Alcoholic extract of the aerial parts of Schinus terebinthifolius exhibit significant antioxidant, antifungal, antialzheimer`s and antileishmanicidal activities. Investigation of the chemical constituents of this plant let to isolate one new naturally occurring compound, synthetically known named (4-aminophenyl) acetic acid, along with the known 2-phenylacetamide, 1-pentadecanol, 3-(4-aminophenyl) prop-2-enoic acid, (E), ethyl 3, 4, 5-trihydroxybenzoate, cinnamic acid and benzamide. The structures of these compounds were established by spectroscopy techniques, including 1D and 2D NMR spectroscopy and comparison with the published data. The structure of (4-aminophenyl) acetic acid has also been confirmed by X-ray diffraction studies. The total alcoholic extract of Schinus terebinthifolius was evaluated for several bioassay activities and the isolated compounds were evaluated for their antifungal and antioxidant activities.
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The Anacardiaceae includes 76 genera with over 600 species. A survey of the literature reveals that 25 of those genera contain poisonous species (Mitchell, 1990).
Schinus terebinthifolius RADDI (COPAL) is an ornamental plant, which belongs to family Anacardiaceae, genus Schinus. It is known as pink peppercorn (Jain et al., 1995), Brazilian pepper tree (Ronald, 1999), Pepper tree, Christmas berry, Faux Poirier, Florida Holly and Warui (Morton, 1978; Williams et al., 2002).
Uses in Traditional Medicine
Antihemorrhagic (reduces bleeding), analgesic (pain-reliever), antiinflammatory, antibacterial, anticancerous, anticandidal, antifungal, antispasmodic, antitumorous, antiviral, laxative, astringent, digestive stimulant, tonic, cardiotonic, hypotensive, wound healer, to stop bleeding and for toothaches. It is taken internally for rheumatism and as a purgative (Sarita Varma, 2002). It is used for many conditions in the tropics, including menstrual disorders, bronchitis, gingivitis, gonorrhea, gout, eye infections, sores, swellings, tuberculosis, ulcers, urethritis, urogenital disorders, venereal diseases and warts. It is also used for colds, flu and other upper respiratory infections (Lloyd et al., 1977).
|Fig. 1:||Structures of isolated compounds|
Previously Isolated Constituents
Phytochemical analysis of Brazilian pepper tree reveals that the plant contains tannins, alkaloids, flavonoids, steroidal saponins, sterols, terpenes and a large amount of essential oil (Lloyd et al., 1977; Stahl et al., 1983; Skopp and Schwenker, 1984; Campello and Marsaioli, 1974; 1975; Kaistha and Kier, 1962b; Hayashi et al., 1990).
New Isolated Constituents
New naturally occurring and synthetically known (4-aminophenyl) acetic acid (Schwartz et al., 1987), 2-phenylacetamide (Giridhar et al., 2003, Manley and Bilodeau, 2004, Firouzabadi et al., 1998, Guranda et al., 2001; Peng et al., 2003), 3-(4-aminophenyl) prop-2-enoic acid, (E) (Aleksi et al., 2001; Ono et al., 1999; Shingo et al., 2003), cinnamic acid (Marco et al., 1978; Ripperger et al., 1981), benzamide (Persinos et al., 1967; Douglas et al.,1997; Buller et al., 1992; Lord et al., 1973; Cook, 1989), Ethyl gallate (Mehta et al., 1988), 1-pentadecanol (Marongiu et al., 2003; Laurence et al., 1999; Dauben, 1948; Ruhoff and Reid, 1933; Kao and Shao-Yuan, 1922) were isolated (Fig. 1).
MATERIALS AND METHODS
Fresh aerial parts of Schinus terebinthifolius RADDI (Anacardiaceae) were collected in June, 2004 from Suez Canal University garden, Ismailia, Egypt. The identity was established by Prof. Dr. Hamdy K. Atta-Alla, Prof. of Floriculture and Medicinal plants, Department of Horticulture, Faculty of Agriculture, Suez Canal University. A voucher specimen (Number AMYM-1001) has been deposited in the Herbarium of Botany Department, Faculty of Science, Suez Canal University, Ismailia, Egypt.
