International Journal of Agricultural and Food Science

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1 Available online at International Journal of Agricultural and Food Science Universal Research Publications. All rights reserved ISSN Original Article ISOLATION AND SEPARATION OF PHENOLIC COMPOUND FROM CORIANDER FLOWERS Dr. P. Nazni * and R. Dharmaligam Department of Food science and Nutrition, Periyar University, Salem, Tamilnadu, India. raiseandrich@gmail.com * Corresponding author: naznifsn@gamil.com Received 16 January 2014; accepted 21 January 2014 Abstract The present study is carried out with coriander flowers; A simple and fast method was developed for simultaneous quantitative determination of three biologically active phenolic compounds such as apigenin, quercetin, keamphferol in flowers of Coriandrum sativum separation using by flash column chromatography then purification were conducted by preparative chromatography and HPLC. The phenolic compound determine with different spectroscopic method such as LC-MS, HC NMR, FTIR and Melting point. The present method is being reported first time and may be used for routine quality control of the flowers of Coriandrum sativum Universal Research Publications. All rights reserved Key words: Apigenin, Quercetin, Keamphferol, Chromatography. Introduction effects as well as their potential estrogenic and anticancer Extracts of aromatic herbs, spices, and medicinal plants are activities (Springob and Saito, 2002). Quercetin belongs to used in food and pharmaceutical processing to impart this group of plant pigments called flavonoids that are flavor or other functional properties, as well as ingredients largely responsible for the colours of many fruits, flowers in the pharmaceutical products. Plant based natural and vegetables. Quercetin works as anti-inflammatory, constituents can be derived from any part of the plant like antioxidant, anticancer agents. (Lamson and Brignale bark, leaves, flowers, roots, fruits, seeds, etc (Gordon and (2000) and Mahesh Chand Meena and Vidya Patni 2008). David, 2001) i.e. any part of the plant may contain active Flavonoids possess a variety of biological activities that are components. The beneficial medicinal effects of plant thought to contribute towards protecting humans against materials typically result from the combinations of degenerative diseases. Quercetin, kaempferol and secondary products present in the plant. The medicinal isorhamnetin have been shown to have an antiinflammatory actions of plants are unique to particular plant species or effect on activated macrophages. In addition, groups and are consistent with this concept as the quercetin and kaempferol show chemopreventive properties combination of secondary products in a particular plant is in brain tumours (medulloblastoma) and synergistically taxonomically distinct (Wink, 1999). They are usually suppress cell proliferation in human gut cancer lines. subdivided according to their substituents into flavanols Quercetin a main aglycone in human nutrition is a potent (kaempferol, quercetin), anthocyanins, flavones, flavonones free radical scavenger and antioxidant, which is why it is and chalcones. These flavonoids display a remarkable array thought to protect humans against several types of cancer of biochemical and pharmacological actions viz.,anti - and cardiovascular diseases. Antioxidant effects of inflammatory, antioxidant, anti -allergic, hepatoprotective, quercetin on the mucosa of the nasal turbinate were antithrombotic, antiviral and anticarcinogenic activities demonstrated. Higher intakes of kaempferol resulted in a (Middleton and Kandaswami, 1993). These compounds lower risk of coronary heart disease. In addition, appear to play vital roles in defence against pathogens and isorhamnetin revealed distinct vasodilatory effects in predators and contribute to physiological functions such as animal models, suggesting vascular protective effects in seed maturation and dormancy (Winkel-Shirley, 2002). human cardiovascular diseases. (Susanne Schmidt1, They are synthesized from phenyl propanoid and acetate Michaela Zietz). derived precursors. Flavonoids are important for human Flavonoids are polyphenolic compounds that occur beings due to their antioxidative and radical scavenging ubiquitously in plant foods. Flavonols and flavones are 13

