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Phytochemical and Biological Investigation of Spergularia marina (L.) Griseb. Growing in Egypt
Phytochemical and Biological Investigation of Spergularia marina (L.) Griseb. Growing in Egypt
Natural Product Sciences. 2014. Sep, 20(3): 152-159
Copyright © 2014, The Korean Society of Pharmacognosy
  • Received : January 15, 2014
  • Accepted : February 16, 2014
  • Published : September 30, 2014
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About the Authors
Omnia Gamal El-Dien
Eman Shawky
shawkyeman@yahoo.com
Amal H. Aly
Rokia M. Abdallah
Nabil A. Abdel-Salam

Abstract
A phytochemical investigation of Spergularia marina (L.) Griseb. growing in Egypt, has been carried out, which resulted in the isolation of seven compounds from the different extracts of the plant namely; β -sitosterol glucoside, tricin (1) dihydroferulic acid (2), vanillic acid (3), 4-hydroxybenzoic acid (4), uracil (5) and 8- hydroxy cuminoic acid (6) Structure elucidation of the isolated compounds was carried out using different spectroscopic techniques. This is the first report for the isolation of these compounds from genus Spergularia . Furthermore, 8-Hydroxy cuminoic acid and uracil were isolated for the first time from family Caryophyllaceae. The chemical composition of the volatile components present in the petroleum ether extract of Spergularia marina (L.) Griseb. using combined gas chromatography-mass spectrometry (GC-MS) is reported here for the first time. Of the 97 components present, 59 were identified including three sulfur containing compounds which represented about 1.8% of the volatiles of the total petroleum ether extract. This prompted us to study and report its possible antimicrobial activity. In addition, the antibacterial and antifungal screening of different extracts of Spergularia marina (L.) Griseb. as well as some isolates have been performed using agar diffusion method.
Keywords
Introduction
Family Caryophyllaceae contains many economic plants, it is the source of many ornamental and fragrant decorative plants. 1 , 2 Genus Spergularia belongs to family Caryophyllaceae, subfamily Paronychioidea, tribe polycarpeae. 3 It includes 25 species distributed especially in cosmopolitan. 4 , 5 Literature survey revealed that some of the Spergularia species are found to have several biological activities. The aerial parts of S. rubra L. have a diuretic effect and have been used internally, as a liquid extract for cases of cystitis, dysuria and urinary calculus. The leaves of S. ramosa Cambess. are used in the form of a decoction, as a remedy for respiratory ailments, tuberculosis, and rickets. 6 For many years, the water extract of the whole plant of S. purpurea has been traditionally used by Moroccan population against many diseases, like diabetes, hypertension, renal disorders and cardiac diseases without much scientific basis of its therapeutic effect. 7 , 8
The work presented in this paper includes the phytochemical investigation of Spergularia marina (L.) Griseb., the most common Spergularia species in Egypt, which resulted in the isolation of seven compounds from the different extracts of the plant. These are β -sitosterol glycoside and tricin ( 1 ) which were isolated from the chloroform extract, dihydroferulic acid ( 2 ), vanillic acid ( 3 ), 4-hydroxybenzoic acid ( 4 ), uracil ( 5 ) and 8-hydroxy cuminoic acid ( 6 ) which were isolated from the ethyl acetate extract. Structure elucidation of the isolated compounds was carried out using different spectroscopic techniques.
The volatile oil analysis of some caryophyllaceous plants are reported in the literature. The volatile constituents of Cerastium candidissimum Correns (Caryophyllaceae) were extracted by steam distillation and analyzed by gas chromatography-electron impact mass spectroscopy “GC- ei MS”. Floral scent of 13 Silene species (Caryophyllaceae) was analyzed by gas chromatography and mass spectrometry; benzenoids together with isoprenoids dominated the scent in all species. 9 Meanwhile, the essential oil of air-dried Minuartia meyeri (Boiss.) Bornm. (Caryophyllaceae) was obtained by hydro-distillation and analyzed by GC-MS. Fifty-two components were identified in the oil. The antimicrobial activity of the isolated essential oil of the plant was also investigated and showed moderate antibacterial activity against Gram-positive and Gram-negative bacteria, but no antifungal activity was observed against two yeast-like fungi. 10
To our knowledge, there are no published reports on the chemical composition of the volatile constituents of Spergularia . In addition the antimicrobial activity of the petroleum ether extract of Spergularia marina (L.) Griseb. has been studied. Furthermore, the antibacterial and antifungal screening of different extracts, as well as some isolates from Spergularia marina (L.) Griseb., has been performed using agar diffusion method.
