significant hepatoprotective, anti-hyperglycemic and antioxidant activities. The study of the polar fractions resulted in isolation and identification of 5 major quinic acid derivatives: 3,5-dicaffoeylquinic acid, 4,5-dicaffoeylquinic acid, 3,4-dicaffoeylquinic acid, 3,4-dicaffoeylquinic acid methyl ester and 3- feruloeylquinic acid. Standardization of the concentration of 3-feruoyl quinic acid (B ) in Artemisia 1 annua L. sample by using it as external standard, also study the influence of seasonal variation on quinic acid derivatives content in A. annua L. leaves was carried out, where the highest concentration of quinic acid derivatives, were found in plant leaves at the pre-flowering stage. Key words: Artemisia annua L., botanical identification, hepatoprotective, anti- hyperglycemic, antioxidant activities, polar fractions, quinic acid derivatives, standardization and seasonal variation. Date of registration: 12-4-2012 Date of thesis defense: 18-3-2015 Period of practical work: 3 years Protocol of present study: 1. Reviewing literature on Artemisia annua L. 2. Collection, identification and authentication of plant material. 3. Botanical study of the different organs of the plant including the macro-and microscopical investigation. 4. Successive extraction of the plant material with different solvents and preparation of different fractions. 5. Biological screening on the different fractions. 6. Biologically active fractions will be subjected to phytochemical investigation followed by isolation of their main constituents by using different chromatographic techniques. 7. Structure elucidation of isolated compounds. 8. Standardization of the bioactive fractions. Introduction The genus Artemisia is one of the largest and most widely distributed genera of family Asteraceae. Generally the genus mainly comprises aromatic herbs and shrubs which includes over 400 species (Mabberley 1990) which are distributed mainly in the Northern hemisphere and temperate zones of Europe, Asia and America. Most of the members of the genus have characteristic scent and taste (Mucciarelli and Maffei 2002; Mohamed et al. 2010; Bora and Sharma 2011). Plants of genus Artemisia produce aromatic oils, or used as flavorings, hallucinogens, vermifuges, and in pharmaceutical industries (Linda et al. 2002). Artemisia herbs have been used worldwide in folk medicine since ancient times. They have been used as tonics, anthelmintic (Bailey 1976) and in treating wounds, bronchitis, and tuberculosis. Medicinally, these species are used for their anti-malarial activity (Zheng 1994; Allen et al. 1998; Tan et al. 1998; Efferth 2007), and found to demonstrate anti-tumor, antiulcerogenic, diuretic (Jung et al. 2004), antioxidant (Kordali et al. 2005; Şura et al. 2012 ), cytotoxic, immunosuppressive activity (Noori et al. 2004) antipyretic, analgesic (Huang et al. 1993), antimicrobial (Şura et al. 2012 ) and antifungal activities (Farzaneh et al. 2005; Kordali et al. 2005). The accumulation of essential oils and other terpenoids in the genus is responsible for the use of its members for flavoring foods or liqueurs, in inflammation and infections by fungi, bacteria and viruses (Kim et al. 2002; Abad et al. 2012; Şura et al. 2012 ). Research interest in genus Artemisia is due to the diverse biology and chemistry of the constituents, for the frequent application in traditional medical practice, and the rich source of the plant material (Wright 2002). Phytochemical reports on Artemisia species deal mainly with terpenoids, flavonoids, coumarines, steroids and polyacetylenes (Wright 2002; Lee et al. 2003). Genus Artemisia is represented in Egypt by A. monosperma, A. judica, A. scorpia and A. herba-alba (Rizk 1986). Artemisia annua L. (Quinghao in Chinese) is considered to be a “cooling herb” in traditional Chinese medicine, also known as sweet Annie, sweet wormwood, annual wormwood (Ferreira et al. 1997; WHO 2006). The plant is native to Asia, mainly China and cultivated in many countries all over the world (Mojarad et al. 2005; Ajah and Eteng 2010) and in high-altitude regions and regions with a pronounced cool period. In Africa, it has been introduced to Cameroon, Ethiopia, Kenya, Tanzania, Uganda, Zambia, Ghana, Rwanda, and South Africa (Allen et al. 1997; Ferreira et al. 1997; Willcox et al. 2004). Artemisia annua L. was firstly appeared in a book entitled “Wu Shi Er Bing Fang” (Prescriptions for Fifty-Two Ailments) more than two thousand years ago and reported for the treatment of hemorrhoids; but “Zhou Hou Bei Ji Fang” (Handbook of Prescriptions for Emergency Treatment), describes the use of A. annua as a treatment for fevers and chills. The first text which specifically identified Artemisia annua L.as a remedy for malaria is “Ben Cao Gang Mu”. (Daniel et al. 1984; Klayman 1985; Hsu 2006). In the modern pharmacopeia of the People’s Republic of China since 1985, A. annua L. is officially listed as the aerial parts of A. annua L. (5g dried herb/1L water for the preparation of a decoction) (Tu 1992). Artemisia annua has become a valuable agricultural crop as a source of raw material and for the production of modern cost-effective antimalarial drug product. It remains the most valuable source for supplying artemisinin and several derivatives. The World Health Organization recommended artemisinin combined therapy (ACTs) in the treatment of chloroquine-resistant Falciparum malaria (Bhakuni et al. 2001; Sriram et al;, 2004, Namdeo et al. 2006; WHO 2006; Ferreira 2007) and artemisinin derivatives; artemether, arteether and artesunate