Vol. 5 | No.3 | 332-337 | July-September | 2012 ISSN: 0974-1496 | CODEN: RJCABP http://www.rasayanjournal.com FLAVONOIDS AND GALLIC ACID FROM LEAVES OF SANTALOIDES AFZELII (CONNARACEAE) SORO Yaya1*, KASSI Amian Brise Benjamin1,2, BAMBA Fanté2, SIAKA Sorho1, TOURE Seikou Amadou2 and COUSTARD Jean-Marie3 1 Laboratoire des Procédés Industriels de Synthèse, de l’Environnement et des Energies Nouvelles, Institut National Polytechnique Félix Houphouët-Boigny, BP 991 Yamoussoukro, Côte d’Ivoire 2 Laboratoire de Chimie Organique Structurale, Université de Cocody, 22 BP582 Abidjan 22, Côte d’Ivoire 3 Laboratoire "Synthèse et Réactivité des Substances Naturelles", UMR 6514 40, Avenue du Recteur Pineau, F-86022 Poitiers, France *E-mail: soro_y@yahoo.fr ABSTRACT Fractionation of the ethyl acetate fraction of the ethanolic extract of the dried powdered leaves of Santaloides afzelii (Connaraceae) on silica gel column chromatography afforded gallic acid and two flavonoids glycosides identified as quercetin-3-O-rhamnoside and myricetin-3-O-rhamnoside. Their structures were elucidated by 1H and 13C-NMR data and UV data. It is the first time that these compounds are reported in the plant. Keywords: Santaloides afzelii, Connaraceae, Polyphenol, Flavonoids glycosides, Gallic acid © 2012 RAS YAN. All rights reserved. INTRODUCTION Santaloides Afzelii (R.Br. ex Planch) G. Schellenb belongs to the plant family Connaraceae. It is a scandent to lianous shrub or small tree, widely dispersed in tropical Africa and used in ethnomedicine for the treatment of diverse ailments1. The Connaraceae family consists of about 20 genera and 350 species of tanniferous tropical trees and shrubs2. The leaves in this family are alternate, without stipules and pinnate compounds. The plants of this family possess analgesic and anti-inflammatory activities3. The aqueous leaves extract of Byrsocarpus coccineus (Connaraceae) contains flavonoids glycosides and may be a potential remedy for the treatment of certain central nervous system disorders in human4-5. Polyphenols compounds, including anthocyanins, flavonols, and phenolic acids, are among the most bioactive natural molecules found in the plants because of their antioxidant activities6. The risk of prostate cancer and pancreatic cancer may decrease at higher dietary flavonoids intakes7-8. Gallic acid can be regarded as a promising candidate for development as a topical anti-HSV-2 agent and inhibited the growth of lung cancer cells9-10. In tropical countries, particularly in Côte d’Ivoire, Santaloides Afzelii is often used in traditional medicine by villagers11. On the other hand macerate of the leaves is used as a wash to stabilize household12. In the present paper, we report the isolation and identification of major phenolic compounds from leaves of Santaloides Afzelii. To our best knowledge, there are no previous reports on the chemical constituents of Santaloides Afzelii (Connaraceae) in the literature. EXPERIMENTAL General Using liquid chromatography with UV photodiodearray detection (LC-UV) and post-column derivatization it is possible to get sure data on polyphenols. Further structural information is provided