Phytochemical and Biological Evaluation of Citrus volkameriana Ten. & Pasq. Growing in Egypt Thesis submitted by Ahmed Mohammed Abdel-Latif Mohammed Tawila Research Assistant, Pharmacognosy Department, National Research Centre For the fulfillment of M.Sc. Degree in Pharmaceutical Sciences (Pharmacognosy) Under the Supervision of Prof. Dr. Nabaweya Moustafa El-Feky Prof. Dr. Ataa Abdel Haleem Said Professor of Pharmacognosy Professor of Pharmacognosy Pharmacognosy Department Pharmacognosy Department Faculty of Pharmacy National Research Centre (NRC) Cairo University Giza Pharmacognosy Department Faculty of Pharmacy Cairo University A. R. E. 2016 Phytochemical and Biological Evaluation of Citrus volkameriana Ten. & Pasq. Growing in Egypt تقييم فيتوكيميائى وبيولوجي لنبات السيترس فولكاميريانا الذي ينمو في مصر. Abstract Citrus volkameriana Ten. & Pasq. (Family: Rutacea), is native to the subtropical regions of the african continent and cultivated in Egypt for the beneficial use as a rootstock for Citrus sinensis.Genus Citrus is reported to have beneficial effects in the traditional system of medicine. The objective of this work is to study the chemical constituents, as well as, the evaluation of th in vivo and in vitro bioactivities of methanolic extract of the leaves and its fractions. Preliminary phytochemical analysis of Citrus volkamerianarevealed the presence of sterols and/or triterpenes, carbohydrates and/or glycosides, coumarins, flavonoids (free and combined) and tannins in different organs under investigation. Volatile constituents were present in leaves, peels, and flowers and in trace amount in the flesh and absent in the seeds. Anthraquinones, alkaloids and/or nitrogenous compounds and saponins were absent in different organs under investigation. GC-MS profile of the prepared essential oil led to the identification of 35 different compounds, representing 99.22% of total essential oil from leaves. The major compounds detected were D- limonene (21.26%), sabinene (14.14%), L-linalool (9.72%), citronellal (7.14%) and terpinene-4- ol (6.28%). The percent of oxygenated compounds were 34.40% including alcohols (20.36%), aldehydes (11.29 %), ketones (1.13%) and esters (1.62%). Phytochemical study on the methanolic extract of Citrus volkameriana leaves extract led to the isolation, characterization and structure elucidation of 15 compounds α-amyrin, β-sitosterol,5-O- demethylnobiletin,4', 5- dihydroxy-6, 7, 8, 3'-tetramethoxyflavone ,Tangeretin ,Nobiletin ,Cirsilineol ,luteolin 7-O-methylether ,Hesperetin,4'-hydroxy- 5, 6, 7-trimethoxyflavon,β- sitosterol 3-O-β-D-glucoside,P-coumaric acid methyl ester,Apigenin,Luteolin and Hesperidin. Methanolic extract70 % of leaves showed no toxicity up to 5 g/kg.bwt. The methanolic extract showed significant anti-inflammatory, analgesic, antiulcer, hepatoprotective effects. Citrus volkameriana leaves extract showed percentages of oedema inhibition higher than or equal to the effect of the reference drug indomethacin in carrageenan- induced paw oedema assay. In ulcer model induced by ethanol, the groups pre-treated with Citrus volkamerianaleaves extract had a significant protection against ulcer formation. In Paracetamol-induced acute hepatopathic encephalopathy model, the extract showed a significant improvement in ALT, AST, GSH, MDA and NO levels indicating a hepatoprotective activity. In-vitro studies revealed the activity of the ethyl acetate fraction as anti-inflammatory through inhibition of inducible nitric oxide. The volatile oil showed cytotoxic effect against MCF-7 breast carcinoma cell line while petroleum ether was cytotoxic against UACC-62 melanoma cell line. All the extracts expressed broad spectrum antimicrobial activity especially against gram positive bacteria. Petroleum ether and the isolated compound luteolin have shown anti HIV-1 activity which is limited by their narrow therapeutic index. Key words:Citrus volkameriana, Preliminary phytochemical screening, Phenolics, Flavonoids, Sterols and triterpenes, , Analgesic, Anti-inflammatory, Antiulcerative, Hepatoprotective, Cytotoxicity, Antimicrobial, Anti HIV-1. :Introduction المقدمة 1- Natural products and medicinal plants tend to provide an open source for drug discovery due to the large biodiversity of their components (Dias et al., 2012). These secondary metabolites were found to play an important role in protection against plant diseases beside having a positive influence on human health (Dias et al., 2012). Family Rutacea, commonly known as rue or Citrus family, is a flowering plants family belongs to order spindales and consists of 1600 species in 155 genera (Chase et al., 1999). They are ever green trees , shrubs and few herbaceous plants which are native to tropical and subtropical regions of the