Phytochemical and biological investigations of Terminalia bellerica Roxb. leaves

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1 Research Article ISSN: Fathalla A. Ayoob et al. / Journal of Pharmacy Research 2014,8(4), Available online through Phytochemical and biological investigations of Terminalia bellerica Roxb. leaves Fathalla A. Ayoob 1, Hanem M. Awad 1 *, Salah M. El-Kousy 2, Khaled N. Rashed 3 and Nabil H. Al-Sayed 1 1 Department of Tanning Materials and Leather Technology, National Research Centre, Dokki, Cairo, Egypt. 2 Chemistry Department, Faculty of Science, Menoufia University, Shibin El-Koum, Menoufia, Egypt. 3 Pharmacognosy department, National Research Centre, Dokki, Cairo, Egypt. Received on: ; Revised on: ; Accepted on: ABSTRACT Background: The extensive literature survey revealed that Terminalia bellerica, is an important medicinal plant with diverse pharmacological spectrum. However, the phytochemical studies on the leaves of Terminalia were very rare. This prompted our interest to carry out an extensive phytochemical study on the leaves of Terminalia grown in Egypt. Methods: In this study we report for the first time phytochemical screening, investigation of antioxidant, antibacterial, cytotoxicity and anti-hiv as well as antifungal activities of seven different successive extracts namely; 70% methanol, petroleum ether, diethyl ether, dichloromethane, ethyl acetate, butanol and aqueous extracts of Terminalia bellerica Roxb. leaves. The bioactive ethyl acetate and n-butanol soluble parts of successive methanol extract of leaves of Terminalia bellerica were subjected to column chromatography for separation of different chemical constituents. Results and Discussion: Three hydrolysable tannins, four flavonol glycoside, one flavone and two flavonolaglycone were identified. The identification of the structures of isolated compounds was occurred using 1D- and 2D-NMR, UV and MS. Compounds 3-10 were reported for the first time from Terminalia bellerica leaves. The 70% methanol, diethyl ether and butanol extracts showed strong antioxidant activity compared to ascorbic acid and rutin as positive controls. nly, 70% methanol extract showed good anti-hiv activity. The 70% methanol, ethyl acetate, butanol and aqueous extracts showed good antibacterial activities against Micrococcus flavus, Staphylococcus aureus and Escherichia coli compared to the antibiotics streptomycin and ampicillin. All extracts except diethyl ether, showed very high antifungal activities against eight investigated fungal strains compared to that of fungicides bifonazole and ketoconazole (positive controls). Conclusion: The results of this study revealed that, this plant may be utilized as promising sources of therapeutics, as strong antioxidant as well as antifungal from natural sources. Key-words: Antibacterial, antifungal, anti-hiv, antioxidant, Terminalia bellerica leaves 1. INTRDUCTIN Medicinal plants have been used in all cultures as a source of medicine, since times immemorial 1. Herbal Medicine is still the mainstay of health care in several developing countries. The widespread use of herbal remedies and health care preparations, as those described in ancient texts such as the Vedas and the Bible, and obtained from commonly used traditional herbs and medicinal plants, has been traced to the occurrence of natural products with medicinal properties. The World Health rganization has estimated that more than 80% of the world s population in developing countries depends primarily on herbal medicine for basic healthcare needs 1. The family Combretaceae is comprised of 20 genera and about 475 species 2, which are found in all tropical regions all over the world and have valuable medicinal activities. After Combretum, Termenalia is the largest second genus of this family that contains 200 species 3, including Terminalia *Corresponding author. Hanem M. Awad, Department of Tanning Materials and Leather Technology, National Research Centre, Dokki, Cairo, Egypt bellerica, T.chebula, T.mulleru, T.arjuna and so many others; among these plants, we put our attention on T.bellerica. This plant is credited with many medicinal properties; and has been introduced by the Arabs from India. It is indigenous in Pakistan and India. In addition, it is used as popular folk medicine in Asian and African countries 4. Its fruits have anti-inflammatory, antihelmintic, expectorant, ophthalmic, antipyretic, antiemetic. Fruits are useful also in cough, asthma, bronchitis, dropsy, dyspepsia, cardiac disorders, skin diseases, leprosy, ulcer and myocardial depressive activity 5, 6. Ripe fruits are used as astringent in combination with chebulic myrobalan (Terminalia chebula) and phyllantus emblica as the famous Triphala drug of Ayurveda. Fruits are also useful in eye diseases and scorpion sting 7. Furthermore, fruits showed anti-hiv, antimalarial and antifungal activities 8, 9. Polyphenolic constituents of its fruits were responsible for antioxidant, antistress and antidiabetic activity 10. Terminalia bellerica Roxb. bark is mildly diuretic and is useful in anaemia and leucoderma. Phytochemically, the fruits of T. bellerica have been reported to contain termilignan, thannilignan, 7-hydroxy-3',4'-(methylenedioxy) flavones, anolignan B 5, gallic acid, ellagic acid, ß-sitosterol 10, 11, arjungenin, belleric acid, bellericoside, cannogenol 3--dgalactopyranosyl-(1 4)--l-rhamnopyranoside sitosterol, ethyl gallate, chebulagic acid, galloyl glucose, mannitol, glucose, galactose,