Experimental
General experimental procedures − Mps. were determined on a Stuart SMP heating stage microscope and are uncorrected. UV spectra were determined on Pye Unicam SP8-100 UV/VIS Spectrophotometer. 1D-NMR and 2D-NMR (COSY, HSQC and HMBC) spectra were recorded on Jeol 500 MHz spectrometer (Japan) and- Bruker 500 MHz spectrometer (Germany). EI/MS were taken at Finnigan-Mat SSQ 7000 at 70 eV (Germany) and JEOL JMS AX 500 mass spectra (Japan). Analytical and preparative TLCs were performed on silica gel (Merck, Kieselgel, 60 F 254 , 0.25 and 0.50 mm, respectively). Spots were visualized by exposure to UV radiation, anisaldehyde/H 2 SO 4 . GC-MS analysis was carried out using Clarus 600 Gas Chromatograph/Mass spectrometer (Perkin Elmer, USA) provided with Turbomass 5.03 software fitted with Rtx ® -5MS (a fused silica, low-polarity phase; 5% diphenyl/95% dimethyl polysiloxane from Restek) column 30 m × 0.32 mm ID and 0.25 μm film thickness. Helium used as carrier gas at flow rate 1.1 mL/min.
Plant material Spergularia marina (L.) Griseb. was collected in April 2006 during flowering stage from Rosetta 40 km East of Alexandria, Egypt. The plant was identified at the Department of Botany, Faculty of Science, University of Alexandria, Egypt.
Extraction and isolation − Fresh aerial parts (10 kg) of S. marina (L.) Griseb., collected at the flowering stage, were chopped and exhaustively extracted with 70% ethanol (40 L) at room temperature. The extract was concentrated under reduced pressure to small volume then fractionated successively with petroleum ether (4 × 500 mL), chloroform (3 × 500 mL), ethyl acetate (3 × 500 mL) and finally n -butanol (3 × 500 mL). The solvents were distilled off and the corresponding residues were weighed. Five grams of the residue of the chloroform extract was chromatographed on a column (64 × 3.5 cm) packed with silica gel (200 g) in chloroform. Elution was started by chloroform with increasing polarity of methanol. Fractions (200 mL each) were collected, screened by TLC systems (chloroform : methanol 9 : 1 and ethyl acetate : methanol : water : acetic acid 60 : 5 : 4 : 1) and similar fractions were combined. β -sitosterol O - β -D-glucopyranoside was identified by direct comparison with reference sample through m.p., mixed m.p., and co-chromatography. Chromatographic separation resulted in the isolation of tricin ( 1 ). Structure elucidation of the isolated compound was carried out using different spectroscopic techniques.
The residue (3 g) of ethyl acetate extract was chromatographed on a column (31 × 9 cm, negative pressure) packed with silica (400 g) in methylene chloride using vacuum liquid chromatography. Elution was started by methylene chloride with increasing polarity of methanol. Fractions, 400 mL each were collected, screened by TLC systems (chloroform : methanol 9 : 1, ethyl acetate : methanol : water 30 : 5 : 4 and ethyl acetate : methanol : water : acetic acid 60 : 5 : 4 : 1) and similar fractions were combined. Chromatographic separation resulted in the isolation of dihydroferulic acid ( 2 ), vanillic acid ( 3 ), 4-hydroxybenzoic acid ( 4 ), uracil ( 5 ) and 8-hydroxy cuminoic acid ( 6 ) from the ethyl acetate extract. Structure elucidation of the isolated compounds was carried out using different spectroscopic techniques.