world. Plants of the family are primarily grown for their economically important fruits and as ornamentals. Among the ornamentals are Poncirus, and Japanese Skimmia (Skimmia japonica Thunb.) and Chinese Skimmia (S. reevesiana Thunb.), which have attractive white flowers and red berries. Important fruits are the genus Citrus, the kumquat (Fortunella), bael (Aegle), elephant apple (Limonia acidissima L.), and Japanese pepper (Zanthoxylum piperitum L.) (Groppo and Pirani, 2012). Citrus is primarily valued for the fruits, which is either eaten alone (sweet orange, tangerine, grapefruit, etc.) as fresh fruits, processed into juice, jams or added to dishes and beverages e.g. Lemons. Citrus species are important in traditional medicine. Citrus has many other uses including animal fodder and craft and fuel wood (Elevitch, 2006). Citrus species are important in traditional medicine. In Samoa, a leaf infusion made from sweet orange is used against mouth sores in infants (Gothesson, 1997). Citrus medica L. leaves are used together with other plant parts to make infusions for treating stomach and skin ailments (Whistler, 1996). Also in Samoa, a sweet orange bark infusion is used to treat postpartum sickness, serious flu, and internal injuries (Whistler, 1996). In Tonga, an infusion of sweet orange leaves, usually together with leaves of mango, Glochidion ramiflorum J.R.Forst. & G.Forst., Diospyros majo Bakh. , and/or the bark of breadfruit, is used to treat “relapse sickness,” mostly affecting postpartum women (Whistler, 1992) .In Tahiti, Citrus leaves are used for internal ailments and fractures. In Fiji, the scraped root of pummelo is used to treat hemorrhoids. In the United States, Citrus is suggested as part of a healthy diet because of its high vitamin C content and its lycopene and flavonoids, which are known to reduce prostate and breast cancer risk, reduce viral effects and inflammation, and improve capillary activity and cholesterol levels (Elevitch, 2006). Citrus species have well-documented pharmacological activities. Results have shown that Citrus extract exerted many bioactivities including anticancer (Visalli et al., 2014), antioxidant, analgesic, anti-inflammatory (Sood et al., 2009), antiulcer (Bhavitavya et al., 2012), hepatoprotective (Abirami et al., 2015), cardioprotective (Lopes et al., 2011), anxiolytic (Carvalho-Freitas and Costa, 2002), antiviral (Balestrieri et al., 2011), anti-microbial (Shende et al., 2015), antiobesity (Cardile et al., 2015) and antidiabetic activity (Parmar and Kar, 2007). These effects were correlated to the wide variety of secondary metabolites produced by Citrus including polyphenolic compounds, alkaloids, limonoids, pectins and dietary vitamin C (Benavente-Garcia and Castillo, 2008; Marti et al., 2009; Murakami et al., 1997; Silalahi, 2002; Tundis et al., 2014; Visalli et al., 2014). Recent studies have been focusing on the effect of methylation of flavonoids on pharmacokinetics and bioactivities of flavonoids. It was found that methylation of flavonoids has positive effects on oral bioavailability and metabolic resistance of flavonoids beside enhancing their cytotoxic activities (Walle, 2007a, b; Walle et al., 2007; Wen and Walle, 2006). One of the rich sources of methylated flavonoids is Citrus genus (Walle, 2007a, b; Walle et al., 2007; Wen and Walle, 2006). Citrus volkameriana Ten. & Pasq. known as volkamer lemon has been known for three centuries. It was first thought of as variant of mandarin lime. More recently it has been identified as a cross of lemon and sour orange. Slightly smaller than lemon trees, it flowers and bears fruits profusely. Fruits are lemon-shaped, wide and with a rough, bright reddish rind. The flesh and juice are yellow-reddish color. The fruits have few seeds, tastes slightly bitter and have a pleasantly fresh taste and aroma. It can be used in cooking instead of lemon (Chapot, 1965). Russo (1956) has brought the attention to volkamer lemon as being a rootstock that can resist mal-secco which is a fungal disease caused by Phoma tracheiphila and can lead to Citrus tree death. Volkamer lemon was also preferred due to its resistance to nematodes and tristeza virus as well as its tolerance to drought (Sofy and El-Dougdoug, 2014; Verdejo-Lucas et al., 2003). It is considered as the best rootstock for growing Citrus in Egypt’s desert areas especially for Valencia sweet orange (Sofy and El-Dougdoug, 2014) and navel orange (Hifny et al., 2012) . Authentic determination of methylated flavonoids of thirteen Citrus species revealed that Citrus volkameriana contains appreciable number of methylated flavonoids (Piattelli and Impellizzeri, 1971; Avula et al., 2005). From previous introduction, phytochemical, as well as, biological investigations of Citrus volkameriana