2 Fathalla A. Ayoob et al. / Journal of Pharmacy Research 2014,8(4), fructose and rhamnose 12, 13. Seeds are reported to contain a new cardenolide, cannogenol 3--D-galactopyranosyl14--L-rhamnopyranoside and phospholipids 14. Terminalia bellerica Roxb. stem bark contains arjungenin and its glycosides, belleric acid and bellericosides 14. The phytochemical studies on the leaves of Terminalia were very rare which resulted in isolation of only hydrolysable tannins; gallic acid and ellagic acid from the water soluble extract of T.bellerica 15. This prompted our interest to carry out an extensive phytochemical study on the leaves of Terminalia grown in Egypt. Furthermore, the total phenol content, antioxidant, anti-hiv, antibacterial as well as antifungal activities of different leaves extracts were studied. 2. MATERIALS AND METHDS 2.1. Materials SRB (Sulforhodamine B), gallic acid (= 95% by HPLC), rutin hydrate (= 94% by HPLC), ascorbic acid (vitamin C, 99%), AZT (3'-azido-3'- deoxythymidine), HEPES (N-2 (2-hydroxyothyl) piperazine-n -(2- ethanesufonic acid), MTT (3,(4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide), DMF (N, N' Dimethyl formamine), penicillin, streptomycin sulfate, glutamine and DPPH (1,1-diphenyl-2-picryl hydrazyl) were purchased from Sigma Chem. Co. (St. Louis, M, USA). The Folin-Ciocalteu s phenol reagent was from Fluka Chemie AG, Buchs, Switzerland. Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were purchased from Gibco, UK. 2-Mercaptoethanol (2-ME) was purchased from Bio-Rad. Dimethyl sulfoxide (DMS) and methanol were of HPLC grade and all other reagents and chemicals were of analytical reagent grade. Polyamide 6S (Riedel de Haen AG, Hannover, Germany); sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden); solvent mixtures, BAW (n-butanol:acetic acid:water, 4:1:5 upper phase), and paper chromatography (PC), Whatman No. 1 & 3 MM (46 57 cm) (Kent, England) Phytochemical screening General UV spectra were run on MM 7070E Shimadzu UV 240 spectrophotometer. 1D-NMR spectrophotometer was recorded at 25 C and TMS as internal standard on Varian JEL-EX 500 MHz in solvent (DMSd6). 2D-NMR spectra were recorded on Bruker DRX 400 MHz spectrophotometer. EI-MS spectra were obtained on JEL JMS-AX Extraction and isolation The air-dried T.bellerica leaves (1.5 kg) were ground to a coarse powder and extracted several times with 70% aqueous methanol at room temperature. The total extract was filtered, and evaporated under reduced pressure at 50 ºC to give brown powder residue (60.0 g). The dry extract was diluted with distilled H 2 and then partitioned successively with petroleum ether, diethyl ether, dichloromethane, ethyl acetate, and n-butanol using separating funnel. Butanol fraction was about (35 g) that chromatographed by polyamide 6S (250 g) column (120 cm L x 3.5 cm D), eluted by mixture of H 2 /MeH with decreasing polarities (250 ml each fraction) to give 60 fractions which collected to six sub-fractions by further examination by PC. Fraction I (5 gm) was subjected to PC (3 MM) using 15% acetic acid/water as eluent. The separated bands were scraped off and eluted with 70% methanol and further purified by Sephadex LH-20 CC eluted with 100% MeH afforded compounds 1 (70 mg), 2 (40 mg) and 3 (35 mg). Fractions II (3 gm) and III (2.8 gm) were subjected to Sephadex LH-20 CC eluted with 100% MeH and MeH : H 2 (8:2) afforded compounds 4 (33 mg) and 7 (45 mg) respectively. Fraction IV (6 gm) was chromatographed by Sephadex LH-20 column with saturated n-buh as eluent afforded compounds 5 (25 mg) and 6 (19 mg). Fraction V after the evaporation of the eluent (60 % MeH) was applied on a polyamide column and eluted by the solvent system n-butanol-water saturated which gave rise to two successive sub-fractions. The first sub-fraction was further applied on smaller Sephadex LH-20 column and eluted by 40 % MeH to afford the purified samples of compound 8 (15mg) and the second sub fraction was eluted on sephadex LH-20 column to give compound 9 (23 mg) after elution by 60 % MeH. Fraction VI afforded compound 10 (25 mg) after elution with 100 % Me-H on Sephadex LH-20 CC Acid hydrolysis Compounds 7 (45 mg), 8 (15 mg), 9 (23 mg) and 10 (25 mg) were dissolved in methanol (10 ml) / 2N HCl (1 ml) and heated under reflux for 2 hours. After cooling, the aqueous solution was extracted with ethyl acetate (3x5 ml) and neutralized with 5% Na 2 C 3. Co-PC was applied to identify the sugar in the aqueous layer using acetic acid (15%) as developing system and aniline hydrogen phthalate as spraying reagent by comparison with standard samples. Co-PC was applied to identify the aglycone in the ethyl acetate layer using n-butanol/acetic acid/water (4:1:5) as developing system and Naturstoff as spraying reagent by comparison with standard samples Plant material Leaves of T.bellerica were collected during November 2011 from Giza Zoo, Cario-Egypt. The botanical identification of the collected plant was authenticated by Dr. T. Labeb, Herbarium of rman garden, Horticulture Research Institute, Giza, Egypt. A voucher specimen of the plant was deposited in the Herbarium of the National Research Centre, El-Bohooth St., Dokki, Cairo, Egypt. The leaves were cleaned, airdried in the shade, and then powdered by laboratory mill to 24 meshes. Powdered materials were maintained in an air tight container at room temperature (28±2ºC), and protected from light until use Spectroscopic data of isolated compounds Gallic acid (1): White amorphous powder, R f values (x100): 67 (BAW) and 54 (15% HAc) on PC, UV at λ max (MeH): H-NMR: 6.94 (s, H-2, 6). 13 C-NMR (125 MHz): 169.3, (C-3 and C-5), (C-4), (C-1), (C-2, 6). Ellagic acid (2): White amorphous powder, R f values (x100): 34 (BAW) and 44 (15% HAc) on PC, UV at λ max (MeH): 255, H- NMR: 7.44 (s, H-4 and H-9). 13 C-NMR: (C-5; and C-10), (C- 3, and C-8), (C-2, and C-7), (C-1a, and C-6a), (C-4b and C-9b), (C-4 and C-9), (C-4a and C-9a).