GC-MS analysis of the petroleum ether extract of Spergularia marina (L.) Griseb. − The fresh flowering plant (250 g) of Spergularia marina (L.) Griseb. was extracted with petroleum ether (1 L) by percolation till exhaustion at room temperature. The solvent was evaporated under reduced pressure at 30 ℃ to give yellowish brown residues (520 mg). A weight of 100 mg of the residue was used for GC/MS analysis. GC/MS analysis was carried out under the following conditions Column Rtx ® -5MS (30 m × 0.32 mm i.d. and 0.25 μm film thickness), carrier gas: helium, mass mode: EI, 70 eV, injection port temperature 280 ℃, column temperature programme 40 ℃ for 0 minute (rate 5 ℃/minute) up to 300 ℃ for 15 minutes. The identity of the components was established by logical and analogical interpretation of their retention times, and mass fragments with those available in the literature in addition to computer searches at the National Research Center, Cairo, Egypt MS Data Library. Components were quantified as relative area percentage of total ion current generated.
Antibacterial and antifungal activities of different extracts of Spergularia marina (L.) Griseb. − Antibacterial and antifungal assays were carried out using the agar diffusion technique 11 against three Gram-positive bacteria; Staphylococcus aureus , Micrococcus luteus and Bacillus subtillus , two Gram-negative bacteria; Esherichia coli and Pseudomonas aeroginosea , and the fungus Candida albicans . The used organisms are local isolates provided from the Department of Microbiology, Faculty of Pharmacy, University of Alexandria. One mL of 24 hours broth culture of each of the tested organisms was separately inoculated into 100 mL of sterile molten nutrient agar maintained at 45 ℃. The inoculated medium was mixed well and poured into sterile 10 cm diameter Petri-dishes, receiving 15 mL. After setting, ten cups, each 8 mm in diameter, were cut in the agar medium (Oxoid). A portion (200 mg) of the volatile fraction of petroleum ether extract of Spergularia marina (L.) Griseb. in addition to the chloroform, ethyl acetate and n -butanol extracts, were accurately weighed, dissolved in 3mL DMSO, the solutions were inserted in the cups and incubated at 37 ℃ for 24 hours. 3 mg of each of tricin, p -hydroxybenzoic acid and 8-hydroxy cuminoic acid were accurately weighed, dissolved in 3 mL DMSO, the solutions were inserted in the cups and incubated at 37 ℃ for 24 hours. The Minimum inhibitory concentration (MIC) and the Minimum bactericidal concentration (MBC) were carried out using the agar dilution method. 12
Tricin (1) − Yellow crystals, mp 291 - 292 ℃, UV λ max , nm (abs.): MeOH 349, 270, MeOH/ NaOMe 419, 263, 278 (sh), MeOH/ AlCl 3 369 (sh), 392, 259 (sh), 278, MeOH/ AlCl 3 /HCl 363 (sh), 389, 257 (sh), 279, MeOH/NaOAc 418, 264, 278 (sh); EI-MS (rel. int) m/z : 331 (14.8), 330 [M] + (62.2), 178 (17.8), 153 (24.4), 152 (8.9), 135 (18.5), 124 (20.0), 119 (11.9), 116 (7.4), 107 (17.0), 108 (31.9), 105 (22.2), 91 (33.3), 74 (22.2); 1 H-NMRδ: 7.21 (2H,s, H-2', 6'), 6.63 (1H,s, H-3), 6.45 (1H,br.s, H-8), 6.19 (1H,br.s, H-6), 3.93 (6H,s, 3',5'-OCH 3 ), 13 C-NMR δ: 182.38 (C-4), 164.75 (C-2), 164.61 (C-7), 161.30 (C-5), 157.80 (C-9), 148.29 (C-3', 5'), 139.70 (C-4'), 120.80 (C-1'), 103.98 (C-2', 6'), 103.84 (C-10), 103.12 (C-3), 98.89 (C- 6, 8), 55.68 (3',5'-OCH 3 ).