2 subclasses of flavonoids (Kühnau J 1976, Markham KR 1989, Herrmann K 1988, and 1979). In 1992 the average daily intake of the flavonols quercetin, kaempferol, and myricetin from the Dutch diet was 16, 4, and 1 mg, respectively; the average intake of the flavones apigenin and luteolin was 1 mg each (. Hertog MGL et al 1993). The intake of these five dietary flavonoids was associated with a reduced risk for ischemic heart disease and stroke in several (Keli SO et al 1996, Knekt P et al 1996 Hertog MGL, ,) although not all (Rimm ER, et al 1996, Hertog MGL, et al 1997 ), epidemiologic studies. Two types of mechanisms have been proposed to explain this protective effect: inhibition of LDL oxidation and inhibition of platelet aggregation (Hertog MGL and Hollman PCH et al 1996). Results obtained by incubation of human platelets or animal cells with isolated isolated flavonoids suggest that flavonoids inhibit platelet aggregation probably by inhibition of cyclooxygenase activity (P Mensink1997). In addition, many studies have demonstrated the potential of plant products as antioxidants against various diseases induced by free radicals (Hou et al., 2003), and it has been determined that the antioxidant effect of plants is mainly attributed to phenolic compounds, such as flavonoids, phenolic acids, tannins, and phenolic diterpenes (Pietta, 2000). The aim of the present study is to deals with the isolation and identification of flavonoid "quercetin" keamphferol and apigenin from coriander flower. Materials and Methods Plant materials The plant materials of C. sativum were collected from the plants grown in and around Salem district, Tamilnadu, India. The plant materials were dried in shade separately. Reagents Reagents and chemicals Methanol and acetic acid were of HPLC grade and were purchased from Merck Company. Deionized water was prepared by a Milli-Q Water Purification system. Extraction and separation of crude sample The dried samples (flower 250 g) were chopped into small pieces and extracted with methanol: water (80:20) by soaking for 48 Hrs at room temperature (25 C). The methanolic extract was decanted, filtered under vacuum, concentrated in a rotary evaporator (40 C.). The resulting crude extract from the flower of CF (25.0g) was fractionated successively with ethyl acetate (EtOAc), n- butanol (BuOH), and the yield of soluble fractions of EtOAc (2.0g), BuOH (1.0g), and water (20.g). (Janmejai K Srivastava et. al 2009, Tereschuk et.al. Naira Nayeem and Karvekar MD 2010). The ethyl acetate (methanol) soluble fraction from flower of CF (2.0g) was loaded into a flash chromatography column (silica gel for mesh revelries grace column silisep) and eluted with different solvent system for gradient programme (like, EtOAc/nhexane and MeOH/chloroform solvent systems). For this separation were separated by 5 sub fractions (EA5 EA10 and methanol: chloroform 7-10). Frequently each fraction was checked with TLC. S. (ĐORĐEVIĆ, M. CAKIĆ, S. AMR 2001, hui-ling cheng et al 2013 and jolanta nazaruk and jan gudej 2001) for different mobile phase to getting single spot using by iodine vapour (solvent system for t1and t2 chloroform: methanol; acetic acid; water 95:5;5:2 and t3). Analysis of Crude Extract by HPLC The resultant flavonoid aglycons were qualified by using reversed-phase HPLC series 6330 from Agilent technologies. Solvent A consisted of 100% water and 0.5% acetic acid; solvent B was 100% acetonitrile. The following gradient was used for eluent B: 5 7% (0 12 min), 7 9% (12 25 min), 9 12% (25 45 min), 12 15% ( min), 15% isocratic ( min), 15 50% ( min), 50% isocratic ( min), 50 5% ( min), 5% isocratic ( min) ( Susanne Schmidt et.al 2010). The flow rate used was 0.8mL/min, and the measured detector wavelengths were set at 280, 320, 330, 340 and 370 nm. Purification of phenolic compound by using Pre-HPLC The fraction sample collected from chloroform methonal solvent system, collected sample purified through semi preparative HPLC (using a PU-2080 pump (Jasco, Japan) equipped with a MD-2010 multi wave length detector (Jasco, Japan) and a mm i.d., 4- lm Synergi Polar-RP column (Phenomenex, Torrance, CA). The mobile phase was solvent A, 100% ACN; and solvent B, ultrapure water. Elution condition was 0 80 min of % A B (linear gradient) at a flow rate of 4 ml/min (Shang-Tse Ho et.al., 2010). The structures of compounds were identified by LC-MS (Agilent 6330 Ion Trap ),FT-IR and NMR (Varian Unity Inova-600, USA), and all spectral data were consistent with the literature (Baderschneider & Winterhalter, 2001; Jiang et al., 2001; Villegas & Kojima, 1985; Yoshida, Ahmed, Memon, Okusa, 1991& Shang-Tse Ho et.al., 2010, Nedime Durust et.al 1999 and). Purified CF flower extract samples were subjected to LC- MS and NMR for the structural elucidation and identification of constituents were performed, three compounds were separated at different retention time. Moreover the particular compounds were determined by FTIR and DSC. Result and discussion The fresh flower harvested to under our study director guidelines and dried with room temperature. The dried material soaking with methanol: water (80:20) for 24hrs. After