3 Fathalla A. Ayoob et al. / Journal of Pharmacy Research 2014,8(4), Methyl gallate (3): White amorphous powder, R f values (x100): 76 (BAW) and 62 (15% HAc) on PC, UV at λ max (MeH): 220, H- NMR: 6.95(s, H-2 and H-6) and 3.72 (s, methyl ester proton). 13 C- NMR: (C-1), (C-2), (C-3), (C-4), (C- 5), (C-6), (C-7), (C-CH 3 ). Luteoline(4): Yellow amorphous powder, R f values (x100): 8 (15% HAc), 78 (BAW),UV spectral data λ max nm (MeH); 242 sh, 252, 266, 348; (+ NaMe): 266, 329 sh, 400; (+ NaAc): 266, 301 sh, 383; (+ NaAc/ H 3 B 3 ): 260, 295, 371; (+ AlCl 3 ): 273, 300 sh, 327, 425; (+ AlCl 3 /HCl): 265 sh, 275, 293 sh, 354, 386. EI-MS m/z (rel.int.): 287 [M + H] +, 1 H-NMR: d ppm (1H, br s, H-5), 7.4 (2H, m, H-2'6'), 6.85 (1H, d, J = 8.5 Hz, H- 5'), 6.6 (1H, s, H-3), 6.4 (1H, d, J = 2.5 Hz H-8), 6.17 (1H, d, J = 2.5 3Hz H-6). Quercetin (5): Yellow amorphous powder, R f values (x100): 7 (15% HAc), 71 BAW. UV at λ max (MeH): 253, 268 sh, 297 sh, 368. [M-H] - m/ z: H-NMR: 7.69 (d, J= 2.1, H-2'), 7.55 (dd, J =2.1 Hz, and 8.4 Hz, H-6' ), 6.90 (d, J= 8.4 Hz, H-5'), 6.42 (d,j= 1.8 Hz, H-8), 6.20 (d, J= 1.8 Hz, H-6). 13 C-NMR: (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), (C-9), (C-10), (C-1'), (C-2'), (C-3'), (C-4'), (C-5'), (C-6' ). Kaempferol (6): Light yellow amorphous powder, R f values (x100): 82 (BAW), 10 (15% HAc). EI-MS m/z: 287, 288, H-NMR: δ 6.12 (1H,d, J=2.3Hz, H-6), 6.32(1H, d, J=2.3 Hz, H-8), 6.86 (2H, d, J=8.6 Hz, H-3' and H-5'),7.97 (2H,d, J=8.6 Hz, H-2' and H-6'). 13 C-NMR: d93.20(c- 8), (C-6), (C-10), (C-3'and C-5'),121.67(C-1' ), (C-2'and C-6' ), (C-3), (C-2), (C-9), (C- 4'),160.65(C-5), (C-7), (C-4). Quercetin3--[6"-a-L rhamnopyranosyl]-(1 6)-ß-Dglucopyranoside (7): Pale yellow powder. R f values (x100): 51 (BAW) and 62 (15%HAc) on PC UV λ max (MeH), 260, 270 sh, 365; (+NaMe): 275, 330 sh, 420; (+AlCl 3 ): 280, 300 sh, 360 sh, 440; (+AlCl 3 / HCl) 270, 300 sh, 360, 400; (+NaAc): 270, 326 sh, 380; (+NaAc/H 3 B 3 ): 263, 304 sh, 380 nm. 1 H- NMR: δ 7.6 (2H, d, J = 8.8 Hz, H-2 and H-6 ), 6.82 (1H, d, J = 8.8 Hz, H- 5'), 6.33 (1H, d, J = 2.0 Hz, H-8), 6.13 (1H, d, J = 2.0 Hz, H-6), 5.05 (1H, d, J = 7 Hz, H-1"), 4.45 (1H, s, H-1'"), 3.75 (1H, d, J = 11.7 Hz, H-6"a), 3.58 (1H, br s, H-2'"), (remaining sugars protons), 0.79 ( 3H, d, J = 6.0 Hz, H-6'"). 13 C-NMR): d (C-4), (C-7), (C-5), (C-4' ), (C-9), (C-2), (C-3' ), (C-3), (C- 2' ), 123.1(C-6' ), 123.5(C-1' ), 116 (C-5' ), (C-10), (C-1" ), 99.9 (C-1'" ), 99.9 (C-6), 94.8 (C-8), 78.1 (C-3" ), 77.1 (C-5" ), 75.7 (C-2" ), 73.9 (C-4'" ), 72.2 (C-3'" ), 72 (C-2'"), 71.3 (C-4"), 69.6 (C-5'"), 68.5 (C-6''), 17.8 (C-6'"). Quercetin-3--a-L-rhamnopyranoside, (8): R f values (x100):20 (H 2 ), 50 (15% HAc), 71 (BAW); UV λ max nm (MeH): 253, 263 sh,344; (+NaMe): 272, 322 sh, 372; (+NaAc): 260, 300 sh, 367; +NaAc/H 3 B 3 ): 272, 382; (+AlCl 3 ): 272, 304 sh, 333 sh, 430; + AlCl 3 /HCl: 272, 303 sh, 353, 401; Negative ESI-Mass: [M-H]- m/z 447.1; 1 H-NMR: δ (ppm) 7.33 (d, J = 2 Hz, H-2' ), 7.28 (dd, J = 2 Hz, and J = 8.5 Hz, H-6' ), 6.90 (d, J =8.5 Hz, H-5' ), 6.42 (d, J = 2 Hz, H-8), 6.23 (d, J = 2 Hz, H-6); 5.29 ( d, J = 1.41 Hz, H-1" of rhamnose), (m, rest of rhamnose protons), 0.85 (d, J = 6.07 Hz, CH 3 of rhamnose); 13 C-NMR: δ (ppm) (C-2), (C-3), (C-4), (C-5), (C- 6), (C-7), (C-8), (C-9), (C-10), (C-1' ), (C-2' ), (C-3' ), (C-4' ), (C-5' ), (C-6' ); (C-1" ), (C-2"), (C-3" ), (C-4"), (C-5"), (C-6" ). Quercetin 3--ß-D- glucopyranoside (9) R f values (x100), 15% HAc (45) and BAW (60), UV Spectral data, λ max (nm): (MeH): 253 nm, 263 sh, 294 sh, 351 nm. (+NaMe): 271 nm, 328 sh, 410 nm. (+NaAc): 273 nm, 321 nm, 375 nm. (+NaAc/ H 3 B 3 ):262 nm, 300 sh, 377 nm. (+AlCl 3 ): 275 nm, 305 sh, 332 nm, 435 nm. (+AlCl 3 / HCl): 275 nm, 305 sh, 361 sh, 403 nm. Negative ESI-MS Data [M- H] = m/z 462.9, 1 H-NMR (ppm): δ 7.67 (1H, dd, J = 2.12 Hz, and J=8.62 Hz, H-6' ), 7.53 (1H, d, J = 2.12 Hz, H-2' ), 6.82 (1H, d, J = 8.62 Hz, H-5' ), 6.4 (1H, d, J = 1.8 Hz, H-8), 6.2 (1H, d, J = 1.8 Hz, H-6), 5.37 (1H, d, J = 7.63 Hz, H-1" ), (5H, m, H-2" - H-6"). 13 C-NMR Spectral Data obtained of δ (ppm): (C-2), (C-3), (C-4), (C- 5), (C-6), (C-7), (C-8), (C-9), (C-10), (C-1' ), (C-2' ), (C-3' ), (C-4' ), (C-5' ), (C-6'), (C-1" ), (C-2"), (C-3" ), (C-4"), (C-5" ), (C-6" ). Kaempferol 3--ß-D-glucopyranoside (10) R f values (x100): 15% HAc (30) and BAW (52), UV Spectral data, λ max (nm) (MeH): 266 nm, 346 nm. (+NaMe): 274 nm, 327sh, 401 nm. (+NaAc): 274 nm, 305 nm, 393 nm. (+NaAc/H 3 B 3 ): 267 nm, 352 nm. (+AlCl 3 ): 274 nm, 304 nm, 349 nm, 396 nm. (+AlCl 3 / HCl): 274 nm, 345 nm, 394 nm. 1 H-NMR (ppm): δ 8.1 (2H, d, J = 8.8 Hz, H-2', H-6'), 6.98 (2H, d,j = 8.8 Hz, H-3', H-5'), 6.54(1H, d, J = 2.0 Hz, H-8), 6.31 (1H, d, J = 2.0 Hz, H-6), 5.57 (1H, d, J = 7.2 Hz, H-1"), (5H, m, H-2"- H-6"). 13 C-NMR (ppm): δ (C-2), (C-3), (C-4), (C-5), (C-6), (C-7), (C-8), (C-9), (C-10), (C-1'), (C-2') , (C-3' ), (C-4' ), (C-5'), (C-6' ), (C-1" ), (C-2" ),74.59 (C-3'' ), (C-4'' ), (C-5'' ), (C-6'' ) Biochemical screening Determination of total polyphenol content (TPC) The content of total phenolic compounds in each extract was determined according to the Folin-Ciocalteu method 16 with some modification to minimize the volume of the reactants used to microlitres 17. An aliquot of 10 µl of each extract (1 mg/ml) was mixed with 50 µl of Folin Ciocalteu phenol reagent (10 x dilutions) and allowed to react for 5 min. Then 40 µl of 20% saturated Na 2 C 3 solution was added and allowed to stand for 1 h in the dark before the absorbance of the reaction mixture was read at 725 nm using a microplate ELISA reader (BioRad). A gallic acid standard curve was obtained for the calculation of phenolic content. The total polyphenol content (TPC) of each