Melting points were determined on Büchi 535 melting point apparatus and are uncorrected. IR spectra were recorded on Bruker FTIR vector 22 spectrophotometer, in KBr disks. UV spectra were obtained on Hitachi UV 3200 spectrophotometer. EIMS (ionization voltage 70 ev) was measured on a Varian MAT 311/A mass spectrometer and HREIMS were taken by MS JEOL-MS route, JMS-600H, Agilent 6890N. Fab-JEOL. JMS-HX 110 Mass spectrometer, glycerol was used as the matrix. 1D and 2D NMR spectra were run on Bruker AMX 400 and AMX 500 MHZ NMR spectrometers. The chemical shifts are given in ppm (δ), relative to TMS as internal standard and coupling constants are in Hz.
Single Crystal Structure Determination
A block shaped yellowish crystal of compound 4 C8H9NO2: Mr 151.4 with dimension 0.61x0.31x0.24 mm was selected for X-ray diffraction studies.
Crystal data for the structure of 4 is presented in Table 1.
Intensity data of compound 4 was collected on a Bruker Smart CCD 1-K area-detector diffractometer using Mo-Kα radiation (λ = 0.7107 Å) (Siemens, SMART and SAINT, 1996). Data reductions were performed using SAINT. The structure was solved by direct methods (Altomare et al., 1993) and refined by full-matrix least squares on F2 using the SHELXTL-PC package (Sheldrick, 1997). The intensity data within the θ range 2.52-24.99 were collected at 293 (2) K. The figure was plotted with the aid of ORTEP (Jhnson, 1976). Crystallographic data for compound 4 has been deposited to Cambridge Crystallographic Data Center (CCDC 610583), 12 Union Road, Cambridge, CB/EZ, UK (Fax: 44-1223-336-033, e-mail: [email protected]).
Column chromatography was carried out on silica gel (70-230 mesh, Merck). TLC was performed on Merck precoated silica gel 60 F254 aluminium foil plates and detection was achieved by UV light (254 nm), p-dimethylaminobenzaldehyde and iodine solution.
Extraction, Isolation and Characterization
Air-dried and powder aerial parts (5.0 kg) of the plant were macerated with ethanol 80% at room temperature till exhaustion. The resulting alcoholic extract was concentrated in vacuo to obtain a crude residue (3.0 kg). A part of this residue (1.0 kg) was dissolved in distilled water (600 mL) and defatted with n-hexane then acidified with glacial acetic acid to pH 3-4. The acidic solution was exhaustively extracted 5 times with CHCl3 (5x500 mL) to yield the acidic chloroform extract (132.2 g). The aqueous solution was basified with 10% NH4OH (pH 8-9) and re-extracted with chloroform to yield the basic chloroform extract (10.17 g) as a brown gummy residue, which represented 1.02% total crude alkaloids of the dry plant material.
|Table 1:||Crystal data, details of the data collection and structure analysis of compound 4|
A portion of the basic chloroform extract (9 g) was chromatographied over 350 g of silica gel and eluted with dichloromethane-methanol with the gradient polarity (0-40%). A total of 155 fractions ca. 150 mL each were collected and combined on the basis of TLC analysis leading to 15 series (A-O). Further purification of these series was achieved by column chromatography and preparative thin layer chromatography. Series A (211 mg), series B (503 mg), series C (240 mg) and series D (504 mg) obtained with 100% dichloromethane, upon examination by TLC (CH2Cl2-MeOH, 97: 3+1 drop of diethyl amine) contained a complex mixture with small amounts, were not investigated. Series E (1500 mg) obtained with CH2Cl2-MeOH (98:2) was purified successively on a silica gel column chromatography with CH2Cl2 and increasing the polarity with MeOH (up to 5%) to furnish compound 1 (869.3 mg).
Series F (728.9 mg) obtained with CH2Cl2-MeOH (96:4) was rechromatographed on a silica gel column chromatography eluting with CH2Cl2 and increasing the polarity with MeOH to yield 253.2 mg of compound 2 and a mixture of two compounds, which was subjected to further purification with preparative thin layer chromatography to afford 95.2 mg of compound 3 and 102.3 mg of compound 7.
Series G (2192.3 mg) obtained with CH2Cl2-MeOH (94:6) was also subjected to column chromatography over silica gel with CH2Cl2 and increasing the polarity with MeOH to afford 569.2 mg of compound 4 and a mixture of two compounds which subjected to further purification using PTLC to yield 456.2 mg of compound 5 and more amount from compound 1 (34.2 mg).