completion of soaking filter to whattmen paper and concentrated by rotary vapor at 40 c. The crude sample subject to inject HPLC and separated to all peak using different gradient programmed based on that separation to developed for column chromatography method. The methanolic extract was subjected to column chromatography on silica gel using solvents of increasing polarities starting from petroleum ether, chloroform, ethyl acetate and methanol in different ratios to yield several Sub-fractions. Fractions (40% chloroform in methanol) were mixed due to their similar TLC pattern this fraction was coded as T1corander flower. The solvent system methanol: acetic acid: water in the ratio 95:5:5. This fraction compound 1 was eluted with chloroform and methanol in different ratios to get fractions of 20ml.Fraction 5 showed a single spot. This fraction was collected dried and was recrystallized using methanol to get 14

3 0.20mg of compound 1 which was identified as keampferol (Jolanta Nazaruk and Jan gudej 2001). Compound 2 was isolated from fractions (10% methanol in ethyl acetate) and subjected to TLC using the solvent system methanol: acetic acid: water in the ratio 5:3:5 which showed a single spot of the targeted compound.the column was then eluted using methanol in ethyl acetate in different ratios and fractions of 10 ml were collected. Fraction 7 and 10 of the second column of compound 2 showed single spot which was sensitive at UV 254. These fractions were collected mixed concentrated and left overnight to obtain 0.12 mg pure compound 2 which was identified as quercitin. (Hao Liu et.al., 2010, Fathy M. Soliman et.al., 2002, Naira Nayeem, Karvekar MD 2010, Jolanta Nazaruk and Jan Gudej2001). Compound 3 was isolated from fractions % (20% methanol in ethyl acetate). The solvent system ethyl acetate: metahnol: formic acid in the ratio 95:5:5showed a major spot.the fractions compound 3 was eluted using the ethyl acetate and methanol in different ratios. Fractions of 20ml each were collected. The compound started to elute at 15% methanol in ethyl acetate. This was then allowed to stand overnight. Yellowish color crystals were obtained. This was further purified and recrystallized the yield of this fraction 0.15 mg. Compound 3 identified as apigenin (jolanta nazaruk and jan gudej 2001). The known flavonoids: Apigenin, keampferol and qucertin were identified by extensive UV spectrum analyses and NMR spectroscopic analyses as well as by comparing their spectroscopic data with those reported in the literature comparison of the melting point(dsc), LC-MS, IR and l3c NMR&1H NMR spectra with literature data. The purified compounds were injected to HPLC. The gradient elution of solvent A [water-acetic acid (25:1 v/v)] and solvent B (Acetonitrile) had a significant effect on the resolution of compounds. As a result, solvent gradients were formed, using dual pumping system, by varying the proportion of solvent A [water-acetic acid (25:1, v/v)] to solvent B (methanol). Solvent B was increased to 50% in 5 min and subsequently increased to 80% in 10 min at a flow rate of 1.0 ml/min. Detection wavelength was 254, 280,360,560 nm (Susanne Schmidt1, Michaela Zietz 2010). LC- Ms parameter jolanta nazaruk and jan gudej Table 1 showed the phenolic compounds identified in coriander flower powder. They are arginine, qucertin, keampferol. The HPLC peak purity per cent of all the 3 compounds are respectively 95%, 96% and 97%. Melting points are found to be , , and This table also provides mass m/2, UV spectrum nm of all the three compounds. In nature apigenin also exists as a dimer, biapigenin, mainly isolated from the buds and flowers of Hypericum perforatum, which has neuroprotective effects (Cheung Z.H et al., 2008). Apigenin is abundantly present in common fruits such as grapefruit, plant-derived beverages and vegetables such as parsley, onions, oranges, tea, chamomile, wheat sprouts and in some seasonings. One of the most common sources of apigenin consumed as single ingredient herbal tea is chamomile, prepared from the dried Flowers from Matricaria chamomilla (McKay DL, Blumberg J.B, 2006 and Sanjeev Shukla and Sanjay Gupta 2010). The structure and the presence of free hydroxyl groups in the various phenolic compounds make them an important class of compounds. The chemical activities of these phenolic compounds in terms of their reducing properties as electron or hydrogen donating is important for their antioxidant activity (Hui-Ling Cheng et al., 2013).The extract possess significant wound healing, analgesic and antiinflammatory activity (Majumdar M et al.,2007, Nayeem N and Karverkar MD 2010). This could be due to the nature of the phytoconstituents present in the plants like phenolic compounds, tannins sterols etc. This resulted in the isolation of apigenin, keampferol and quercitin. For this study was analysed melting point of the compound and The DSC peak purity compare with HPLC peak purity. The figures (1a-1f) of the compound were identified and determined by using LC-MS, FTIP, NMR and DSC methods. The obtained compound was off white powder with a melting point of C. Analysis by mass spectroscopy (ESI) gave molecular mass at m/z. The IR, NMR, melting point and the chemical test of compound 1 can be correlated to the available literature data of the flavonoid keampferol. Figure -1a - Compound-1 a LC-MS Figure 1b -Compound-1 b FTIR 15