4 Fathalla A. Ayoob et al. / Journal of Pharmacy Research 2014,8(4), extract was expressed as mg gallic acid equivalents per gram of plant extract on a dry-weight basis DPPH radical scavenging assay The antioxidant activity of each extract and standard were assessed based on the radical scavenging effect of the stable DPPH free radical 18. Weighed quantities of each extract were dissolved in distilled DMS and used. 10 µl of each extract or standard (from 0.0 to 300 µg/ ml) was added to 90 µl of a 100 µm methanolic solution of DPPH in a 96-well microtitre plate (Sigma-Aldrich Co., St. Louis, M, US). After incubation in the dark at 37 C for 30 min, the decrease in absorbance of each solution was measured at 520 nm using an ELISA micro plate reader (Model 550, Bio-Rad Laboratories Inc., California, USA). Absorbance of blank samples containing the same amount of either water or DMS and DPPH solution was also prepared and measured. The scavenging potential was compared with a solvent control (0% radical scavenging) and ascorbic acid. The percentage of DPPH bleaching was utilized to calculate the IC 50 (half-maximal inhibition concentration). Radical scavenging activity was calculated by the following formula: % Reduction of absorbance = [(A B - A A ) / A B ] x 100, where: A B absorbance of blank sample and A A absorbance of tested extract solution (t = 30 min) 17, Antiviral activity Cells and virus C8166 cells and HIV-1 IIIB were kindly donated by Medical Research Council, AIDS Regent Project (Serbia). The cells were maintained at 37 o C in 5% C 2 in RPMI-1640 medium supplemented with 10% heatinactivating FBS. HIV-1 IIIB was prepared from the supernatants of H9/ HIV-1 IIIB cells. The 50% HIV-1 tissue culture infectious dose (TCID 50 ) in C8166 cells was determined and calculated by Reed and Muench method 20. Virus stocks were stored in small aliquots at -70 o C. The titer of virus stock was TCID 50 per ml Cytotoxicity assay All samples were dissolved in DMS. AZT was dissolved in RPMI and stored at -20 o C. The cellular toxicity of each extract on C8166 cells was assessed by MTT colorimetric assay. Briefly, 100µl of cells were plated into 96-well plates, 100 µl of various concentrations of each extract was added and incubated at 37 o C in a humidified atmosphere of 5% C 2 for 72h. 100 µl supernatant were discarded, MTT reagent was added and incubated for 4 h, 100µl 50% DMF-15% SDS was added. After the formazan was dissolved completely, the plates were read on a Bio-Tek ELx 800 ELISA reader at 570 nm/630 nm. 50% cytotoxicity concentration (CC 50 ) was calculated 20, Inhibition of syncytia formation The effect of samples on acute HIV-1 infectivity was measured by the syncytia formation assay. In the presence or absence of various concentrations of each extract, C8166 cells were infected with HIV- 1 at a multiplicity of infection (MI) of 0.015, and cultured in 96-well plates at 37 o C in 5% C 2 for 3 days. AZT was used as a positive control. At 3 days post-infection, the cytopathic effect (CPE) was measured by counting the number of syncytia (multinucleated giant cell) in each well of 96-well plates under an inverted microscope (100 ). The inhibitory percentage of syncytia formation was calculated by the percentage of syncytia number in sample-treated culture compared to that in infected control culture. 50% effective concentration (EC 50 ) were calculated. Formula: According to the method described by Reed and Muench 20, 50% cytotoxic concentration (CC 50 ) and 50% effective concentration (EC 50 ) was determined from dose-response curve. Therapeutic index (TI) of anti-hiv activity is CC 50 /EC Cell viability (% of control) = (D test -D blk )/(D ctrl - D blk ) CPE inhibition (%) = (1-CPE test /CPE ctrl ) Antibacterial activity The following Gram (-) (Enterobacter cloacae human isolate, Escherichia coli ATCC 35210, Pseudomonas aeruginosa ATCC and Salmonella typhimurium ATCC 13311) and Gram (+) bacteria (Bacillus cereus clinical isolate, Listeria monocytogenes NCTC 7973, Micrococcus flavus ATCC and Staphylococcus aureus ATCC 6538) were used. The organisms were obtained from Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research Sinisa Stankovic, University of Belgrade, Serbia. The antibacterial assay was carried out by microdilution method 22, 23. The bacterial suspensions were adjusted with sterile saline to a concentration of 1.0 x 10 5 CFU/ml. The inocula were prepared daily and stored at 4 C until use. Dilutions of the inocula were cultured on solid medium to verify the absence of contamination and to check the validity of the inoculum. All experiments were performed in duplicate and repeated thrice. The minimum inhibitory and bactericidal concentrations (MICs and MBCs) were determined using 96-well microtitre plates. The bacterial suspension was adjusted with sterile saline to a concentration of 1.0 x 10 5 CFU/ml. The compounds and extracts for testing were added (1 and 10 mg/ml) in broth Triptic Soy broth (TSB) medium (100 µl) with bacterial inoculum (1.0 x 10 4 CFU per well) to achieve the wanted concentrations. The microplates were incubated at rotary shaker (160 rpm) for 24 h at 37 C. The lowest concentrations without visible growth (at the binocular microscope) were defined as concentrations that completely inhibited bacterial growth (MICs). The MBCs were determined by serial sub-cultivation of 2 µl into microtitre plates containing 100 µl of broth per well and further incubation for 24 h. The lowest concentration with no visible growth was defined as the MBC, indicating 99.5% killing of the original inoculum. The optical density of each well was measured at a wavelength of 655 nm by microplate manager 4.0 (Bio-Rad Laboratories) and compared with a blank and the positive control. The antibiotics streptomycin and ampicillin were used as positive controls (1 mg/ml in sterile physiological saline). All experiments were performed in duplicate and repeated thrice.