by the combination of HPLC (LC) with mass spectrometry (MS). The structures were established on the basis of one and two-dimensional NMR spectral experiments and ultraviolet (UV) spectrometry. FLAVONOIDS AND GALLIC ACID FROM CONNARACEAE SORO Yaya et. al Vol. 5 | No.3 | 332-337 | July-September | 2012 A Brucker Avance 400 spectrometer was used for 1H and 13C- NMR spectra recorded at 400 and 100 MHz, respectively. The spectra were recorded at 23°C using an external reference (TMS) in MeOH-d4 or DMSO-d6 in a sealed capillary tube placed inside the NMR cell. Chemical shifts are reported relative to Me4Si for 1H and 13C. The reproducibility of 13C NMR shift was about ±0.05 ppm. Chemical assignments were made using either DEPT 135, or HMBC or HSQC techniques or common chemical shift assignments rules. Flash column chromatography was performed on Macherey-Nagel Silica gel 60 (1540µm). TLC plates (Macherey-Nagel, ALUGRAM® SILG/UV254, 0,2mm silica gel 60Å) were visualized under UV light at 254nm and/or by dipping the TLC plates in a solution of phosphomolibdic acid (3g) in EtOH (100mL) followed by heating with a heat gum. ESI-MS was recorded on a Shimadzu GC MS-QP 2010 with electron-impact ionization (70 eV). HRMS in the positive ion mode was performed using a QTOF Ultima Global hybrid quadrupole time-of-flight instrument (Waters-Micromass). High-Performance Liquid Chromatography (HPLC) Analysis Analytic HPLC was performed using a RP-18 (5µm) Lichro CART® 150-4,6mm at 25°C. The binary elution system was composed with acetonitrile (solvent A) and 0.2% TFA/water (solvent B). Separations were performed at room temperature by solvent gradient elution: 10-20% B during 40 min, 20-30% B during 5 min, 30-40% B during 5 min, 40-45% B during 5 min and then return to the initial conditions (10% B) in 5 min to re-equilibrate the column. The flow rate for both analysis and washing cycles was 0.8 mL/min. The concentration of each sample was 0.1 mg/mL in methanol and detection wavelengths were 254, 280, 325 and 530 nm. Plant material The leaves of Santaloides Afzelii were collected in November 2009 at the beginning of the dry season from Korhogo in north of Côte d’Ivoire. A voucher sample was identified by Prof. Aké-Assi Laurent, Faculty of Science and Technology, Cocody-Abidjan University where a specimen was deposited. The collected plant materials were washed under running and shed dried. Phytochemical screening Phytochemical screening was performed to establish the type of secondary metabolites present in the plant. Air-dried leaves of Santaloides Afzelii were tested for the presence of flavonoids, anthraquinones, alkaloids, terpenoids and steroids, tannins and saponins using Harborne method13. It shows the presence of flavonoids, triterpenes, steroids, tannins and alkaloids. Extraction procedure The air-dried powdered sample (450 g) was exhaustively extracted with hexane at room temperature by constant stirring. The residue was extracted with 70% EtOH (3 x 500mL) at room temperature by constant stirring during 24 hours. After filtration on cotton then watmann paper, the extract was concentrated under reduced pressure at 40°C to afford a brown