which exhibit an improved solubility and greater potency than artemisinin towards drug-resistant Plasmodium infections (Sriram et al. 2004; Abad et al. 2012), also artemisinin appears to be selective for human breast cancer. (Linda et al. 2002). Accordingly, several trials were carried out in different countries for cultivation of A. annua L. with high artemisinin content. (El-Askary et al. 2004) succeeded to cultivate A. annua L. in Egypt with a high yield of artemisinin (1.0 % dry weight; at the pre-flowering stage, when compared with that in the Vietnamese cultivar; 0.86% dry weight) (Ferreira et al. 1995). In Pakistan, a decoction of A. annua herb is used for the treatment of malaria, while the leaves are used for fever, cough and common cold and to treat diarrhea. The oil of A. annua L. is used in perfumes due to its pleasant fragrance (Hayat et al 2009). Recent researches found that artemisinin and its derivatives are effective against cancer, leishmania (Yang and Liew 1993; Sen et al. 2007), Trypanosoma (Mishina et al. 2007) and have antiviral activities (Abid Ali Khan et al. 1991; Li et al. 2005). Leaves and crude extracts of A. annua L. have been reported to demonstrate antioxidant activities which is due to the high content of flavonoids (Zheng and Wang 2001l; Cai et al. 2004; Bilia et al. 2006). Artemisia annua L. has attracted our attention due to its diverse biological actions, ranging from anti-malarial to anticancer activities (Woerdenbag et al. 1993; Beckman et al. 1998). Previous reports focused mainly on terpenes of non-polar fractions, especially artemisinin; the sesquterpene lactone. However, little attention was paid to the phenolic compounds of the polar fraction. Accordingly, this work aimed at exploring the polar fractions of A. annua to identify their biologically active compounds. Therefore, it is deemed of interest to carry out:  Botanical study of the plant cultivated in Egypt.  Authentication of the plant by DNA fingerprinting.  Successive fractionation of the alcoholic extract of the leaves.  Biological study of the polar fractions using: 1- CCl Induced liver damage rats. 4 2- Alloxan-induced diabetic rats.  Cytotoxic study of polar fraction using different cell lines.  Isolation of the active principles and identification of main constituents in the polar fractions. Summary Artemisia annua L. (Quinghao in Chinese) is a traditional Chinese medicinal plant, known as the only source of artemisinin; the effective anti- malarial drug. The plant is native to Asia, mainly China and cultivated in many countries all over the world. Artemisia annua L. firstly appeared for the treatment of hemorrhoids fevers and chills more than two thousand years ago, and on 1985, A. annua L. has officially been listed as a remedy for malaria. Studies conducted on A. annua L. reported the isolation of terpenes from non- polar fractions, especially artemisinin; the sesquiterpene lactone. However, little attention was paid to the phenolic compounds in the polar fraction. Accordingly, this work aimed at exploring the polar fractions of A. annua L. leaves to identify their biologically active compounds. The present study includes: Part I: Botanical identity of the plant, Part II: Biological study of polar fractions of the alcoholic extract of Artemisia annua L. leaves, Part III: phytochemical study of Artemisia annua L. leaves, Part IV: Standardization and seasonal variation of the total quinic acid derivatives in A. annua L. leaves Part I: Botanical identity of the plant Chapter 1  Macromorphology: Artemisia annua L. is an annual herb, cultivated in Experimental station of medicinal plant of the Faculty of Pharmacy, Cairo University, Giza. The herb is attaining a height of 1-2.5 m. It has a single stem with alternating branches. 1. The stem Stem is herbaceous, erect, cylindrical, single, longitudinally ribbed with alternate branches. The stem is light green in color, slight hairy with a faint aromatic odor and bitter in taste. 2. The leaf The leaf is green in color being glabrous with a thin brittle, papery texture. The leaf is simple, lobed with pinnatipartite to pinnatisect having acute apex and serrate margin. The leaf possesses aromatic odor and bitter in taste. 3. The flower The flower head is tiny, yellow in color, occurred as hemispherical capitula attached to a short peduncle (stalk). Each individual capitulum measured 1 mm long and 3 mm in diameter. They are arranged in racemose manner. The capitulum is imbricated with 2-3 rows of involucular bracts. Ray florets are sessile, zygomorphic, pistillate; corolla is narrow tubular rounded upwards with 2-3 teeth. Disc florets are sessile, actinomorphic, and hermaphrodite; corolla is tubular with five sympetalous corolla, anthers have five syngenecious epipetalous stamens.  Micromorphology Chapter 2 1. The stem A transverse section in the stem is rounded in outline showing numerous ridges. It is formed of an outer epidermis with few hairs, followed by a cortex. The cortex consists of 2-3 rows of collenchyma then several rows of parenchyma cells. The endodermis is indistinct. The pericycle consists of large patches of lignified fibers interrupted by parenchyma cells. The vascular tissue forms separated vascular bundles arranged in a circle surrounding wide pith. The central pith is relatively wide formed of parenchyma cells. The pith occupied about two third of the stem radius. The epidermis Epidermal cells are polygonal, elongated cells with straight anticlinal walls and smooth cuticle; it shows few trichomes arising from one epidermal