5 Fathalla A. Ayoob et al. / Journal of Pharmacy Research 2014,8(4), Antifungal activity The used fungi; Aspergillus fumigatus (ATCC 1022), Aspergillus versicolor (ATCC 11730), Aspergillus ochraceus (ATCC 12066), Aspergillus niger (ATCC 6275), Trichoderma viride (IAM 5061), Penicillium funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC 9112) and Penicillium var. cyclopium (ATCC 46511), were obtained from Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research Sinisa Stankovic, University of Belgrade, Serbia. The micromycetes were maintained on malt agar and the cultures were stored at 4 C and sub-cultured once a month 24. The antifungal assay was carried out by modified microdilution technique 25, 26. The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile saline to a concentration of approximately 1.0 x 10 5 in a final volume of 100 µl per well. The inocula were stored at 4 C for further use. Dilutions of the inocula were cultured on solid malt agar to verify the absence of contamination and to check the validity of the inoculum. Minimum inhibitory concentration (MIC) determinations were performed by a serial dilution technique using 96-well microtiter plates. Each examined extract was added in concentration of 10 mg/ml in broth Malt medium (MA) with inoculum. The microplates were incubated at rotary shaker (160 rpm) for 72 h at 28 C. The lowest concentrations without visible growth (at the binocular microscope) were defined as MICs. The fungicidal concentrations (MFCs) were determined by serial subcultivation of 2 µl of tested extracts dissolved in medium and inoculated for 72 h, into microtiter plates containing 100 µl of broth per well and further incubation 72 h at 28 C. The lowest concentration with no visible growth was defined as MFC indicating 99.5% killing of the original inoculum. The fungicides bifonazole and ketoconazole were used as positive controls ( µg/ml). All experiments were performed in duplicate and repeated thrice. 3. RESULTS AND DISCUSSIN 3.1. Phytochemical screening Chromatographic investigation of the 70% methanolic extract of T. bellerica leaves led to isolation of ten compounds, three hydrolysable tannins; gallic acid (1), ellagic acid (2) and methyl gallate (3); one flavone; luteoline (4); two flavonol aglycones; quercetine (5) and kampferol (6); and four flavonol glycosides; quercetin 3--[6"- α-l rhamnopyranosyl]-(1 6)- ß-D- glucopyranoside (rutin, 7), quercetin-3--α-l-rhamnopyranoside(8),quercetin3--ß-dglucopyranoside (9) and kaempferol 3--ß-D-glucopyranoside (10) (Figure 1). CR H R 4 H Ellagic acid (2) R 3 R 2 H R 1 H H 4 R 1 = R 4 = H; R 2 = H; 5 R 2 = R 4 = H; R 1 = H 6 R 1 = R 2 = R 4 = H; 7 R 1 = R 4 = H; R 2 = -rutinoside. 8 R 1 = R 2 = H; R 4 = -α-l rhamnopyranoside. 9 R 1 = R 2 = H; R 4 = -ß-D-glucopyranoside. 10 R 3 = R 4 = H; R 1 = H; R 2 = -ß-D-glucopyranoside. Figure 1. Strucutres of isolated compounds from the bioactive ethyl acetate and n-butanol soluble parts of successive methanol extract of leaves of Terminalia bellerica Inspection of UV and 1 H-NMR of compound 7, revealed that it was a flavonol attached to two sugar moieties 27, 28. The presence of the anomeric proton of ß-D-glucose at δ 5.05 (J = 7Hz) and the anomeric proton of α-l-rhamnose at δ 4.45 ppm suggested that glucose is the internal sugar moiety and rhamnose is the terminal sugar moiety. The structure was deduced as quercetin 3--[6"-α-L rhamnopyranosyl]- (1 6)-ß-D- glucopyranoside (rutin). An evidence for the 1 6 linkage between α-l-rhamnpyranose and ß-D-glucopyranose was obtained from the 13 C-NMR, where a substantial downfield shift for C-6" at δ 68.5 ( δ C-6" = 8 ppm) relative to the non substituted glucopyranose moiety. The attachment of disaccharide part to the quercetin aglycone was observed by the absence of a signal of H-3 in 1 H-NMR, in addition to the downfield shift of C-3 in 13 C-NMR to be at δ Acid hydrolysis of compound 7 afforded glucose, rhamnose and quercetin after direct comparison with standard samples on Co-PC 29. This compound was isolated for the first time from the leaves of T.bellerica. H H H R= H Gallic acid (1) R= CH 3 methyl gallate (3) Compound (8) obtained as yellow amorphous powder of chromatographic properties and color reactions similar to those reported for quercetin-3--glycoside. This assumption was primarily supported by UV spectral data of (8) in methanol and different diagnostic shift reagents 27. Complete acid hydrolysis of compound (8) yielded quercetin and rhamnose, identified by CoPC with authentic samples in

6 Fathalla A. Ayoob et al. / Journal of Pharmacy Research 2014,8(4), different solvents. Compound (8) exhibited a Molecular weight of 448 in Negative ESI-MS spectrum analysis, which showed [M-H] - at m/z: Structure confirmation of (8) was achieved through 1 H-NMR spectroscopic analysis whereby the spectrum revealed the characteristic pattern of quercetin proton resonances besides the anomeric proton resonance appearing as doublet signal of coupling constant (J=1.41 Hz), at δ 5.29 ppm assignable to the rhamnoside proton H-1"; with the methyl rhamnose proton resonance revealed at δ 0.85 ppm (d, J= 6.07 Hz) 30. Final confirmation of the identity was achieved through 13 C-NMR 31 and it was isolated for the first time from T. bellerica. Compound (9) was isolated as a yellow amorphous powder, appeared on PC as dark purple under UV light, turning bright yellow when fumed with ammonia vapor, changing orange with Naturstoff reagent, which suggested a flavonol 3-glycoside structure. Compound (9) exhibited R f -values, UV spectral data and a molecular weight of 464 in negative ESI-MS analysis ([M-H] - at m/z: 463.9). Complete acid hydrolysis which was found identical with those of quercetin-3-glucoside 32. This structure was confirmed by 1 H- and 13 C-NMR analyses. The 1 H-NMR spectrum (DMS-d6), revealed the presence of signals at δ 7.53 (d, J = 2.34 Hz, H-2'), 7.67 (dd, J = 2.12 Hz and 8.5 Hz, H-6' ), 6.82 (d, J = 8.62 Hz, H-5' ), 6.40 (d, J = 1.83 Hz, H-8), 6.20 (d, J = 1.98 Hz, H-6) and 5.37 ( d, J = 7.63 Hz, H-1" of glucose), (m, rest of glucose protons) 33. Further confirmation of the structure of compound (9) as quercetin-3--glucoside, was achieved through 13 C-NMR spectrum (DMS-d 6 ). The presence of ß- glucopyranoside moiety in the compound (9) followed from the anomeric carbon resonance at δ ppm and from the chemical shift values of the remaining sugar resonances at δ (C-2"), (C-3"), (C-4"), (C-5"), (C-6"). Resonances of the carbons of the flavonoid moiety were assigned by comparison with the corresponding signals in the published spectrum of