residue. The residue (20g) was suspended in water and partitioned successively with CH2Cl2 (3x 200mL) and AcOEt (3x 200mL). The obtained extracts were separately dehydrated with anhydrous sodium sulfate and evaporated under vacuum after filtration to give dichloromethane extract (1.82g) and ethyl acetate extract (5.10g). Isolation and purification The ethyl acetate fraction (5g) of Santaloides Afzelii leaves was subjected to column chromatography on silica gel 60 with solvents gradients CH2Cl2-AcOEt and AcOEt-MeOH to give 12 fractions (F1-F12). Fractions F5 and F8 were purified by flash column chromatography on silica gel 60, eluting with CH2Cl2MeOH (10-1) to afford compound 1 (342mg) and compound 2 (219mg) respectively. The different fractions were checked by TLC and HPLC. Another aliquot of each compound was dissolved in CD3OD or DMSO and analyzed by NMR for chemical structure determination. FLAVONOIDS AND GALLIC ACID FROM CONNARACEAE 333 SORO Yaya et. al Vol. 5 | No.3 | 332-337 | July-September | 2012 Compound 1: Quercetin-3-O-rhamnoside ; Yellow powder; HPLC Rt 48.63 min ; UV vis max 256.348 nm (methanol) ; HREI-MS (m/z) 448.0905; C21H20O11 (calcd 448.0903). 1H NMR (400 MHz, DMSOd6): 12.00 (1H, s, OH-5); 7.34 (1H, d, J = 2Hz, H-2’); 7.32 (1H, dd, J = 8.3Hz/2Hz, H-6’); 6.93 (1H, d, J = 8.3Hz, H-5’); 6.22 (1H, d, J = 2Hz, H-6); 6.39 (1H, d, J = 2Hz, H-8); 5.25 (1H, d, J = 1.2Hz, H-1’’); 4.24 (1H, dd, J = 3.3Hz/1.2Hz, H-2’’); 3.78 (1H, dd, J = 9.3Hz/3.3Hz, H-3’’); 3.40 (1H, m, H-4’’); 3.17 (1H, m, H-5’’); 0.82 (3H, d, J = 6.1Hz, H-6’’). 13C NMR (100 MHz, DMSO-d6): 179.7 (C-4); 166.0 (C7); 163.2 (C-5); 159.3 (C-2), 158.6 (C-9); 149.8 (C-4’); 146.4 (C-3’); 136.3 (C-3); 123.0 (C-6’); 122.9 (C1’); 116.9 (C-2’); 116.4 (C-5’); 105.9 (C-10); 103.6 (C-1’’); 99.8 (C-6); 94.7 (C-8). 73.3 (C-5’’); 72.1 (C3’’ and C-4’’); 71.9 (C-2’’); 17.7 (C-6’’). 2 DAD-CH3 280 n m KAP-F AcOEt-2 45 45 1 40 40 30 25 25 Fraction AcOEt m A U 35 30 m A U 35 20 20 3 15 15 10 10 5 5 0 0 -5 -5 0 5 10 15 20 25 30 35 40 45 50 55 Compound 1 60 Minut es DAD-CH4 KAP-3 450 325 nm 450 800 400 400 350 350 300 300 250 250 200 200 150 150 100 100 800 48.63 Min KAP-3 348 Lambda Max mAU 200 568 m A U 200 0 50 0 400 0 256 50 0 -50 250 -50 0 5 10 15 20 25 30 35 40 45 50 55 300 350 400 450 500 550 600 Compound 2 60 nm Minutes D AD -C H 4 K AP-62-2 140 600 400 mAU m A U 600 325 nm 140 120 100 100 42.70 Min KAP-62-2 260 120 40 20 20 200 100 524 100 0 0 mAU 60 40 m A U 60 mAU 80 m A U 80 349 Lambda Max 200 0 0 250 0 5 10 15 20 25 30 35 40 45 50 55 300 350 400 450 500 550 600 60 nm Minutes DAD-CH3 KAP-AG Compound 3 280 nm 1400 1400 1200 1200 2000 2000 270 4.63 Min KAP-AG Lambda Max 1000 400 400 200 200 1000 1000 563 mAU 600 m A U 800 600 m A U 800 mAU 1000 0 0 0 221 0 250 0 5 10 15 20 25 30 35 40 45 50 55 300 350 400 450 500 550 600 60 nm Minutes Fig.-1: HPLC and UV analysis of sample. HPLC column:Lichro CART® RP-18 (5µm)150x4.6mm; gradient elution: acetonitrile (solvent A) and 0.2% TFA/water (solvent B), detection wavelength: 280 nm. Flow-rate: 0.8mL/min.The ethyl acetate fraction of Santaloides Afzelii leaves was purified by flash chromatography on silica to afford 1 and 2 as crystalline compounds. Compound 2: Myricetin-3-O-rhamnoside; Yellow powder; HPLC Rt 42.70 min ; UV vis max 260.349 nm (methanol) ; The HREI-MS (m/z) 464.0851, C21H20O12 (calcd 464.0853). 