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 , , ,Cirsilineol , 7-O-methylether ,Hesperetin,4'-hydroxy- 5, 6, 7-trimethoxyflavon,β- sitosterol 3-O-β-D-glucoside,P-coumaric acid methyl ester,,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 Ten. & Pasq. remained necessary. 2- منهج البحث

Isolation and identification of the main constituents as well as investigation of the biological activities of Citrus volkameriana Ten. & Pasq. extract for chosen organ(s) and correlation of the results with that of edible Citrus.

Material: Plant Material:

Different organs of Citrus volkameriana Ten. and Pasq. were collected from the Horticulture Research Institute, Giza, Egypt in June 2011. All trees aged 11-13 years during collection. The plant was kindly authenticated by senior botanist Dr. Mohammed El- Gibali. A dried specimen was placed at the museum of the pharmacognosy department, faculty of pharmacy, Cairo University with voucher number 130501. The collected plants were separated into different organs (leaves, flowers, peels, fleshy part and seeds). The fleshy part was made into juice while the others were air-dried, powdered and kept in tightly-closed container. Methanolic extracts (70%) for different organs under investigation were prepared by percolation followed by filtration and concentration under reduced pressure (45 οC).

Material for Chromatography:

 Silica gel G (E. Merck, Darmstadt, Germany) 60 mesh for column chromatography.  Sephadex LH-20 for CC (Pharmacia, Uppsala, Sweden).  Silica gel aluminium sheets G 60 (F254-Merck) for TLC.  Sheets of Whatmann filter paper No.1 for PC (Whatmann Ltd., Maidstone, Kent, England).

Solvents:

Petroleum ether (b.p. 60-80oC), n-hexane, acetone, ethyl acetate, dichloromethane, methanol (absolute and 95%), ethanol (absolute and 95%), n-butanol, acetic acid,formic acid, distilled water and DMSO all of analytical grade.

Solvent Systems for Chromatography (Stahl, 1969): For thin layer chromatography (TLC). S1: Dichloromethane: Methanol (10:90 v/v). S2: Ethyl acetate: Dichloromethane (10:90 v/v). S3: n-Hexane: Acetone (70:30 v/v). S4: Dichloromethane: Hexane (6:4 v/v). S5: n-Hexane: Ethyl acetate (85:15 v/v). S6: Ethyl acetate: Formic acid: glacial acetic: water (100:11:11:27 v/v). S7: n-Hexane: Ethyl acetate (1:1 v/v) S8: Ethyl acetate / n-hexane (1:9 v/v) E9: ethyl acetate: acetic acid: water (12:1:1 v/v) for Paper chromatography (PC). Solvent system 1: n- Butanol - acetic acid - water (4: 1: 5v/v) Solvent system 2: Acetic acid - water (15: 85 v/v). Solvent system 3: Benzene- Acetic acid - water (125: 72: 3 v/v)

Reagents for Phytochemical Screening: (Evans, 2009; Harborne, 1999 and Qadir et al,. 2015)

Aluminium Chloride (for Flavonoids).

Aluminium chloride 1% solution in methanol.

Ferric Chloride (for tannins).

Ferric chloride 9 g in 100 mL water.

Mayer’s (for alkaloids).

Mercuric chloride 1.353g dissolved in 60 mL water, mixed with a solution of 5 g of potassium iodide in 20 mL water and then diluted to 100 mL with water.

Molisch’s (for carbohydrates and/or glycosides).

α-naphthol 20 g was dissolved in 100 mL. Absolute ethanol and 2 mL of sulphuric acid is added.

Modified Dragendorff's (for alkaloids and other nitrogenous bases)

. Solution A: 0.85 g Bismuth sub-nitrate was dissolved in a mixture of 10 mL acetic acid and 40 mL distilled water. . Solution B: 8 g Potassium iodide was dissolved in 20 mL water. . Stock solution: Equal volumes of A and B were mixed (can be stored for a long time in a dark vessel). . Spray reagent: 1 mL Stock solution was mixed with 20 mL acetic acid and 10 mL water before use.