quercetin-3- -ß-D-glucopyranoside 33. Compound (9) was identified as quercetin-3--ß-d-glucopyranoside (9) and it was isolated for the first time from T.bellerica leaves. Compound (10) was isolated as yellow powder; dark purple under UV light gave bright yellow with ammonia vapor and orange yellow with AlCl 3 reagent. UV spectral data of compound (10) showed two major absorption bands in MeH band I at 346 nm and band II at 266 nm, which is a flavonol with an occupation at position 3 34, 35. The additions of NaMe make a bathochromic shift (+ 55) in band I with an increase in intensity. This means that position 4' has free H group 27. The addition of NaAc makes a bathochomic shift in band II ((+ 8 nm) compared with the same band in MeH and the presence of a shoulder at 327 nm in NaMe suggested the presence of a free hydroxyl group at position 7. After the addition of H 3 B 3 to NaAc, the hypthochromic shift in band I (- 49) suggested the absence of any catecholic hydroxyls. The bathochromic shift in band I (+ 50) on addition of AlCl 3 which is still stable even after the addition of HCl indicated the presence of free hydroxyl group at position 5 and absence of catecholic hydroxyls 27. Acid hydrolysis of compound (10) gave kaempferol as aglycone and, glucose, which identified via CoPC with kaempferol authentic and sugar moiety. The 1 H NMR of compound (10) showed aromatic signals at δ 8.1 and 6.9 assignable H-2', 6' and H-3', 5', respectively, together with two meta coupled protons at δ 6.3 and δ 6.5 assignable H-6 and H-8, respectively. The anomeric proton of the sugar appeared at δ 5.57 indicating its ß configuration of glucose at position C- NMR spectral data displayed 21 carbon, 15 of kaempferol with 3- substituted and 6 carbons of glucose unit which indicated by the anomeric carbon atom of glucose at δ and the other carbon signals appeared at their proper position (C-5" at δ 77.83; C-3" at δ 74.59; C-4" at δ 76.80; C-2" at δ and C-6" at δ 61.23). From the previous data compound (10) is identified as kampferol-3--ß-dglucopyranoside and was isolated for the first time from T. bellerica leaves. Gallic acid 35 and ellagic acid 35, 36 were isolated previously from the leaves of T.bellerica. However, the other eight compounds (3-10) were isolated for the first time from the leaves of T. bellerica and proved according to authentic samples and discussed according to their previous data (Figure 1) Biological activities screening Plant drugs are considered to be less toxic and free from side effects than synthetic drugs. Terminalia bellerica is a large deciduous tree. The selection of this genus is based on uses in traditional medicine, since Terminalia species are widely used medicinal plants both in Africa and in Asia. The extensive literature survey revealed that Terminalia bellerica, is an important medicinal plant with diverse pharmacological spectrum. Different parts of Terminalia bellerica exhibited many pharmacological activities. It has been reported that fruit extract of Terminalia bellerica exhibited no toxicity in vivo 41, 42, strong antioxidant activity 42, antidiarrheal activity 43, 44, significant activity on the immune system in vitro 45, antihypertensive activity 46, significantly effective in relaxation of spontaneous contractions 47, showed high activity as hepatoprotective and the activity increased by the presence of its active constituent s gallic acid 48, possesses potential broad spectrum antimicrobial activity against S. aureus, B. subtilis, E. coli, P. aeruginosa, proved to have antisalmonella activity 42, 49-51, have a significant activity on wound healing 52, antidiabetic activity 53, 54 and analgesic activity 55. In addition, it was reported that benzene and ethanol extracts of Terminalia bellerica barks increased the total cholesterol content and decreased protein content and epididymal sperm count 56. However, only limited research has been performed on the leaves of Terminalia bellerica. including, antifungal activity against A. flavus, A.niger, A.brassicicola, A.alternata and H.tetramera 57. In this study we report for the first time phytochemical screening, investigation of antioxidant, antibacterial, cytotoxicity and anti-hiv as well as antifungal activities of seven different successive extracts namely; 70% methanol, petroleum ether, diethyl ether, dichloromethane, ethyl acetate, butanol and aqueous extracts of Terminalia bellerica Roxb. leaves DPPH radical scavenging activity The seven extracts of Terminalia bellerica Roxb. were assayed for radical scavenging activity using the DPPH colorimetric test. The stable radical DPPH has been used widely for the determination of primary antioxidant activity, that is, the free radical scavenging activities of pure antioxidant compounds, plant and fruit extracts and food materials. This assay is based on the reduction of DPPH radicals in methanol, which causes an absorbance drop at 520 nm. Figure 2

7 Fathalla A. Ayoob et al. / Journal of Pharmacy Research 2014,8(4), shows the antioxidant activities of the seven extracts compared to that of ascorbic acid and rutin (standard antioxidants). The antioxidant activities of these extracts ranged from 4.2 µg/ml to 77.4µg/ml as shown in Table 1. The highest DPPH radical scavenging effect was detected in the 70% methanol extract with an IC 50 of 4.2 µg/ml. This antioxidant activity is compared to that of ascorbic acid (IC µg/ ml) and rutin (IC µg/ml) which are often used as a positive control because of their high antioxidant activity. The diethyl ether extract showed an IC 50 of 6.5 µg/ml. The butanol extract showed an IC 50 of 7.9 µg/ml. The ethyl acetate extract showed an IC 50 of 8.9 µg/ ml. The aqueous extract showed an IC 50 of 21.9 µg/ml. The dichloromethane extract showed an IC 50 of 33.4 µg/ml, while the petroleum ether extract showed the least antioxidant activity with IC 50 of 77.4 µg/ml. In examining radical scavenging capacity, it can be observed that the more polar protic solvents are more effective at extracting the antioxidant components in the Terminalia bellerica Roxb. extract, except for the diethyl ether extract which showed strong antioxidant activity as well. These results are in agreement with the previously reported data 17, 19. Table 1: Antioxidant activities and total phenol contents of seven different successive extracts of Terminalia bellerica Roxb. leaves. Extract DPPH-IC 50 TPC mggae/g (µg/ml) plant extract Mean ± SD Mean ± SD (n = 3) (n = 3) 70% Methanol 4.20 ± ± 2.26 Petroleum Ether (60-80 o C) ± ± 1.59 Diethyl Ether 6.47 ± ± 3.02 Dichloromethane ± ± 2.83 Ethyl Acetate 8.85 ± ± 