Series H (552.2 mg), obtained with CH2Cl2-MeOH (90:10) was separated using the same conditions as above, afforded more amount from compound 1 (210.3 mg) and a mixture of two compounds which applied on PTLC for further purification to yield more amount of compound 5 (23.4 mg).
Series I (2032.2 mg), obtained with CH2Cl2-MeOH (88:12) was applied to a silica gel column chromatography and eluted with CH2Cl2: MeOH of increasing polarity, to furnish 1343.7 mg of compound 6.
(4-aminophenyl) acetic acid (4). Rf; (0.35, CH2Cl2:MeOH, 95%, 1 drop of diethyl amine), yellow needle crystals in dichloromethane-methanol; m.p. 170-172°C; UV λmax nm (MeOH) (log ε): 202 (4.26), 211 (3.99), 251 (3.08), 226 (4.17), 278 (3.60), 366 (2.40), 390 (2.52); IR bands (KBr) vmax: 3394, 3217, 2925, 2862, 2673, 2574, 2492, 1895, 1660, 1606, 1512, 1446, 1294, 1232, 1178, 1109, 1026, 864, 798, 682, 657, 567, 526 cm-1. 1H NMR (400 MHZ, pyridine-d5): δ 11.33 (1H, s, OH-10), 7.74, 7.88 (1H each, 2x br s, NH2), 7.43 (2H, d, J 8.39, H-3, H-5), 7.13 (2H, d, J 8.36, H-2, H-6), 3.74 (2H, s, H-8); 13C NMR (125 MHZ, CD3OD): 177.6 (C-9), 157.4 (C-1), 127.6 (C-4), 131.1 (C-3, C-5), 116.3 (C-2, C-6), 42.6 (C-8); HR EIMS m/z: 151.0637 (calcd. for C8H9O2N, 151.0633); EIMS m/z (rel. Int.): 151 (28), 107 (100), 90 (5), 77.0 (26), 55 (4), 51 (15).
2-phenylacetamide (1). Rf; (0.60, CH2Cl2:MeOH, 95%, 1 drop of diethyl amine), Beige crystals in dichloromethane-methanol; m.p. 156-158°C; UV λmax nm (MeOH) (log ε): 206 (3.52), 241 (2.32), 258 (2.46), 330 (1.84), 351 (2.24), 366 (2.08), 390 (2.40); IR bands (KBr) vmax: 3357, 3178, 2925, 2856, 2806, 1949, 1639, 1450, 1415, 1286, 1180, 1130, 1072, 1029, 910, 871, 746, 698, 584, 536, 474 cm-1. 1H NMR (500 MHZ, CDCl3): δ 5.50, 5.88 (1H each, 2x br s, NH2), 7.24-7.34 (5H, Ar-H), 3.55 (2H, s, H-7); 13C NMR (125 MHZ, CD3OD): δ 176.96 (C-8), 130.14 (C-2, C-6), 129.56 (C-3, C-5), 127.8 (C-4), 136.90 (C-1), 43.42 (C-7); HR EIMS m/z: 135.0672 (calcd. for C8H9ON, 135.0684); EIMS m/z (rel. Int.): 135 (20), 107 (4), 92 (100), 91 (96), 77 (2).
Ethyl gallate (2). Rf; (0.52, CH2Cl2:MeOH, 95%, 1 drop of diethyl amine), pink needle crystals in dichloromethane-methanol; reacted positively with FeCl3 reagent and give green color; m.p.146-148°C; UV λmax nm (MeOH) (log ε): 218 (4.59), 241 (3.50), 275 (4.20), 340 (2.38), 341 (2.53), 365 (2.33), 389 (2.62); IR bands (KBr) vmax: 3454, 3305, 2968, 2927, 2857, 1705, 1618, 1533, 1455, 1409, 1317, 1254, 1197, 1098, 1035, 967, 867, 762, 609 cm-1. 1H NMR (500 MHZ, CD3OD): δ 7.03 (2H, s, H-2, H-6), 4.25 (2H, q,-CH2), 1.33 (3H, t,-CH3), 11.58 (3OH, s, OH-3, OH-4, OH-5, in pyridine-d5); 13C NMR (125 MHZ, CD3OD): δ168.5 (C-7), 146.4 (C-3, C-5), 139.7 (C-4), 121.8 (C-1), 110.0 (C-2, C-6), 61.6 (C-8), 14.6 (C-9); HR EIMS m/z: 198.0516 (calcd. for C9H10O5, 198.0528); EIMS m/z (rel. Int.): 198 (86), 183 (10), 169 (3), 153 (100), 125 (28), 107 (5), 79 (11).