Lieberman-Burchard (for sterols and/or triterpenes).

Acetic anhydride 1 mL and sulphuric acid 1 mL.

Reagents for UV Spectroscopic Analysis of Flavonoids, (Harborne, 1995).

i) Sodium methoxide solution: 2.5 g freshly cut sodium metal was added cautiously in small pieces to 100 mL dry methanol. ii) Aluminium chloride: 5g anhydrous aluminium chloride was cautiously added to 100 mL of methanol. iii) Hydrochloric acid: Hydrochloric acid (50 mL) was mixed with distilled water (100 mL). iv) Sodium acetate: anhydrous coarsely powdered. v) Boric acid: coarsely powdered.

Spray Reagents.

i) Aluminium Chloride for Flavonoids (Markham, 1982).

Aluminium chloride 1% solution in ethanol, the chromatograms were sprayed with the reagent, air-dried, then examined in visible and under UV light to notice changes in color. Flavonoids yield yellow fluorescence at long wavelength UV light (365nm).

ii) Ferric Chloride for Phenolics (Markham, 1982)

Ethanolic solution of ferric chloride 1% is used to spray chromatograms to detect the presence of phenolics.

iii) P-Anisaldehyde Sulfuric Acid for Sterols, Triterpenes and phenyl propanoids. (Stahl, 1969)

P-Anisaldehyde (0.5 ml) was added to 10 mL glacial acetic acid, 85 ml methanol and 5mL H2SO4. Dried chromatograms were sprayed and heated at 110°C until the spots attained maximum color intensity.

iv) Sulphuric Acid for Sterols and Triterpenes (Stahl, 1969).

Sulphuric acid (20%) in ethanol, dried plates were sprayed and heated at 110°C to maximum color intensity of the spots.

v) Aniline Hydrogen Phthalate for Sugars (Stahl, 1969)

Aniline (0.93 g) and O-phthalic acid (1.66 g) were dissolved in 100 mL n-butanol saturated with water. The chromatogram was sprayed and heated for 5-10 min. at 110°C. The color developed ranges from dark brown to yellow.

vi) Iodine/Potassium Iodide for Coumarins (Stahl, 1969).

One gram iodine and 1g potassium iodide dissolved in 100 mL ethanol. Material for In-vivo Biological Study: Experimental Animals:

Wister albino rats of both sex, weighing ranged from 125-150g and Swiss mice of 20-30g body weight were used throughout the experiments. The animals were obtained from the animal house colony of the National research centre, Dokki, Giza, Egypt.The animals were housed in standard metal cages in an air conditioned room at 22 ± 3˚C, 55 ± 5% humidity and provided with standard laboratory diet and water ad libitum. Rats were used for determination of the anti-inflammatory, gastric ulcerogenic and hepatoprotective activities, while mice were used for determination of the median lethal doses (LD50) and analgesic study. Rats and mice were divided into groups each of six (n=6). All experimental procedures were conducted in accordance with the Ethics Committee of the National Research Centre and following the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals.

Material for In-vitroylSiS laci oloiB : Cell Lines for Macrophage growth and anti-inflammatory activity: Raw murine macrophage (RAW 264.7) was purchased from the American Type Culture collections (LGC Standards GmbH Mercatorstr.5146485Wesel, Germany).

Cell Lines for Cytotoxic Activity: The human cell lines human kidney adenocarcinoma TK10, non epithelial melanotic melanoma UACC62 and human breast adenocarcinoma MCF7 were previously obtained from National Cancer Institute (NCI) in the framework of a collaborative research program between The Council for Scientific and Industrial Research (western Cape, South Africa) and National Cancer Institute (NCI), USA.