1.08 Butanol 7.94 ± ± 4.02 Aqueous ± ± 2.59 Ascorbic Acid 82.79* ± 2.34 Rutin 40.62* ± 1.92 *Concentrations are in µm/ml % Inhibition of DPPH Concentration (µg/ml) Aqueous Butanol Ethyl Acetate Dichloromethan Diethyl Ether Petroleum Ether Rutin Ascorbic Acid 70%Methanol Figure 2. Radical scavenging activities of seven different successive extracts of Terminalia bellerica Roxb. leaves compared to ascorbic acid and rutin as positive controls Total polyphenol contents (TPC) As plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators, the total amount of phenolic compounds in the selected plant extracts was determined using the Folin-Ciocalteu method. Phenolic compounds undergo a complex redox reaction with phosphotungstic and phosphomolybdic acids present in this reagent. The total phenol content of these extracts ranged from 79.4 mggae/g extract to mggae/ g extract as shown in Table 1. The 70% methanol extract exhibited the highest level of polyphenols content (167.6 mggae/g extract). The aqueous extract exhibited the second highest level of polyphenols (156.4 mggae/g extract). The butanol extract exhibited the third level of polyphenols content (141.2 mggae/g extract) followed by the diethyl ether extract, which exhibited the fourth level of polyphenols content (129.7 mggae/g extract). The ethyl acetate extract exhibited the fifth level of polyphenols content (121.9 mggae/g extract). The dichloromethane extract exhibited the sixth level of polyphenols content (109.3 mggae/g extract), while, the petroleum ether showed the least level of polyphenolic content (79.4 mggae/g extract) among all the seven investigated extracts. From the above results, we can estimate that 70% methanol extract, which showed the highest antioxidant activity, also had the highest amount of polyphenols. Furthermore, the petroleum ether that showed the lowest antioxidant activity has the lowest amount of polyphenols Statistical analysis of TPC against the antioxidant activity With reference to the above mentioned results, it is obvious that, the correlations of TPC against the antioxidant activity based on the DPPH assay involving all seven extracts were very low reflected by the negative correlation coefficient (-0.87), confirming that, not only the phenolic compounds contribute to the radical scavenging activity of these extracts 17 but there may be other compounds than phenolics are responsible for these high antioxidant activities. This may explain our data which showed that only minor differences are found in total phenol content, but major differences in antioxidant activity: E.g., by comparing the 70% methanol extract against the petroleum ether extract (Table 1), a more than eigtheen-fold difference in DPPH- IC 50 between the 70% methanol extract (4.2) and the petroleum ether extract (77.41) could not be based on a less than 50% difference in TPC ( vs ) Anti-HIV activities The seven extracts of Terminalia bellerica Roxb. were assayed for their anti HIV activities compared to AZT (positive control) in C8166 cells. The cellular toxicity of each extract on C8166 cells was assessed by MTT colorimetric assay. The effect of samples on acute HIV-1 infectivity was measured by the syncytia formation assay using AZT as positive control. According to the method described by Reed and Muench 20, 50% cytotoxic concentration (CC 50 ) and 50% effective concentration (EC 50 ) was determined from dose-response curve. Therapeutic index (TI) of anti-hiv activity, which is CC 50 /EC 50, is shown in Table 2. Butanol, ethyl acetate and aqueous extracts of Terminalia bellerica showed mild anti-hiv-1 activity, whose TI values were all below 10. However, 70% methanol extract of Terminalia bellerica showed a good anti-hiv-1 activity, whose TI value was over 10 but less than

8 Fathalla A. Ayoob et al. / Journal of Pharmacy Research 2014,8(4), Table 2: The summary of cytotoxicity and anti-hiv-1 activities of the active successive extracts of Terminalia bellerica Roxb. leaves in C8166 cell. Extract Fraction Experiment Method CC 50 (µg/ml) EC 50 (µg/ml) Therapeutic index (TI) 70% Methanol Cytotoxicity assay MTT ± ± 2.6 Inhibition of syncytium formation CPE ± 0.2 Butanol Cytotoxicity assay MTT 22.2 ± ± 0.1 Inhibition of syncytium formation CPE ± 0.9 Ethyl Acetate Cytotoxicity assay MTT ± ± 0.4 Inhibition of syncytium formation CPE ± 0.6 Aqueous Cytotoxicity assay MTT ± ± 0.8 Inhibition of syncytium formation CPE ± 1.2 AZT Cytotoxicity assay MTT ± ± 5.6 Inhibition of syncytium formation CPE ± 0.3 ng/ml Table 3: Antibacterial activity of six different successive extracts of Terminalia bellerica Roxb. leaves (mg/ml). Bacteria Aqueous Dichloromethane Butanol MeH 70% Ethyl acetate Pet. Ether Streptomycin Ampicillin extract MIC MIC MIC MIC MIC MIC MIC MIC MBC MBC MBC MBC MBC MBC MBC MBC Bacillus cereus 0.30± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.01 Micrococcus flavus 0.50± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.01 Staphylococcus aureus 0.15± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.01 Listeria monocytogenes 1.25± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.01 Escherichia coli 0.75± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.03 Pseudomonas aeruginosa 0.60± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.00 Enterobacter cloacae 0.75± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.01 Salmonella typhimurium 0.75± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± The other extracts did not show any significant activity against HIV Antibacterial activity The seven extracts of Terminalia bellerica Roxb. were assayed for their antibacterial activities against four Gram (-) bacteria (Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium) and four Gram (+) bacteria (Bacillus cereus clinical isolate, Listeria monocytogenes, Micrococcus flavus and Staphylococcus aureus) using microdilution method. The minimum inhibitory and bactericidal concentrations (MICs and MBCs) of these extracts, compared to the antibiotics streptomycin and ampicillin (positive controls), are shown in Table 3. Diethyl ether did not show any activity against all the tested bacteria. All extracts did not show good antibacterial activities against Bacillus cereus, Listeria monocytogenes, Pseudomonas aeruginosa, Enterobacter cloacae and Salmonella typhimurium compared to the antibiotics streptomycin and ampicillin. The 70% methanol and ethyl acetate extracts showed much better MIC activity, however, the 70% methanol and butanol extracts showed comparative MBC compared to the antibiotics streptomycin and ampicillin against Micrococcus