1H NMR (400 MHz, DMSO-d6): 12.68 (1H, s, OH-5); 6.38 (1H, d, J = 2Hz, H-6); 6.20 (1H, d, J = 2Hz, H-8); 6.90 (2H, d, J = 2Hz, H-2’/H-6’); 5.20 (1H, d, J = 1.8Hz, H-1’’); 3.89 (1H, dd, J = 3.2Hz/1.8Hz, H-2’’); 3.55 (1H, dd, J = 10.6Hz/3.2Hz, H-3’’); 3.17 (1H, m, H-4’’); 3.34 (1H, m, H-5’’); 0.84 (3H, d, J = 6.1Hz, H-6’’). 13C NMR FLAVONOIDS AND GALLIC ACID FROM CONNARACEAE 334 SORO Yaya et. al Vol. 5 | No.3 | 332-337 | July-September | 2012 (100 MHz, DMSO-d6): 177.7 (C-4); 164.0 (C-7); 161.2 (C-5); 157.4 (C-9); 156.4 (C-2); 145.7 (C-5’ and C-3’); 136.4 (C-4’); 134.2 (C-3); 119.5 (C-1’); 107.8 (C-2’); 107.8 (C-6’); 103.9 (C-10); 101.9 (C-1’’); 98.7 (C-8); 93.5 (CH-6); 71.7 (C-4’’); 70.9 (C-5’’); 70.7 (C-3’’); 70.4 (C-2’’); 17.9 (C-6’’). Compound 3: Gallic acid; HPLC Rt 4.52 min; UV vis max 270 nm (methanol); The ESI-MS m/z 169 [M - H]. RESULTS AND DISCUSSION The HPLC analysis (Figure 1) of the ethyl acetate fraction of the ethanolic extract of the dried powdered leaves of Santaloides afzelii indicated the presence of three (3) majors compounds 1, 2 and 3. A preliminary study of the UV spectra (Figure 1) of compounds 1, 2 and 3 showed absorbance bands at 256/260, 348/349 271 nm respectively, characteristics of phenolic compounds14. Compound 1 The HREI-MS spectrum of compound 1 revealed a molecular ion peaks M+ at m/z 448.0905 corresponding to the molecular formula C21H20O11 (calcd 448.09033). The 1H-NMR spectrum of compound 1 showed an ABX spin coupling system at ppm 7.34 (d, J = 2Hz), 7.32 (dd, J = 8.3Hz/2Hz) and 6.93 (d, J = 8Hz) assigned to H-2’ , H-6’ and H-5’. It also showed an AB spin coupling system of two protons at ppm 6.39 (d, J = 2Hz) and 6.22 (d, J = 2Hz) assigned to H-8 and H-614. The signal at 12.00 showed the presence of the proton of OH group only on carbon C-5. The signal of the anomeric proton of rhamnose at ppm 5.25 showed a doublet with coupling constant J = 1.2 Hz, indicating configuration. The 13C NMR spectrum of compound 1 showed 21 resonances. The DEPT NMR experiment revealed 10 quaternary carbons and 11 primary or tertiary carbons. It showed six carbon signals of a sugar moiety at ppm 103.56, 71.92, 72.12, 72.05, 73.27 and 17.67 assigned to C-1”, C-2”, C3”, C-4”, C-5” and C-6” respectively. The position of rhamnose was also confirmed in HMBC spectrum by observation of a peak between H 5.25 (H-1”) and C 136.25 (C-3). Compound 2 The HREI-MS spectrum of compound 2 revealed a molecular ion peaks M+ at m/z 464.0851 corresponding to the molecular formula C21H20O12 (calcd 464.08525). The 1H-NMR spectrum of compound 2 showed a system of two proton at ppm 6.90 (d, J = 2Hz) corresponding to H-2’ and H-6’. It also showed an AB spin coupling system of two protons at ppm 6.38 (d, J = 2Hz) and 6.20 (d, J = 2Hz) attributed to H-8 and H-6. The signal at 12.68 showed the presence of the proton of OH group only on carbon C-5. The signal of the anomeric proton of rhamnose at ppm 5.20 showed a doublet with coupling constant J = 