77 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) ISSN (Print) 2313-4410, ISSN (Online) 2313-4402 © Global Society of Scientific Research and Researchers http://asrjetsjournal.org/ Isolation of Taxifolin from Trichilia Emetica Whole Seeds Abdullahi Usmana*, Vera Thossb, Muhammed Nur-e-Alamc aDepartment of Chemistry, Nasarawa State University, Keffi. Nigeria. b,cSchool of Chemistry, Bangor University, LL57 2UW, UK. aEmail: ausman2015@yahoo.com Abstract Taxifolin was isolated from the boiled water extract of Trichilia emetica whole seeds. The structure was determined by IR, 1H and 13C NMR and mass spectroscopic methods, as well as comparison of the data obtained with that reported in the literature. Keywords: Trichilia emetica; Taxifolin; Meliaceae; Spectroscopy; Limonoids. 1. Introduction Trichilia emetica Vahl (Also known as Cape, Natal or Woodland Mahogany) belongs to the Meliaceae (Mahogany family). They are evergreen tree reaching 20-35 m in height and are widely distributed in the tropics and the subtropical regions of Africa [1]. Phytochemical studies of the root and stem bark of this plant have led to the isolation of compounds with complex and interesting structures called limonoids (a tetranortriterpenoids) with common name trichilin, others are tannin and kurubasch aldehyde [2, 3, 4, 5, 6, 8]. The leaves, stem and root of T. emetica have been used in African folk medicine for the treatment of chronic wounds, fever, pneumonia, jaundice, gonorrhea, intestinal disorder such as hepatitis, and also to induce labour in women. Additionally, the seed oil is used in combination with Cyathula natalensis Sond to treat leprosy [2, 7, 8]. To the best of our knowledge, the isolation of Taxifolin from the whole seeds of T. emetica is been reported for the first time. ------------------------------------------------------------------------ * Corresponding author. http://asrjetsjournal.org/ American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 21, No 1, pp 77-82 78 2. Materials and Methods Flash chromatography (RevelerisTM Flash Chromatographic System) fitted with Reveleris® C18 column with silica flash cartridges of 18 g (RevelerisTM SRC Cartridges); detection, ELSD (Evaporation Light Scattering Detection) and photodiode array detector (254-280 nm); mobile phase, linear gradient of methanol/water (containing 0.1% formic acid) at a flow rate of 18 mL/min was used for the isolation of the compound. Thin layer chromatography (TLC) and preparative thin layer chromatography (PTLC) was performed on (silica gel 60 F254, 25 Glass plates 20 × 20 cm, E. Merck, Germany). Thermo Instruments HPLC system mass spectrometer with electron spray ionization (ESI) source was used for recording of the mass spectra. Visualization of the compound was done using UV lamp UVL-14 EL hand held 220V 50Hz 4W 254nm white light by UVP. The IR spectrum (KBr) was recorded on a Perkin Elmer 100 FT-IR spectrometer. 1H and 13C NMR spectra were recorded on a Bruker spectrometer at 400.13 and 100.62 MHz using tetramethylsilane (TMS) as an internal standard. 2.1. Preparation of the material 2.1.1. Collection of the material T. emetica seeds were collected from Kumasi, Ghana, in February 2013 and identified by a botanist Mr Martin A. Akoh of Kwame Nkruma University of Science and Technology, Kumasi, Ghana. A voucher specimen TBG- 2014-1 was deposited at the herbarium of Treborth Botanical Garden Bangor, UK. 2.1.2. Extraction T. emetica whole seeds 1.0 kg was reflux in water at 100oC for 30 minute, filtered under gravity using Whatman No. 1 filter paper. The solvent was removed using rotary evaporator at 40oC to give 72.14 g dark viscous residue. The residue was used as the crude extract for the experiment. 2.1.3. Purification The crude extract 72 g was subjected to (450 × 95 mm) column containing 200g of silica gel (20-120 mesh) using the wet packing method in hexane. Subsequently, the column was eluted with a mixture of hexane/chloroform (100:0 and 50:50 V/V) and chloroform/methanol (100:0 to 0:100 V/V). Ninety six (96) fractions were collected, based on their TLC profiles were combined into five primary fractions A-E. All the combine fractions were dried down under vacuum at 40 oC. Fraction B was further purified by column chromatography and prep-TLC. Final purification was carried out by Flash system using 50% methanol in water to give taxifolin (5 mg). 