Benzamide (3). Rf; (0.64, CH2Cl2:MeOH, 90%, 1 drop of diethyl amine), white powder; m.p. 131-133°C; UV λmax nm (MeOH) (log ε): 204 (3.93), 271 (3.69), 235 (3.50), 738 (3.10), 746 (3.15), 818 (3.04), 823 (3.07), 839 (3.04), 843 (3.06); IR bands (KBr) vmax: 3365, 3175, 3029, 2922, 2853, 1952, 1660, 1637, 1605, 1601, 1495, 1451, 1415, 1286, 1286, 1246, 1183, 1117, 1074, 968, 936, 863, 747, 700, 584, 532, 475 cm-1. 1H NMR (500 MHZ, CD3OD): δ7.44 (2H, dd, J 7.62Hz, H-3, H-5), 7.85 (2H, d, J 7.44Hz, H-2, H-6), δ7.53 (2H, dd, J 7.43Hz, H-4), 8.36, 8.34 (1H each, 2x br s, NH2, in pyridine-d5); 13C NMR (125 MHZ, CD3OD): δ134.98 (C-1), δ132.92 (C-4), δ128.85 (C-3, C-5), δ128.64 (C-2, C-6), δ172.43 (C-7); HR EIMS m/z: 121.052764 (calcd. for C7H7ON, 121.05275); FABMS (+ve) [M+1]+: 122; EIMS m/z (rel. Int.): 121 (62), 105 (88), 77 (100).
3-(4-aminophenyl)prop-2-enoic acid (5). Rf; (0.33, CH2Cl2:MeOH, 95%, 1 drop of diethyl amine), white crystals in dichloromethane-methanol; m.p.153-155°C; UV λmax nm (MeOH) (log ε): 209 (4.22), 226 (4.37), 249 (3.42), 285 (4.11), 303 (4.04), 308 (4.05), 368 (2.43), 389 (2.50); IR bands (KBr) vmax: 3396, 3220, 2922, 1899, 1659, 1609, 1601, 1514, 1411, 1291, 1232, 1176, 1108, 989, 943, 890, 859, 826, 798, 682, 566, 528, 448 cm-1. 1H NMR (300 MHZ, CD3OD): δ 7.40 (2H, d, J 8.28, H-3, H-5), 6.78 (2H, d, J 8.29, H-2, H-6), 7.46 (2H, d, J 15.99, H-8), 6.43 (2H, d, J 15.76, H-9), 11.41 (1H, s, OH-10 in pyridine-d5), 7.99, 7.88 (1H each, 2x br s, NH2, in pyridine-d5); 13C NMR (75 MHZ, CD3OD): 171.63 (C-10), 160.67 (C-1), 127.58 (C-4), 130.68 (C-3, C-5), 116.74 (C-2, C-6), 142.93 (C-8), 117.84 (C-9); HR EIMS m/z: 163.0627 (calcd. for C9H9O2N, 163.0633); EIMS m/z (rel. Int.): 163 (32), 162 (19), 120 (9), 119 (28), 107 (100), 94 (4), 93 (3), 77 (53).
1-pentadecanol (6). white needle crystals in dichloromethane-methanol; m.p. 44-46°C; IR bands (KBr) vmax: 3453, 2920, 2851, 1740, 1656, 1601, 1469, 1381, 1249, 1219, 1083, 1018, 995, 972, 916, 833, 765, 722, 668, 634, 560, 471 cm-1. 1H NMR (500 MHZ, CD3OD): δ 0.88 (3H, t, J 6.86,-CH3), 1.28-1.36 (2H each, m, 12-CH2), 1.64 (2H, p, J 6.98, CH2-2), 3.97 (2H, t, J 6.59, CH2-1); 13C NMR (125 MHZ, CD3OD): δ 69.15 (C-1), 33.05 (C-2), 30.37-30.76 (C-4-13), 26.89 (C-3), 23.70 (C-14), 14.39 (C-15); FABMS (+ve) [M+1]+: 229; (calcd. for C15H32O, 228.245303); EIMS m/z (rel. Int.): 196 (9.1), 168 (39.1), 140 (16.6), 126 (10.1), 112 (17.2). 98 (25.8), 84 (38.8), 70 (49.8), 56 (53).