Bacterial Strains for Antimicrobial Activity:

Antimicrobial activity tests were carried out against four gram positive bacteria, Bacillus subtilis NRRL-B-4219, Micrococcus luteus B-287, Staphylococcus aureus ATCC 29213, Streptococcus faecalis ATCC 19433, four gram negative bacteria, Alcaligenes faecalis B-170, Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 10131, Pseudomonas aeruginosa ATCC 27953, gram stain resistant microbe Mycobacterium tuberculosis. All clinical isolates were obtained and identified from Chemistry of Natural and Microbial Product Department, National Research Centre, Egypt.

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Standards, Kits and Chemicals for In-vivo and In-vitro Bioactivity Studies:

 Indomethacin capsules (Kahira Pharmaceutical and Chemical Company, Cairo, Egypt).  Carrageenan (Sigma –Aldrich chemical company, USA).  Silymarin (SEDICO, Cairo, A.R.E) is used as a standard hepatoprotective drug.  Paracetanmol Sigma (St. Louis, MO, USA) is used as hepatotoxic agent. Acetaminophen was prepared freshly by dissolving in sterile distilled water and stirred well until all crystals were dissolved.  Emetin (DOX) (Pharmacia, Belgium): used as a reference cytotoxic agent.  Dexamethasone(Ameria, Cairo, A.R.E): used as a reference nitric oxide inhibitory agent.  Dipenacid (CID, Cairo, A.R.E): as a reference wide spectrum antimicrobial drug.  Azidothymidine (Sigma-Aldrich chemical company, USA): as a reference HIV-1 anti-infectivity agent.  Biodiagnostic kits are used for the assessment of serum AST and ALT.  Sulphorhodamine B (SRB) (Sigma-Aldrich chemical company, USA) for colorimetric cytotoxicity assay.  Ethyl alcohol (ethanol), BDH-Chemical, England.  Saline Solution (GIBCO, USA).  General methods for phytochemical study: Thin layer Chromatography (Stahl, 1969):

Readymade chromatographic plates (20 x 20 cm) coated with silica gel F254 or cellulose were used for analytical separation and completed as described for PC.

Column Chromatography (Stahl, 1969):

Glass columns of different sizes were packed with Sephadex LH-20, polyamide or silica gel. The Sephadex gel was left to swell in methanol prior to use by adding enough methanol and left overnight before adding additional solvent to produce the pourable slurry. Silica gel was activated before packing at 100 °C for three hours. After packing the column thoroughly with the adsorbent, a concentrated solution of the material to be fractionated was mixed with a small amount of silica gel and was applied to the top of the column. Elution was then started with the eluent stated in each case. Bands formed during the elution process were traced in both visible and UV regions. Each fraction desorbed from the column was then evaporated under reduced pressure; similar fractions were pooled and investigated.

Spectroscopic Analysis:

Ultraviolet spectrophotometric analysis was carried out in 4 mL capacity quartz cells (1 cm thickness). The isolated pure compounds were dissolved in HPLC grade methanol. NaOMe, AlCl3, AlCl3/HCl, NaOAc, NaOAc/ H3BO3 reagents were separately added to the prepared methanolic solution and UV measurements were carried out.

Typical conditions of nuclear magnetic resonance spectrometer with spectral width 300, 400 and 500 MHz for 1HNMR and 75, 100 and 125 MHz for 13CNMR were adopted.

EI/MS spectroscopic analysis for pure compounds was done.

Apparatus:

Apparatus for spectral analysis:  UV-Visible Spectrophotometer: UV-Visible Jasco V-670 spectrophotometer.  EI-MS were measured using Jeol JMS-Ax500 spectrometer.  NMR spectra were measured using JOEL-FT NMR-AL 300 MHz spectrophotometer, JOEL ECX-400p NMR spectrometer and JEOL ECA 500 MHz spectrometer for 1HNMR and 75, 100 and 125 MHz for 13CNMR.  Apparatus for GC-MS analysis:  GC-Ms analysis of the prepared essential oil was carried out using gas chromatography – mass spectrometry instrument stands at the laboratory of medicinal and aromatic plants, National Research Centre with the following specifications. Instrument: a TRACE GC Ultra gas chromatograph (THERMO Scientific Corp., USA), coupled with a thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer). The GC-MS system was equipped with a TG-WAX MS column (30 m x 0.25 mm i.d., 0.25 μm film thickness). Analysis was carried out using helium as carrier gas at a flow rate of 1.0 mL/min at a split ratio of 1:10 and the following temperature program: 400C for 1 min; rising at 40C/min to 1500C and held for 6 min; rising at 40C/min to 2100C and held for 1min. The injector and detector were held at 210 and 2000C, respectively. Diluted sample (1:10 hexane, v/v) of 0.2 μL of the mixture was injected. Mass spectrum was obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 40-450.

Laboratory Equipments:  Ultraviolet Lamp For localization of spots on paper and thin layer chromatograms.  Rotary evaporator: (Heidolph, Germany).  Chromatoplates: glass plates 20 cm ×20 cm and 20 cm ×10 cm and 0.2 mm thickness.  Glass columns for chromatography (1 cm x 100 cm) and (2.5 x 80 cm).  Flash columns for chromatography (5 cm x 100 cm) , (2.5 cm x 80 cm) and (15 cm x 60 cm) .  Medium pressure liquid chromatography (MPLC) column (5 cm x 20 cm)  6- Glass tanks 10 cm x 20 cm x24 cm for TLC, 25 cm x 60 cm x 60 cm for PC.  Micro-pipette, 0.1 mL for spotting.  Atomizer for spraying the chromatograms.  Sensitive electric balance: Precisa 600 C, Switzerland.  ELISA reader for cytotoxic activity test.  Laminar flow cabinet biosafety class II level for in-vitro cytotoxic activity test, (Baker, SG403INT, Sanford, ME, USA).  Water jacketed Carbon dioxide incubator for incubation of cytotoxic cells, (Sheldon, TC2323, Cornelius, OR, USA).  Microplate multi-well reader for measuring the absorbance in in-vitro cytotoxic activity test, (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA).  Tail Flick, USA for radiant heat stimuli (infrared heat) (Ugo Basile).  Melting points were determined in open capillary tubes on Electrothermal 9100 digital melting point apparatus (Electrothermal Engineering Ltd, Serial No. 8694, Rochford, UK).  Clevenger's apparatus for hydro-distillation of the essential oil.  Plethysmometer (UGO Basile 21025 Comerio, Italy) for measuring rat's hind footpad thickness (paw volume).

3-خطة البحث : 1- Surveying literature concerning the plant constituents and the reported biological activities. 2- Authentication of the collected plant by a botanist. 3-A specimen will be kept at Pharmacognosy Dept., National Research Centre and Pharmacognosy Dept. museum, Faculty of Pharmacy, Cairo University. 4-Preliminary phytochemical screening of the plant organs under investigation. 5- Extraction of the chosen organ(s) using methanol 70% and fractionation using different organic solvents 6- Isolation of the main constituents using paper, thin layer and column chromatographic tools. 7- Identification of the isolated compounds using physical, chemical and different spectroscopic tools (UV, NMR and MS). 8-Biological screening of the total methanolic extract, its fractions as well as the isolated compounds. 9-Comparison of the obtained results with that reported for some species of Citrus.

Rseoile

1. Preliminary Phytochemical Screening:

The results revealed 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.

Leaves were the organ of choice due to their availability and being rich in phenolic compounds; flavonoids and tannins and volatile constituents.

2. Analysis of Volatile Constituents:

The essential oil was obtained by hydro-distillation of fresh leaves (500 g) for 6–8 h in Clevenger's apparatus. The yield of oil was recorded (0.4 ml/100 g Leaves). The major constituents were analyzed by GC-MS.

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%).

3. Isolation of Phytoconstituents:

Phytochemical study on the methanolic extract of Citrus volkameriana Ten. & Pasq. leaves led to the isolation, characterization and structure elucidation of 15 compounds according to the following method.