3. Result and discussion Taxifolin was obtained as yellow needles. It has a molecular formula of C15H12O7 which was established on the basis of ESI-HRMS at m/z 305.0660[M + H]+(Calcd for 305.0662) (Fig. 1). The IR spectrum of this compound has a broad band at 3369 cm-1 region corresponding to phenolic and alcoholic O-H stretching. Another band at American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 21, No 1, pp 77-82 79 1638 cm-1 is due to carbonyl and aromatic C=C stretching. The DEPT spectrum (Fig. 2) showed fifteen carbon signals, which consist of seven methine and eight quaternary carbon. Also, the resonance for the keto group appeared at C-4 (δc 198.4). Figure 1: Mass spectrum of taxifolin The 1H NMR spectrum (Fig. 3) showed that the resonance of proton H-2 had been split by proton H-3 resulting in a doublet at δH 4.91 (d, 11.5 Hz), while proton H-3 was split by proton H-2 yielding a doublet at δH 4.51 (d, 11.5 Hz). These protons signals H-2 and H-3 were seen to couple to each other in COSY correlation (Fig. 4). The down field position of the C-2 chemical shifts (δC 85.1) observed in the DEPT spectrum and the appearance of a doublet at 4.91 (d, 11.1 Hz) attributed to position 2, indicate that the flavan moieties possessed the 2,3-trans configuration [9, 10, 11]. The proton spectrum also showed that H-2’ was split by H-6’ in a meta position resulting in a doublet at δH 6.98 (d, 1.3 Hz), proton H-5’ was split by proton H-6’ yielding a doublet at δH 6.81(d, 8.2 Hz) while proton H-6’ was split by proton H-5’ and H-2’ resulting in a doublet of doublet at δH 6.87 (dd, 1.8, 8.2 Hz) attributed to ring B. The proton at H-6 and H-8 split each other in a Meta position and each yielding a doublet at δH 5.90 and 5.94 (d, 1.3 Hz, each) assigned to ring B. 5060708090100110120130140150160170180190200 f1 (ppm) 0 500 1000 1500 2000 250048 .36 48 .57 48 .79 49 .00 49 .21 49 .28 49 .43 49 .50 49 .64 49 .71 73 .64 85 .11 96 .55 97 .19 10 1.8 3 11 5.8 6 11 6.0 7 12 0.8 9 12 9.8 2 14 6.2 8 14 7.1 1 16 4.4 3 16 5.2 2 16 8.6 0 19 8.4 2 C3 C2C2'C5' C6' C1'C3' C4' C4 Figure 2: DEPT spectrum of Taxifolin in CD3OD American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 21, No 1, pp 77-82 80 4.44.54.64.74.84.95.05.15.25.35.45.55.65.75.85.96.06.16.26.36.46.56.66.76.86.97.07.17.2 f1 (ppm) -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 4. 51 4. 54 4. 91 4. 94 5. 90 5. 94 6. 81 6. 83 6. 85 6. 85 6. 87 6. 87 6. 98 6. 98 H3 H2 H8 H6 H5' H6' H2' Figure 3: 1H NMR spectrum of Taxifolin in CD3OD The structure was further confirmed by long-range heteronuclear multiple bond correlation (HMBC) as observed in (Fig. 4), where the ring C methine protons H-2 (δH 4.91) correlated with C-3, C-4, C-1’, C-2’and C- 6’ (δc 116.1); and H-3 (δH 4.51) correlated with C-2, C-4, C-1’ (δc 129.8). Ring B also showed correlations of H-2’ to C-2, C-4’, and C-6’ (δc 116.1); H-5’ to C-2, C-1’ and C-3’ (δc 144.9); and H-6’ to C-2, C-1’ and C-3’ (δc 146.2). The 1H and 13C NMR value obtained in this study is comparable to literature value [14] (Table 1). COSY HMBC OHO OH OH 3 2 45 6 7 8 9 10 1' 3' 4' 5' 6' O OH OH2' A C B Figure 4: HMBC and COSY correlations of Taxifolin This compound has been isolated from the stem of Quercus acuta Thunberg [12], whole plant and fruit of Cayratia japonica [13]. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 21, No 1, pp 77-82 81 Table 1: 1H and 13C NMR data of Taxifolin in deuterated methanol (CD3OD) Source: Wang et al., 2012 [14] 4. Conclusion This is the first report of the isolation and identification of taxifolin from T. emetica. The work was carried out by using different chromatographic separation and spectroscopic techniques. Acknowledgement This study was supported by the Nigerian Tertiary Education Trust Fund (TETFUND). References [1] C. Orwa, A. Mutua, R. J. R. Kindt, A. Simons, 2009. Trichilia emetica. Agroforestree database: a tree reference and selection guide version 4.0. Available at http://www.worldagroforestry.org/af/treedb/. [Accessed July 15, 2013]. [2] B.M. Komane, E.I. Olivier, A.M. Viljoen, Trichilia emetica (Meliaceae) - A review of traditional uses, biological activities and phytochemistry. Phytochem. Lett. 4, 1–9, 2011. [3] M. Traore, L. Zhai, M. Chen, C.E. Olsen, N. Odile, G.I. Pierre, O.J. Bosco, G.T. Robert, S.B. Christensen, Cytotoxic kurubasch aldehyde from Trichilia emetica. Nat. Prod. Res. 21, 13–17, 2007. [4] M. Nakatani, J.C. James, K. Nakanishi, Structure of Limonoid Antifeedant from Trichilia roka. Phytochemistry 24, 195–196, 1985. [5] M. Nakatani, J.C. James, K. 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