1.8Hz, indicating configuration. The 13C NMR spectrum of compounds 2 showed 21 resonances. The DEPT NMR experiment revealed 11 quaternary carbons and 10 primary or tertiary carbons. It also showed six carbon signals of a sugar moiety which appeared at ppm 101.86, 70.42, 70.73, 71.72, 70.91 and 17.88 assigned to C-1”, C-2”, C-3”, C-4”, C-5” and C-6” respectively. The position of rhamnose was also confirmed in HMBC spectrum by observation of a peak between H 5.20 (H-1”) and C 134.20 (C-3). The flavones and flavonols present two major absorption bands in the UV analysis in the ranges of 320385 nm (Band I) and 250-285 nm (Band II)15. The substitution of the proton of hydroxyl (in position 3) of a flavonol leads to a hypsochrom effect on band I which shifted to 345 and 365 nm16. The UV spectra of compounds 1 and 2 showed low absorption bands which confirmed the position of rhamnose. The comparison of the UV, H-NMR and EI-MS spectra data with reported values leads to the identification of compound 1 and 2 as quercetin 3-O- -rhamnoside and myricetin 3-O- -rhamnoside respectively17-20. Compound 3 The HPLC-MS-ESI analytical technique showed UV vis max absorbance band at 270 nm (methanol) (Figure 1) and ESI-MS (negative mode) m/z 169 [M - H]. Compound 3 was identified as gallic acid by comparing its retention time, ESI-MS and UV data with standard or reported literature values21-22. FLAVONOIDS AND GALLIC ACID FROM CONNARACEAE 335 SORO Yaya et. al Vol. 5 | No.3 | 332-337 | July-September | 2012 5' HO OH OH 4' 6' 5' 8 9 7 O 3' 2 1' OH 2' 3 10 6 5 OH 4' 6' O 1'' HO 4 OH O O 2'' 5' ' 8 HO 9 7 O 3' 2 1' 4' ' OH 2' 6'' 3'' OH 6 5 OH 4 OH O 1 5 '' 3 10 O HO 1'' O 2' ' 6' ' 3'' 4' ' OH OH 2 HO O HO OH OH 3 Fig.-2: Structure of compounds 1, 2 and 3 isolated from Santaloides Afzelii leaves ACKNOWLEDGEMENTS The authors are very grateful to the “Laboratoire "Synthèse et Réactivité des Substances Naturelles", UMR 6514” of the University of Poitiers (France), the “Programme d’Appui Stratégique à la Recherche Scientifique (PASRES)” for material supports and to the Embassy of Côte d’Ivoire in France for financial support. REFERENCES 1. M. Arbonnier, Trees, Shrubs and Lianas of West African Dry Zones, CIRAD, 279 (2009). 2. C. Wiart, Medicinal Plants of Asia and the Pacific, CRC Press, Taylor and Francis Group, 240-246 (2006). 3. I.O. Ishola, A.J. Akindele and O.O. 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Mohamed, L. Driss, M. Yoko and M. Kazumoto, Actes Inst. Agron.Vet., 21 (3), 157 (2001). 21. A. Mahajan and N. Pai, Journal of Chemical and Pharmaceutical Research, 2 (5), 97 (2010). 22. Y.-Y. Soong and P. J. Barlow, Food Chemistry, 97, 524 (2006). [RJC-965/2012] www.ijcepr.com ISSN: 2229-3892(Print); ISSN: 2229-5283(Online) [Abstracted in : Chemical Abstracts Service , American Chemical Society, USA and CAB(I) , UK] ________________________________________________________________________________________________________ ijCEPr widely covers all fields of Chemical, Environmental and Pharmaceutical Research. Manuscript Categories: Full-length paper, Review Articles, Short/Rapid Communications. 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