Full-Text (PDF)
Total Page：16
File Type：pdf, Size：1020Kb
Journal of Medicinal Plants Research Vol. 3(5), pp. 424-428, May, 2009 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875© 2009 Academic Journals Full Length Research Paper New acylated flavonol glycoside from Ceratonia siliqua L. seeds A. Gohar*, S. R. Gedara and H. N. Baraka Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt. Accepted 7 May, 2009 New flavonol glycoside, 4`-p-hydroxybenzoylisorhamnetin-3-O--L-rhamnopyranoside named cerato- side (5), together with the known kaempferol-3-O--L-rhamnopyranoside (afzelin) (3), quercetin-3-O-α-L- arabinofuranoside (auriculain) (4) quercetin-3-O--L-rhamnopyranoside (6), -sitosterol (1) and - sitosterol-3-O-β-D-glucoside (2) were isolated for the first time from Ceratonia siliqua L. seeds. The identity of these compounds was established through UV, IR, MS and NMR spectral data. The antioxidant activity of the organic solvent fractions together with some isolated compounds were carried out and showed significant results. Key words: Ceratonia siliqua, carob, flavonol glycosides, antioxidants INTRODUCTION Ceratonia siliqua L. Fabaceae, the carob tree, is a Medi- while simple phenols, mainly cinnamic acid, made up teranean plant. Components of the carob pod have been about 2% of the total (Avallone et al., 1997; Corsi et al., used as a sweetener, as a food ingredient in the produc- 2002; Balban, 2004; Joslyn et al., 2006; Ayaz et al., tion of confectionery, beverages, bread or pasta (Mara- 2007). The butanol soluble fraction of the methanol ex- kis, 1996; Avallone et al., 1997). Many recent activities tract of the carob cotyledons contain five C-glycosylfla- are reported for carob pods such as antihyperglycemic vones including schaftoside, isoschaftoside, neoschafto- (Jung-In et al., 2005), antioxidant (Cantalejo, 2001; Fouly side, isoschaftoside-4'-O-glucoside and schaftoside-4`-O- et al., 2004; Makris and Kefalas, 2004), immunomodulat- glucoside (Batista and Gomes, 1993). Flavonoids repre- ing (Cantalejo, 2001) and antiproliferative on mouse he- sented 26% of the polyphenols and the major compo- patocellular carcinoma cell line (Corsi, et al., 2002). nents were identified as the glycosides myricetin and The nutritive value of the carob pods was attributed to quercetin-3-O--L-rhamnoside (Joslyn et al., 2006; Vaya its high levels of carbohydrates (45%), appreciable amo- and Mahmood, 2006). The antioxidant activity of the ca- unts of protein (3%), and low levels of fat (0.6%) (Aval- rob pod is attributed to the presence of such phenolic lone et al., 1997; Ayaz et al., 2007). The green carob compounds (Owen et al., 2003; Ayaz et al., 2007). pods contain a major leucoanthocyanins in highly poly- To date it is still unknown whether other compounds merised leucodelphinidins and proanthocyanidin together may be responsible for this activity. The aim of this study with hydrolysable tannins, crystalline galloyl glucose com- was therefore to investigate in addition to the antioxidant pounds. In the ripe pods as well as the leaf and the wood activity of the seeds, its chemical constituents. extracts, gallic acid, epigallocatechin-3-gallate and epica- techin-3-gallate were detected. A total of 24 polyphenol compounds were identified with a yield of 3.94 g/kg (dry MATERIALS AND METHODS weight). Gallotannins 1,6-di-, 1, 2,6-tri- and 1,2,3,6-tetra- General O-galloyl--D-glucose were isolated from the Carob fi- bre. The profile was dominated by gallic acid in various Melting points were measured by Hot-Stage melting point forms: free gallic acid (42% of polyphenols by weight), microscope (Sybron, USA). UV spectra were measured in spectro- gallotannins (29%), methyl gallate (1%) and catechin scopic methanol using Beckmann DU-7 Spectrophotometer. IR (KBr-discs) spectra were measured by Nicolet MX-1 FT-IR spectro- 1 13 meter. H- and C-NMR experiments were run in DMSO-d6 at 400 MHz for 1H-NMR and 100 MHz for 13C-NMR on JOEL TNM- IR (KBr-discs) spectra were measured by Nicolet MX-1 FT-IR spectro- 1 13 *Corresponding author. E-mail: [email protected]. meter. H- and C-NMR experiments were run in DMSO-d6 at 400 Gohar et al. 425 MHz for 1H-NMR and 100 MHz for 13C-NMR on JOEL TNM-LA, FT- ric acid, mixed with an equal volume of ethanol and refluxed on a NMR system, Japan, using TMS as internal standard. Chromato- steam bath for two hours. The solvent distilled off and the hydroly- graphic separation was performed using silica gel 60 for column (E- sate shacked with ether. The ether extract was dried over anhy- Merck, Germany). TLC was performed on silica gel GF254 (E-Merk, drous sodium sulphate and the solvent was distilled off. The residue Germany, 0.2 mm thickness). was dissolved in methanol and kept for identification of the agly- cone moiety. The acidic mother liquor containing the sugar was neutralized with silver oxide