Cinnamic acid (7). White crystals in dichloromethane-methanol; m.p. 132-134°C; 1H NMR (500 MHZ, CD3OD): δ7.24 (2H, dd, J 8.32Hz, H-3, H-5), δ7.55 (2H, d, J 7.84Hz, H-2, H-6), δ7.37 (2H, dd, J 7.47Hz, H-4), δ7.54 (2H, d, J 16.09Hz, H-7), δ6.63 (2H, d, J 15.80Hz, H-8); 13C NMR (75 MHZ, CD3OD): δ164.98 (C-9), δ136.20 (C-1), δ130.95 (C-4), δ129.57 (C-3, C-5), δ128.91 (C-2, C-6), δ142.73 (C-7), 121.43 (C-8); HR EIMS m/z: 148.05243 (calcd. for C9H8O2, 148.0524262); FABMS (+ve) [M+1]+: 149; EIMS m/z (rel. Int.): 148 (9), 147 (61), 146 (100), 104 (12), 103 (96), 91 (93), 90 (6), 77 (75).
The microorganisms used in the antifungal assays Trichphton longifusus, Candida albicans, Aspergillus flavus, Microsorum canis, Fusarium solani and Candida glabrata have been maintained from Microbiology department, Karachi university, Karachi, Pakistan. Stock solutions of each sample were freshly prepared in 1 mL dimethylsulfoxide (DMSO). These solutions were diluted into sterile molten Sabouraud dextrose agar (SDA) medium to reach a final concentration of 200 μg mL-1 separately. Test tubes were kept at room temperature for solidification. Medium containing DMSO was used as negative control. Fungal cultures were cut to 4x4 mm from 1 week grown plates and then inoculated onto the slant. After an incubation period of 7-10 days at 29°C, tubes were examined for the growth inhibition. Growth on the media containing compound was determined by measuring the linear growth (mm) of fungal culture (Atta-ur-Rahman et al., 2001). Growth inhibition (%) was calculated with reference to the negative control.
Antioxidant Assay (DPPH (1, 1-diphenyl-2-picryl Hydrazyl) Free Radical Scavenging Activity)
The reaction mixture containing 5 μL of test sample (1 mm in DMSO) and 95 μL of DPPH (Sigma, 300 μm) in ethanol the reaction mixture was taken in a 96-well micro titer plate (Molecular Devices, USA) and incubated at 37°C for 30 min. The absorbance was measured at 515 nm. Percent radical scavenging activity determined by comparison with a DMSO containing control. IC50 values represent concentration of compounds to scavenge 50% of DPPH radicals. BHA (3-t-Butyl-4-hydroxyanisole) was used as a positive control. All the chemicals used were of analytical grade (Sigma, USA) (Gulcin et al., 2004).
In vitro Cholinesterase nhibition Assay
Electric-eel acetylcholinesterase (EC 188.8.131.52), horse-serum butyrylcholinesterse (E.C 184.108.40.206), acetylthiocholine iodide, butyrylthiocholine chloride, 5,5Â´-dithiobis [2-nitrobenzoic acid] (DTNB) and galanthamine were purchased from Sigma (St.Louis, MO, USA). All other chemicals were analytical grade. Acetylcholinesterase and butyrylcholinesterase inhibiting activities were measured by the spectrophotometric method developed by Ellman et al. (1961). Acetylthiocholine iodide and butyrylthiocholine chloride were used as substrates to assay acetylcholinesterase and butyrylcholinesterase, respectively. The reaction mixture contained 150 μL of (100 mM) sodium phosphate buffer (pH 8), 10 μL of DTNB, 10 μL of test-compound solution and 20 μL of acetylcholinesterase or butyrylcholinesterase solution, which were mixed and incubated for 15 min (25°C). The reaction was then initiated by the addition of 10 μL acetylthiocholine or butyrylthiocholine, respectively. The hydrolysis of acetylthiocholine and butyrylthiocholine were monitored by the formation of yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine, respectively at a wavelength of 412 nm (15 min). Test extract and the positive control (Galanthamine and Eserine) were dissolved in EtOH. All the reactions were performed in triplicate in 96-well micro-plate in SpectraMax 340 (Molecular Devices, USA). The percentage (%) inhibition was calculated as follows (E-S)/E x 100, where E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound (Tougu, 2001).