flavus. Both butanol and aqueous extracts showed better MIC; in addition, both butanol and ethyl acetate extracts showed better MBC compared to the antibiotics streptomycin and ampicillin against Staphylococcus aureus. Both 70% methanol and ethyl acetate extracts showed better MIC activity, however, their MBC were less than the antibiotics streptomycin and ampicillin against Escherichia coli Antifungal activity The seven extracts of Terminalia bellerica Roxb. were assayed for their antifungal activities against eight strains (Aspergillus fumigatus, Aspergillus versicolor, Aspergillus ochraceus, Aspergillus niger, Trichoderma viride, Penicillium funiculosum, Penicillium ochrochloron and Penicillium var. cyclopium) using modified microdilution technique. Table 4 shows the MIC and MCF of each extract compared to that of fungicides bifonazole and ketoconazole (positive controls). Diethyl ether did not show any activity against all the tested strains. The six other extracts all show much better MIC against all the tested strains. The six extracts showed MIC activities ranged from 2.5 to 7.5 fold and from 3.3 to 10 fold, in addition to MCF values ranged from 0.4 to 1.6 fold and 1 to 4 fold higher than that of bifonazole and ketoconazole, respectively, against Aspergillus fumigates. The six extracts showed MIC activities ranged from 1.6 to 10 fold and from 3.3 to 20 fold, in addition to MCF values ranged from 1.6 to 5 fold and 4 to 12.5 fold higher than that of bifonazole and ketoconazole, respectively, against Aspergillus versicolor. The six extracts showed MIC activities ranged from 2.5 to 15 fold and from 2.5 to 15 fold, in addition to

9 Fathalla A. Ayoob et al. / Journal of Pharmacy Research 2014,8(4), Table 4: Antifungal activity of six different successive extracts of Terminalia bellerica Roxb. leaves (mg/ml). Fungi Aqueous Dichloromethane Butanol MeH 70% Ethyl Pet. Ether bifonazole ketoconazole extract acetate MIC MIC MIC MIC MIC MIC MIC MIC MFC MFC MFC MFC MFC MFC MFC MFC Aspergillus fumigatus 0.02± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.06 Aspergillus versicolor 0.06± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.05 Aspergillus ochraceus 0.02± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.02 Aspergillus niger 0.02± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.04 Penicillium var. cyclopium 0.02± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.09 Penicillium ochrochloron 0.02± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.04 Penicillium funiculosum 0.02± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.09 Trichoderma viride 0.02± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.01 MCF values ranged from 1.6 to 5 fold higher than that of both bifonazole and ketoconazole against Aspergillus ochraceus. The six extracts showed MIC activities ranged from 1.2 to 7.5 fold and from 1.6 to 10 fold, in addition to MCF values ranged from 0.8 to 5 fold and 2 to 12.5 fold higher than that of bifonazole and ketoconazole, respectively, against Aspergillus niger. The six extracts showed MIC activities ranged from 2.5 to 7.5 fold and from 16.6 to 50 fold, in addition to MCF values ranged from 0.8 to 5 fold and 4 to 25 fold higher than that of bifonazole and ketoconazole, respectively, against Penicillium var. cyclopium. The six extracts showed MIC activities ranged from 5 to 10 fold, in addition to MCF values ranged from 1 to 6.25 fold and 2 to 12.5 fold higher than that of bifonazole and ketoconazole, respectively, against Penicillium ochrochloron. The six extracts showed MIC activities ranged from 5 to 10 fold and from 62.5 to 125 fold, in addition to MCF values ranged from 2 to 6.25 fold and 28 to 87.5 fold higher than that of bifonazole and ketoconazole, respectively, against Penicillium funiculosum. The six extracts showed MIC activities ranged from 2.5 to 10 fold and from 5 to 20 fold, in addition to MCF values ranged from 0.4 to 10 fold and 0.6 to 15 fold higher than of bifonazole and ketoconazole, respectively, against Trichoderma viride. In summary, Terminalia bellerica, is an important medicinal plant with diverse pharmacological spectrum. Different parts of Terminalia bellerica exhibited many pharmacological activities. However, only limited research has been performed on the leaves of Terminalia bellerica. In this study we report for the first time phytochemical screening, investigation of antioxidant, antibacterial, cytotoxicity and anti-hiv as well as antifungal activities of seven different successive extracts namely; 70% methanol, petroleum ether, diethyl ether, dichloromethane, ethyl acetate, butanol and aqueous extracts of Terminalia bellerica Roxb. leaves. The results of this study revealed that Terminalia bellerica leaves exhibited a strong antioxidant activity, especially its 70% methanol, diethyl ether and butanol extracts. nly, 70% methanol extract showed good anti-hiv activity. The 70% methanol, ethyl acetate, butanol and aqueous extracts showed good antibacterial activity against Micrococcus flavus, Staphylococcus aureus and Escherichia coli compared to the antibiotics streptomycin and ampicillin. All extracts except diethyl ether, showed very high antifungal activities against all the used eight fungal strains compared to that of fungicides bifonazole and ketoconazole (positive controls). ACKNWLEDGEMENTS This work was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant No 260. Conflict of Interest The authors have declared that there is no conflict of interest. 4. REFERENCES 1. Yip PY, Chau Mak CF, Kwan CY, DNA methods for identification of Chinese medicinal materials, Chin Med, 2007, 2, 9. doi: / Thiombiano A, Schmidt M, Kreft H, Guinko S, Influence du gradient climatique sur la distribution des espèces de Combretaceae au Burkina Faso (Afrique de l ouest), Candolleaa, 2006, 61, McGaw LJ, Rabe T, Sparg SG, Jäger AK, Eliff JN, Van SJ, An investigation on the biological activity of Combretum spp, J Ethnopharmacol, 2001, 75, The wealth of India; (Raw material). (1st edn). Vol. X, PID, CSIR, New Delhi, 1952, Valsaraj R, Pushpangadan P, Smitt UW, Adsersen A, Christensen SB, Sittie A, Nyman U, Nielsen C, lsen CE, New anti-hiv-1, antimalarial, and antifungal compounds from Terminalia bellerica, J Nat Prod, 1997, 60, The wealth of India. A dictionary of Indian raw materials and industrial products. National Institute of Science Communication and Information Resources, New Delhi, 2004, 5: R Z, P Int. 7. Chopra RN, Nayar SL, Chopra IC, Glossary of Indian medicinal plants. CSIR, New Delhi, India, 1956, 106, Umukoro S, Ashorobi RB, Evaluation of anti-inflammatory