and filtered. The precipitate was wash- Chemicals ed several times with few ml of water. The filtrate and the washing were combined and evaporated to dryness. The residue dissolved Authentic sample, -sitosterol, -sitosterol-3-O-glucoside, kaemp- in one ml of pyridine and used for identification of sugar moiety ferol and quercetin were obtained from isolated and identified mate- (Kikuchi and Matsuda, 1996). Sugars were identified though TLC rials in the Department of Pharmacognosy, Faculty of Pharmacy, and GLC Mansoura University. Authentic sugars, glucose, galactose, rham- nose, xylose and arabinose were obtained from El-Nasr Pharma- ceutical and Chemical Company, Egypt. Shift reagents viz., sodium GLC Analysis of the trimethylsilyl ether derivatives of the methoxide, aluminum chloride and HCl were prepared according to sugars the standard methods (Mabry et al., 1970). Chromatograms were visualized under UV light (Ultra-Violet Lamp 254 and 366 nm, De- It was performed on Pachard Gas Chromatograph Model 427 saga, Germany) and by spraying developed plates with 1% vanillin- equipped with dual flame ionization detector. The stationary phase was SE-30 (10%) on Chromosorb W, 80 – 100 mesh, packed in a H2SO4 for -sitosterol, -sitosterol-3-O-glucoside, 5% aluminum chloride and alcoholic potassium hydroxide for flavonoids and ani- coiled glass column 150 cm long and 4 mm internal diameter. The line hydrogen phthalate for sugars. For TLC analysis, the following flow rates of H2, air and N2 were 40, 300 and 35 ml/min respec- tively. The temperature of the column, injector port and the detector solvent systems were used: pet. ether- ether 6:4 (system I), chloro- o form - methanol 9.5:0.5 (system II), chloroform - methanol 9:1 (sy- were 205, 250 and 260 C respectively in isothermal technique. The stem III) and chloroform - methanol 8:2 (system IV). pyridine solution of the sugar was mixed with 0.2 ml hexamethyl- disilazane and 0.1 ml trimethylchlorosilane, shaken for 30 s. and al- lowed to stand for 5 min. Plant material, extraction and chromatographic isolation The Ceratonia siliqua L. pods were purchased from a local market Measuring the antioxidant activity in El Mansoura, Egypt in 2006. The seeds separated from the pods ABTS [2, 2-azinobis(3-ethylbenzothiazoline 6-sulphonate] radical and grounded in a suitable mill. + cation (ABTS ) scavenging activity was measured according to the method described by Re et al., 1999. ABTS was dissolved in water Extraction and isolation to a 7 mM concentration and the ABTS radical cation was produced by adding potassium persulfate to a final concentration of 2.45 mM. Dried powdered seeds (75 g) were extracted with cold methanol (3 The completion of radical generation was obtained in the dark at L) and the extract was evaporated to dryness in a rotary evaporator room temperature for 12–16 h. This solution was then diluted with at 40oC. The residue (22 g) was suspended in distilled water and ethanol to adjust its absorbance at 734 nm to 0.50 ± 0.02. Different extracted with chloroform followed by ethyl acetate. plant extracts, total extract and compound C-6 and C-5 were pre- The chloroform extract (9.5 g) was chromatographed on silica gel pared by dissolving 1 mg each in 0.5 ml methanol and 0.5 ml phosphate buffer. To determine the scavenging activity, 1 ml of dilu- column. Elution was started with hexane, hexane- ethyl acetate gra- + dient. The eluted fractions (250 ml each) were collected, concen- ted ABTS solution was added to 50 l of plant extract or tested iso- trated and screened by TLC. Similar fractions pooled together. lated compounds (or water for the control), and the absorbance at Fractions 14 - 16 eluted with hexane - ethyl acetate (95:5) were cry- 734 nm was measured 6 min after the initial mixing, using ethanol stallized from methanol to afford compound 1 (30 mg). Fractions 32 as the blank. The percentage of inhibition was calculated by the - 34 eluted with hexane- ethyl acetate (50:50) gave compound 2 (20 equation: mg). Inhibition percentage (%IP) = [Ac − As /Ac] × 100 The ethyl acetate soluble fraction (5 g) was chromatographed on silica gel column, using ethyl acetate followed by ethyl acetate- me- Where Ac and As are the absorbance of the control and of the thanol gradient. The eluted fractions (250 ml each) were collected, tested samples respectively. concentrated and screened by TLC. Similar fractions pooled toge- ther. Fractions 11 – 14, eluted with 10% ethyl acetate methanol mixture, contained two flavonoid spots and so, fractions 15 – 18 Identification of the isolated compounds contained another two-flavonoid spots. Rechromatography of the o first fraction on silica gel column and elution with CH2Cl2 – CH3OH -Sitosterol (1): White needles (CH3OH). - M.p. 135-137 C. - Rf = mixture (gradient), 100 ml fractions were collected, afforded com- 0.59 (system I).