Air dried leaves (1.9 kg) of Citrus volkameriana were exhaustively extracted using 70% methanol. The total methanolic extract was then filtered off, concentrated to yield 142 g of a viscous residue. The residue was fractionated with petroleum ether (60–80 °C), dichloromethane, ethyl acetate and n-butanol saturated with water, respectively. The obtained fractions, as well as, the remaining aqueous phase were concentrated to give five fractions (7.8 (oily), 15.2 (dark green), 25.3 (brown viscous), 41.2 (brown viscous) and 47.5 g (brown viscous)), respectively.

Petroleum ether fraction (7 g) was subjected to silica gel flash column chromatography using gradient elution. Four main subfractions were obtained (P-I ( 942 mg), P-II (2.1 g), P-III (7.1 mg), P-IV (33.4 mg)). Subfractions P-I was further purified through silica gel column using gradient elution. Two compounds were obtained.

Compound C1: α-amyrin.

Compound C2: β-sitosterol.

Subfractions P-II eluted with 10% acetone were further purified through silica gel column using gradient elution. Two compounds were obtained.

Compound C3: 5-O-demethylnobiletin

Compound C4: 4', 5- dihydroxy-6, 7, 8, 3'-tetramethoxyflavone (7- methylsudachitin).

Subfractions P-III eluted with 15 % acetone was crystallized from methanol to afford one compound.

Compound C5: Tangeretin

Subfractions P-IV eluted with 25 % acetone was purified using preparative thin layer chromatography to afford one compound.

Compound C6: Nobiletin

Dichloromethane fraction (14 g) was chromatographed on a flash column adopting gradient elution. Three main subfractions were obtained (C-I (825 mg), C-II (3.83 g), and C- III (3.43 g)).

Subfractions C-II (3.83 g) eluted with 20 % acetone were purified through sephadex LH20 column to give two sub-subfractions A and B.

Sub-subfractions C-II-A on concentration yield one compound.

Compound C7: Cirsilineol

Sub-subfractions C-II-B were chromatographed on silica gel column using gradient elution where two compounds were obtained.

Compound C8 : Luteolin 7-O-methylether

Compound C9 : Hesperetin

Subfractions C-III eluted with 40 % acetone were further purified through medium pressure liquid chromatography column using gradient elution yielding two compounds. Compound C10: 4'- hydroxy- 5, 6, 7-trimethoxyflavone

Compound C11: β-sitosterol 3-O-β-D-glucoside

Ethyl acetate fraction (23 g) was subjected to silica gel column chromatography using gradient elution. Four main subfractions were obtained (E-I (32 mg), E-II (970 mg), E-III (5.4 g) and E-IV (2.4 g).

Subfraction E-I eluted with 2 % methanol / dichloromethane was purified using preparative thin layer chromatography affording one compound.

Compound C12: P-coumaric acid methyl ester

Subfractions E-II eluted with 5 % methanol / dichloromethane was further purified by crystallization from 20 % methanol / chloroform yielding one compound.

Compounds C13: Apigenin

E-III eluted with 10 % methanol / dichloromethane were further purified by crystallization from 20 % methanol / chloroform yielding one compound.

Compounds C14: Luteolin

. Subfractions E-IV eluted with 20% methanol /dichloromethane was then subjected to reversed phase MPLC. Elution was performed using acetone: acetonitril: water (1: 1: 1) then purified by preparative thin layer chromatographic separation affording one compound.

Compound C15: Hesperidin

4. Bioactivity Study:

4.1. In-vivo bioactivity study: It was carried out on the methanolic extract 70% of Citrus volkameriana Ten. & Pasq. leaves using the following doses 250, 500, 1000 mg/kg.

4.1.1. Determination of Median Lethal dose (LD50):

The methanolic extract 70% of Citrus volkameriana Ten. & Pasq. leaves showed no toxicity up to 5 g/kg.bwt.