Dihydroferulic acid (2) − Orange crystals, mp 89 - 90 ℃,; UV λ max , nm (abs.) MeOH: 280 (0.978); EI-MS (rel. int) m/z : 197 (8.35), 196 [M] + (30.47), 195 [M + - H] (33.99), 180 [M + - H -CH 3 ] (42.9), 178 [M + - H 2 O] (35.31), 151 [M + -COOH] (41.83), 138 [M + +H-CH 2 COOH] (100), 123 [M + - CH 2 CH 2 COOH] (38.01), 119 [M + - H 2 O – CH 2 COOH] (61.6), 107 [M + + H –OCH 3 - CH 2 COOH] (68.59), 91 [C 7 H 7 ] (23.63), 78 [C 6 H 6 ] (50.24); 1 H NMR, δ: 6.80 (1H, d, H-2), 6.66 (1H, d, H-5), 6.63 (1H, dd, H-6), 3.82 (3H, s, 3-OCH 3 ), 2.80 (2H, t, H-7), 2.42 (2H, t, H-8); 13 C- NMR, δ: 177.3 (s, C-9), 147.7(s, C-3), 144.7 (s, C-4), 134.1(s, C-1), 120.6 (d, C-6), 115.6 (d, C-5), 112.9(d, C-2), 55.9 (q, 3-OCH 3 ), 32.3 (t, C-7), 29.5 (t, C-7).
Vanillic acid (3) − Pale orange crystals, mp 210 - 212 ℃,; UV λ max , nm (abs.) MeOH: 287(0.994); EI-MS (rel. int) m/z : 169 (28.38), 168 [M + ] (100), 154 [M + H-CH 3 ] (16.24), 139 [M-H-CO] (44.92), 93[M-CO 2 -OCH 3 ] (34.07); 1 H NMR, δ: 7.44 (1H, br.d, H-2), 7.37 (1H, dd, H-6), 6.74 (1H, d, H-5), 3.76 (3H, s, 3-OCH 3 ); 13 C- NMR, δ: 171.35 (s, C-7), 149.25 (s, C-4), 147.15 (s, C-3), 126.53 (s, C-1), 115.06 (d, C-5), 123.66 (d, C-6), 113.07 (d, C-2), 55.85 (q, 3-OCH 3 ).
4-Hydroxybenzoic acid (4) − Pale yellow crystals, mp 213 - 214 ℃; UV λ max , nm (abs.) MeOH: 278 (1.064); EIMS (rel. int) m/z : 139 (21.0), 138 [M + ] (100), 121 [M - OH] (86.41), 105 [M + H-OH-OH], (35.58), 93 [M-COOH] (44.28), 78 [C 6 H 6 ] (93.43); 1 H NMR, δ: 7.73 (2H, d, H-2, 6), 6.72 (2H, d, H-3, 5); 13 C- NMR, δ: 169.22 (s, C-7), 159.46 (s, C-4), 130.63 (d, C-2, 6), 127.55 (s, C-1), 113.96 (d, C-3, 5).
Uracil (5) − Yellowish white amorphous residue, mp 335 ℃; UV λ max , nm (abs.) MeOH: 278 (1.189); IR ν max cm −1 (KBr): 3410.1(N-H), 3103.8 (=C-H), 1722.4 (C=O); CHN: C% 42.47, H%3.33, N%18.93, O% 35.27; EI-MS (rel. int) m/z :112 [M + ] (48.7%), 96 (20.5), 86 (21.8), 73(100); 1 H NMR, δ: 10.93 (2H, br.s, NH), 7.39 (1H, d, H-6), 5.45 (1H, d, H-5); 13 C- NMR, δ: 164.26 (s, C-4), 151.45 (s, C-2), 142.09 (d, C- 6), 100.18 (d, C-5).