Determination of IC50 Values
The concentrations of test compounds that inhibited the hydrolysis of substrates (acetylthiocholine and butyrylthiocholine) by 50% (IC50) were determined by monitoring the effect of increasing concentrations of these compounds in the assays on the inhibition values. The IC50 values were then calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA).
Leishmania major prmastigotes (DESTO), cultivated in bulk were aseptically be sedimented down at 3000 rpm, counted with the help of improved Neubavr chamber under the microscope and diluted with the fresh medium to a final concentration of 1x106 parasites/mL. The compound to be checked were dissolved to a final concentration of 1.0 mg in 0.1 mL of PBS (Phosphate Buffered Saline, pH 7.4 containing 0.5% MeOH, 0.5% DMSO). In 96-well microtiter plate, 180 μL of the parasite culture (1.0x106 parasites/mL) was added in difference wells. Twenty microliter of the experimental compound was added in culture and serially diluted so that minimum concentration of the compound is 0.1 μg mL-1. Ten microliter of PBS was added as negative control while glucantime, amphotericin B, pentaamidine and ampicilline to a final concentration of 0.1 mg mL-1 was added separately as positive control. The plate was incubated between 21-22°C in dark for 2 h. The culture was examined microscopically on an improved neubaver chamber and IC50 values of compounds possessing antileishmanial activity were calculated (Habtemariam, 2003). All assays were run in duplicate.
RESULTS AND DISCUSSION
The basic chloroform extract of the finely powder aerial part of Schinus terebisifolius was subjected to column chromatography. The fractions obtained were subjected to different sub-columns and preparative thin layer chromatography, afforded one new naturally occurring compound, synthetically known named (4-aminophenyl) acetic acid, along with the known 2-phenylacetamide, 1-pentadecanol, 3-(4-aminophenyl) prop-2-enoic acid, (E), ethyl gallate cinnamic acid and benzamide. The known compounds were identified by comparison of their spectral analysis data with the published ones (Schwartz et al., 1987; Giridhar et al., 2003; Manley and Bilodeau, 2004; Firouzabadi et al., 1998; Guranda et al., 2001; Peng et al., 2003; Aleksi et al., 2001; Ono et al., 1999; Shingo et al., 2003; Marco et al., 1978; Ripperger et al., 1981; Persinos et al., 1967; Douglas et al.,1997; Buller et al., 1992; Lord et al., 1973; Cook, 1989; Mehta et al., 1988; Marongiu et al., 2003; Laurence et al., 1999; Dauben, 1948; Ruhoff and Reid, 1933; Kao and Shao-Yuan, 1922). (4-aminophenyl) acetic acid was obtained as yellow needle crystals, m.p. 170-172°C. Its showed violet color under UV-light (λ 254 nm) and reacted positively with p-dimethylaminobenzaldehyde reagent. The molecular formula was determined as C8H9O2N by HR EIMS [M+] m/z: 151.0637, in conjunction with the NMR spectra. The IR spectrum showed hydroxyl group (3394 cm-1), amino group (2673-2492 cm-1), carbonyl group (1660 cm-1). In 1H-NMR spectrum we observed a singlet of two protons at 3.74 corresponding for a methylene group and in the region of aromatic proton, a typical AA`BB` system at 7.43 and 7.13 (2H each, d, J 8.39) can also been observed. The mass spectrum showed a base peak at 107 corresponding to the loss of one carboxylic group. This was confirmed in the 1H-NMR spectrum, which displayed a singlet of one hydroxyl group at δ11.33. In addition, the 1H-NMR spectrum showed two broad singlet of one proton each at δ7.74 and 7.88 due to the amine group. This group was deduced to be in Para position with the methylene group according to the HMBC spectrum in which we observed the 3J correlation between the proton signal at δ 3.74 and C-2. On the other hand, the signal at δ7.88 was assigned for C-3. The assignment of all the carbons was possible from HMBC and COSY correlation experiment.