4.1.2. Anti-inflammatory effects:

The most effective dose was 1000 mg / kg .It showed significant inhibition of oedema with potency more than reference drug indomethacin in the 1st, 2nd and 3rd hour while it showed equal potency in the 4th hour .The dose 500 mg /kg also showed significant inhibition of oedema with potency higher than indomethacin in the 3rd and 4th hour. The dose 250 mg /kg showed significant inhibition of oedema at with potency 1.1 and 0.9 in the 3rd and 4th hour. The dose 250 mg/kg is preferred to reduce any possible adverse effects.

4.1.3. Analgesic Activity (Antinociceptive):

The most effective dose was 500 mg/kg and showed significant prolongation in the reaction time by 59 ,65, 86 and 90% at zero , 30 , 60, 120 min respectively as compared with reference drug indomethacin at the same time. The dose 250 mg/kg also showed a significant prolongation in reaction time by 55, 72, 78% at 30, 60, 120 min respectively. The dose 250 mg/kg is preferred to reduce any possible adverse effects.

4.1.4. Anti-ulcerative activity:

The number and the severity of gastric mucosal lesions evoked by oral administration of 60 % ethanol were significantly reduced by given methanol extract in doses (250,500 and 1000 mg/ kg). The percentage of reduction in number -52.9 , - 62.4, -62.4 % while in the severity were -61.8, -89 and -80.5% , respectively as compared with ethanol treated group.The most effective dose was 500 mg/kg.

4.1.5. Hepatoprotective study:

The most effective concentration was 500 mg /kg which significantly decreased both ALT and AST concentrations by 91 and 105% while the dose 250 mg/kg also showed a significant decrease in ALT and AST concentrations by 68 and 84% as compared with silymarin. The dose 500 mg/Kg also increased the level of GSH and NO by 77.5 and 133% and decreased the concentration of lipid peroxide by 98% as compared with the reference silymarin.

4.2. In- vitro Bioactivity Study It was carried out on methanolic extract (70%), its fractions, prepared essential oil and some of the isolated compounds.

4.2.1. Macrophage growth and anti-inflammatory activity:

The treatment with the methanolic extract, its fractions and prepared essential oil revealed that ethyl acetate fraction is promising inhibitors of NO as concluded from its inhibitory percentages, 80.2 % as compared with reference drug dexamethason.

4.2.2. Cytotoxic Assay:

Volatile oil has potent cytotoxic activity on MCF7 cell line (135%) with respect to the reference drug emetin. Petroleum ether fraction has potency (58%) on UACC-62 melanoma cell line with respect to the reference drug emetin. The isolated compound apigenin also showed a significant cytotoxic activity on UACC-62 melanoma cell line.

4.2.3. Antibacterial activity:

The methanolic extract 70% of Citrus volkameriana Ten. & Pasq. leaves, fractions , volatile oil and isolated compounds Apigenin and luteolin showed a broad antimicrobial spectrum and exerted a much stronger antimicrobial effect against gram-positive bacteria than gram-negative bacteria. The most susceptible bacteria was Micrococcus luteus B-287 whereas the highest MIC value was 31.25 μg/ml for petroleum ether fraction and the oil.

4.2.4. Anti HIV-1 activity: The petroleum ether fraction inhibited the syncytia formation with EC50 of 17.40 μg/ml. However, CC50 of petroleum ether fraction on C8166 cell line was 37.72 μg/ml. The therapeutic index (TI) of petroleum ether fraction was about 2.17. Isolated compound luteolin also, showed inhibition of the syncytia formation with EC50 of 9.29 μg/ml. However, CC50 of luteolin on C8166 cell line was 13.59 μg/ml. The therapeutic index (TI) was about 1.46. The therapeutic index must be more than 10 folds therefore methanolic extract and its fractions and luteolin did not match this criteria.

4- فترة دراسة الموضوع 4 سنوات و 7 شهور 5-تاريخ البحث من 9 مايو 1111 حتى 17 يناير 111I.