8-Hydroxy cuminoic acid (6) − Pale yellow crystals, mp 156 - 157 ℃; UV λ max , nm (abs.) MeOH: 280 (1.028); EI-MS (rel. int) m/z : 180 [M] + (7.66%), 166 (100%), 165 (18.21%),163 (13.17%), 150 (14.87%), 135 (14.79%), 121 (35.22%), 118 (16.26%), 105 (51.14%); 1 H NMR, δ: 7.83 (2H, d, H-2, 6), 7.43 (2H, d, H-3, 5), 1.43 (6H, s, CH 3 -9, 10); 13 C- NMR, δ: 169.12 (s, C-7), 152.16 (s, C-4), 134.78 (s, C-1), 128.42 (d, C-2, 6), 123.47 (d, C-3, 5), 70.44 (s, C-8), 31.64 (q, C-9, 10).
Result and Discussion
The phytochemical investigation of Spergularia marina (L.) Griseb. resulted in the isolation of seven compounds ( Fig. 1 ) from the different extracts of the plant. They were identified as β -sitosterol glycoside and tricin ( 1 ) 13 which were isolated from the chloroform extract. Meanwhile, dihydroferulic acid ( 2 ), 14 vanillic acid ( 3 ), 15 4-hydroxybenzoic acid ( 4 ), 16 uracil ( 5 ), 17 and 8-hydroxy cuminoic acid ( 6 ) 18 were isolated from the ethyl acetate extract. Structure elucidation of the isolated compounds was carried out using different spectroscopic techniques. It is worth mentioning that this is the first report for the isolation of these compounds from genus Spergularia . Furthermore, 8-hydroxy cuminoic acid and uracil were isolated for the first time from family Caryophyllaceae.
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Compounds isolated from Spergularia marina (L.) Griseb.
EI-MS spectrum of uracil ( 5 ) showed a molecular ion peak at m/z 112 [M] + calculated for a molecular formula C 4 H 4 O 2 N 2 which indicated four degree of unsaturation, supported by data from the elemental analysis. The 1 H, 13 C-NMR and DEPT experiment spectral data revealed the presence of 2, 4-pyrimidinedione through the appearance of two aromatic doublets each integrated for one proton, at 5.45 ( J = 7.5 Hz) and 7.39 ( J = 7.5 Hz), and two N-H proton through the appearance of a highly deshielded broad singlet integrated for 2 protons at δ 10.93. 13 C-NMR spectrum and DEPT experiment showed two carbonyl functional groups at δ 151.45 and 164.26, and two methines carbon at δ 100.18 and 142.09. The HMQC and the HMBC experiments allowed the assignment of the protons to the corresponding carbons. From the previously mentioned discussion, the structure of 5 is identified as 2,4(1H, 3H)-pyrimidinedione (uracil) which was confirmed by comparing its spectral data with those reported in the literature. 17
The EI-MS spectrum of 8-hydroxy cuminoic acid showed a molecular ion peak at m/z 180 suggesting a molecular formula C 10 H 12 O 3 which indicated five degree of unsaturation, supported by data from UV spectrum. The 1 H and 13 C-NMR spectral data revealed the presence of one 1, 4-disubstituted benzene ring through the appearance of two aromatic doublets each integrated for two protons, at δ 7.43 ( J = 7.5 Hz) and 7.83 ( J = 7.5 Hz), two tertiary methyls through the appearance of a singlet integrated for six protons at δ 1.43, one hydroxylated quaternary carbon at δ 70.44 and one carboxyl functional group at δ 169.12. The DEPT experiment confirmed the presence of two methyl, four methines and four quaternary carbons. The HMQC and the HMBC experiments allowed the assignment of the protons to the corresponding carbons. From the previously mentioned discussion the structure of ( 6 ) is identified as 4-(1-hydroxy-1-methylethyl) benzoic acid (8-hydroxy cuminoic acid) which was confirmed by comparing its spectral data with those of 8-hydroxycuminyl β -D-glucopyranoside reported in the literature. 18
The chemical composition of the volatile components present in the petroleum ether extract of Spergularia marina (L.) Griseb. was investigated by combined GC/MS spectrometry. The GC chromatogram ( Fig. 2 ) showed 97 peaks corresponding to 97 components. Only 59 Components were identified by GC/MS that almost constitutes about 86.25% of the volatiles of the total petroleum ether extract of Spergularia marina (L.) Griseb. The identification of the previous peaks was established through National Institute of Standards and Technology (NIST) mass data search libraries and the highest REV similarity index hits. Results of qualitative and quantitative analysis of petroleum ether extract of Spergularia marina (L.) Griseb. are given in Table 1 . Results showed that the percentage of: the hydrocarbons constituted about 44.2%, the oxygen-containing compounds constituted about 39.9%, the sulphur-containing compounds constituted about 1.8%, the nitrogen-containing compounds constituted about 0.51% and the unidentified compounds was 13.75%. The main components in the volatile fraction of Spergularia marina (L.) Griseb. were 6,9-pentadecadien-1-ol (8.47%), tetratetracontane (6.33%), eicosanoic acid (5.61%), 1-hentetracontanol (5.21%), tetratriacontane (4.82%), 1,2-benzenedicarboxylic acid- mono (2-ethylhexyl) ester (4.44%), 3-micosene (3.65%), 3-octadecene (3.47%), 9-eicosene (3.31%), 1-nonadecanol (2.87%), 3-hexadecene (2.82%) and heptadecane, 2, 6, 10, 15-tetramethyl (2.2%), whereas, three sulfur-containing compounds were identified and represent about 1.8% of the volatiles of the total petroleum ether extract of Spergularia marina (L.) Griseb. It is important to mention that this is the first report for the GC/MS analysis of the volatile constituents of Spergularia marina (L.) Griseb.
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GC chromatogram of the volatile fraction of petroleum ether extract of Spergularia marina (L.) Griseb.
Results of GC/MS analysis of the volatile fraction of petroleum ether extract ofSpergularia marina(L.) Griseb.
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Results of GC/MS analysis of the volatile fraction of petroleum ether extract of Spergularia marina (L.) Griseb.
continued
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continued
Multiple drugs resistance has become a very real issue in pharmaco-therapeutics as there are an increasing number of diseases which are exhibiting various levels of drug resistance, including bacterial infections. The search for new drugs to combat this problem is receiving much attention. The presence of sulfur containing compounds in the volatile fraction of Spergularia marina (L.) Griseb. prompted us to study its possible antimicrobial activity. The results of antibacterial and antifungal activities screening ( Tables 2 and 3 ) showed that the volatile fraction of petroleum ether extract of Spergularia marina (L.) Griseb. has only antibacterial activity against Grampositive bacteria; Staphylococcus aureus , Micrococcus luteus and Bacillus subtillus .
Results of antibacterial and antifungal screening of the volatile fraction of petroleum ether extract ofSpergularia marina(L.) Griseb.
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DMSO and commercial discs of ciprofloxacin, clotrimazole and rifampicin were used as negative and positive controls.
Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of the volatile fraction of petroleum ether extract ofSpergularia marina(L.) Griseb.
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Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of the volatile fraction of petroleum ether extract of Spergularia marina (L.) Griseb.
Antimicrobial activity testing was carried out for the different fractions in an attempt to define the biologically active fraction. The results of antibacterial and antifungal activities screening ( Tables 4 and 5 ) showed that all the tested extracts and isolates didn’t exhibit antibacterial activity against the Gram-negative Esherichia coli and Pseudomonas aeroginosea . All the tested extracts of Spergularia marina (L.) Griseb. exhibited activity against all the tested Gram-positive bacteria, except the n -butanol which didn't exhibit activity against Staphylococcus aureus . On the other hand, all the tested extracts except, n -butanol, didn't exhibit activity against the fungus Candida albicans . The ethyl acetate and chloroform fractions were the most active and therefore they were chosen for further investigation which resulted in the isolation of the previously mentioned seven compounds. The major com-pounds isolated were also tested to compare their results with the extracts. The extracts were more active. Tricin and p -hydroxybenzoic acid showed activity against Gram-positive bacteria: Bacillus subtillus , Micrococcus luteus and the fungus Candida albicans . 8-hydroxy cuminoic acid showed activity only against the fungus Candida albicans .