X-ray Crystal Structure Analysis of (4)
X-ray structure analysis was possible for compound 4. This compound crystallizes from dichloromethane-methanol at room temperature as yellow block in the orthorhombic space group P212121, with a = 509.68(5), b = 951.77(10), c = 1538.91(16) pm, α = β = γ = 90°, V = 746.52(13) x106 pm3 and Z = 4. The crystallographic data are listed in Table 1. The solid-state structure of 4 is shown in Fig. 2.
In the crystal lattice of compound 4, one molecule interacts with four nearest neighbors, which results in a net motif, as depicted in Fig. 2b. It is interesting to note that the intermolecular aryl groups are directly stacked over one another for every two molecules. The dotted lines in Fig. 2b represent distances of 296.5(5), 276.2(4) and 292.8(4) pm for the N1 .O2, N1 .O1 and O2 .O1 interactions, respectively (Table 2).
From the above mention the structure of compound 4 was deduced to (4-aminophenyl) acetic acid. This compound was already synthesized by Schwartz et al. (1987), but it is the first time to isolate from any plant, so it is new naturally compound.
|Fig. 2a:||ORTEP drawing (50% probability level) of compound 4. Selected bond lengths [pm]: N(1)-C(4) 136.0(5), C(1)-C(7) 151.3(6), C(7)-C(8) 151.9(6); O(1)-C(8) 125.3(5); O(2)-C(8) 131.6(5); selected bond angels [°]: N(1)-C(4)-C(3) 118.3(3), N(1)-C(4)-C(5) 122.2(4), C(2)-C(1)-C(7) 120.2(4); C(6)-C(1)-C(7) 121.9(4); C(1)-C(7)-C(8) 112.7(3); O(1)-C(8)-C(7) 120.9(4); O(1)-C(8)-O(2) 121.7(4); selected torsion angles [°]: C(2)-C(3)-C(4)-N(1) 179.3(4), N(1)-C(4)-C(5)-C(6)-179.6(4), C(2)-C(1)-C(7)-C(8)-68.6(5), C(6)-C(1)-C(7)-C(8) 111.7(4)|
|Fig. 2b:||The crystal packing of compound 4 along a-axis, showing H-bonding in dashed-lines|
Total alcoholic extract of Schinus terebinthifolius showed significant activity against Trichophyton logifusus and Candida albicans. Compound 1 and 2 showed moderated activity against Trichphyton longifusus and Microsporum canis, compound 4 showed good activity against Microsporum canis and moderate activity against Trichphyton longifusus, compound 5 showed moderate activity against Trichophyton logifusus and Microsporum canis and compound 6 showed good activity against Trichophyton logifusus, Microsporum canis and Aspergillus flavus, while compounds 3 and 7 were not investigated because they have small amounts (Table 3).
On the other hand, total alcoholic extract of Schinus terebinthifolius showed very significant activity (92.8%) against DPPH Radical at 200 μg mL-1 (Table 3) and the isolated compound 2 showed very significant activity (96.1%) at 1 mM; compound 4 displayed moderate activity (50%) and the other compounds showed low activity. Compound 1 showed low antibacterial activity against Shigella flexnari, while the other compounds showed non-significant antibacterial activities (Table 4).
The total alcoholic extract gave significant inhibition for alzheimer`s as shown in Table 5, good antileishmanicidal activity. On the other hand, it gave low antibacterial activity against Pseudomonas auruginosa and Salamonella typhi, non-significant inhibition against insecticidal activity, non-significant phytotoxicity and non-significant cytotoxicity.
|Table 2:||Intermolecular hydrogen bond distances [pm] and angles [°] of the participating moieties of 4|
|Symmetry codes (I)-1+x,y,z (ii)-½-x,-y,1/2+z (iii)-x,1/2+y,3/2-z|
|Table 3:||In vitro antifungal bioassay (agar tube dilution protocol)|
|MIC: Minimum Inhibitory Concentration, C: Compound|
|Table 4:||DPPH (1, 1-Diphenyl-2-picryl hydrazyl) free radical scavenging activity|
|aC : Compound, bSEM: Standard error of mean, cIC50: The concentration of sample required to inhibit 50% of DPPH radical|
|Table 5:||Bioassay results of the total alcoholic extract of Schinus terebinthifolius|
The first author is grateful to HEJ Institute of chemistry, Karachi University, Karachi, Pakistan for the Fellowship it has provided her with to undertake this work. The authors thank the bioassays lab members for bioassay analysis.
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