Results of antibacterial and antifungal screening of the different extracts ofSpergularia marina(L.) Griseb. and some of its isolates
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Results of antibacterial and antifungal screening of the different extracts of Spergularia marina (L.) Griseb. and some of its isolates
Minimum inhibitory concentration (MIC) and inimum bactericida concentration (MBC) of different extracts ofSpergularia marina(L.) Griseb. and some of its isolates
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Minimum inhibitory concentration (MIC) and inimum bactericida concentration (MBC) of different extracts of Spergularia marina (L.) Griseb. and some of its isolates
References
Atta-ur-Rahman 2002 Studies in Natural Products Chemistry; Volume 26 1st Edition Elsevier Amesterdam 3 - 4
Hickey M. , King C. 1988 100 Families of Flowering Plants 2nd Edition Cambridge University Press Cambridge 75 - 78
Flora of North America Editorial Committee 2005 Flora of North America; Volume 5 Oxford University press New York 9 - 10
Boulos L. 1999 Flora of Egypt; Volume 1 Al Hadara publishing Cairo 50 -
Kubitzki K. , Rohwer J.G. , Brittrich V. 1993 The Families and Genera of Vascular Plants; Volume II Spinger-Verlag Berlin Heidelberg Germany 214 -
De Tommasi N. , Piacente S. , Gacs-Baitz E. , De Simone F. , Cosimo P. , Aquino R. 1998 J. Nat. Prod. 61 323 - 327    DOI : 10.1021/np970398l
Jouad H. , Lacaille-Dubois M. A. , Eddouks M. 2001 J. Ethnopharmacol. 75 219 - 223    DOI : 10.1016/S0378-8741(01)00193-3
Jouad H. , Lemhadri A. , Maghrani M. , Zeggwagh N. A. 2003 J. Ethnopharmacol. 87 43 - 49    DOI : 10.1016/S0378-8741(03)00102-8
Jürgens A. , Witt T. , Gottsberger G. 2002 Biochem. Syst. Ecol. 30 383 - 397    DOI : 10.1016/S0305-1978(01)00106-5
Yayli N. , Gulec C. , Ucuncu O. , Yasar A , Ulker S. , Coskuncelebi K. , Terzioglu S. 2006 Turk. J. Chem. 30 71 - 76
Jain S. R. , Kar A. 1971 Planta Med. 20 118 - 123    DOI : 10.1055/s-0028-1099675
Andrews J. M. 2001 J. Antimicrob. Chemother. 48 5 - 16    DOI : 10.1093/jac/48.suppl_1.5
Kwon Y. S. , Kim C. M. 2003 Arch. Pharm. Res. 26 535 - 539    DOI : 10.1007/BF02976877
Huang Z. , Dostal L. , Rosazza J. P. 1993 Appl. Environ. Microbiol. 59 2244 - 2250
Sakushima A. , Coskun M. , Maoka T. 1995 Phytochemistry 40 257 - 261    DOI : 10.1016/0031-9422(95)00059-G
Cho J. Y. , Moon J. H. , Seong K. Y , Park K. H. 1998 Biosci. Biotechnol. Biochem. 62 2273 - 2276    DOI : 10.1271/bbb.62.2273
Bednarek E. , Dobrowolski J. Cz. , Dobrosz-Teperek K. , Kozerski L. , Lewandowski W. , Mazurek A. P. 2000 J. Mol. Struct. 554 233 - 243    DOI : 10.1016/S0022-2860(00)00670-0
Ishikawa T. , Takayanagi T. , Kitajima J. 2002 Chem. Pharm. Bull. 50 1471 - 1478    DOI : 10.1248/cpb.50.1471