4 Figure 1 c -Compound-1 c 1H NMR Figure 1 d- Compound-1 d 13C NMR band is found, at approx. 3425cm-1, and the other at approx that is most probably the result of νoh vibrations of phenol OH groups. The other most important set of bands are the aromatic ring vibrations centered around 1600 and 1500 cm 1, which usually appear as a pair of band structures, often with some splitting. the C=C band vibrations of C=C group from the aromatic ring,the C=C vibration should occurs at approxy.1508,1454 and 1442( stretch)cm (for confirming aromatic C-H peaks slightly above 3000 cm. its occurring in spectrum rang 2995 and 2926 cm ). The appearance and ratio of these band structures is strongly dependent on the position and nature of substituents on the ring. The above spectral characteristics indicate with high probability that the compound is kaempferol(gangwal A. et al., 2010 and Shafaghat, F. Salimi 2008) The figures (2a-2f) of the compound were identified and determined by using LC-MS, FTIP, NMR and DSC methods. The obtained compound was off white powder yellow colour with a melting point of C. Analysis by mass spectroscopy (ESI) gave molecular mass at m/z. The IR, NMR, melting point and the chemical test of compound 2 can be correlated to the available literature data of the flavonoid Qucetcetin. Figure 1 e -Compound-1 e DSC Melting point and Purity by DSC FTIR (KBr):3425, 3322,2955, 2926,2853, 2616,1660, 1613, 1569, 1508, 1454, 1442,UVmax (methanol) : 366,264,266 nm. 1H-NMR(dmso-d6 400 MHz)δ 6.1 (1H,d, J=2.0 Hz,H-6),6.4(1H,d, J=2.0 Hz,H-8),8.0 (2H,dd, J1=2,j2=8, Hz,H-3 and H-5 ),6.9 (2H,dd, J=2,j=8.0 Hz,H- 2 and H-6 ),12.4(1h,s,h-4 h-7),10.1(1h,s,h-5),9.4(1h,s,h- 4). 13C-NMR: (MeOH 300 MHz)δ158(C-8), 160 (C-6), 150(C-10), 130(C-3 and C-5 ), 109(C-1 ), 116(C-2 andc- 6 ), 145(C-3), 139(C-2), 165(C-9), (C-4 ), 123(C-5), 162(C-7), 177(C-4) (Fatma A. Ahmed et al 2011,v 2013). The 1H-NMR spectrum of compound V displayed the characteristic signals of the kaempferol nucleus: two doublets at δ 6.1 and 6.4 ppm (J =2.3Hz), assigned to the H-6 and H-8 protons respectively and a pair of A2B2 aromatic system protons at δ 6.86 and 7.97 ppm (J =8.0 Hz), assigned to H-3 and 5 and H-2,6 respectively. A 100% base peak [M]- for compound was observed at m/z in the mass spectrum indicating the compound as kaempferol. 13CNMRsignals were found to be in agreement with reported values. The molecular formula was deduced from 1H, 13C-NMR and mass spectrometry. On the basis of spectral evidence, the structure was decided to be kaempferol (C15H10O6, 286.6). FTIR spectr m cm that is probably the result C=C vibrations of olifinic structure from that spectrum occurring approx. at 1660 cm( strong C=C stretch absorption band). In the spectrum of νoh vibrations two a Figure 1 f- Compound-1 f HPLC Chromatogram Figure 2 a- Compound-2 a LC-MS Figure 2 b - Compound-2 b FTIR 16

5 Figure 2 c - Compound-2 c DSC thermogram Melting point and Purity by DSC Figure 2d - Compound-2 d HPLC Chromatogram range between 1600 and 1400 cm-1, which denotes the axial deformation of the aromatic C=C. IR spectrum showed characteristic peak position of active ingredientquercetin.3411 cm :O-H stretching vibration of phenol,1663 cm :C=O aryl ketonic stretch,1612 cm 1522cm,1462,1450,1408:c-c aromatic ring stretch, cm band region occurring for Phenol or tertiary alcohol, OH bend. the intensive band its showing approx. at 1383 cm :in plane bending of O-H bond in aromatic hydrocarbon,1264 cm :C-O stretch of aryl ether, 1200cm:c-o stretch of phenol, 1169 cm: C-O-C stretch and bending in ketone,942,824,681,604cm:out of plane C-H bending of aromatic hydrocarbon group of alcohol respectively, present in the molecular of quercetin(. Z uhal g uvenalp, l. om ur demirezer 2005, Aarti chourasiya et al.,2012, K.Selvaraj et al., 2013, Milena Kalegari et al., 2011, silverstein et al., 1994). This is an important fact, because the chemistry and oxidation stability of the alcohol are strongly influenced by the degree of substitution. Whether an alcohol is primary (1 ), secondary (2 ) or tertiary (3 ), may be reflected in the position of the OH stretch absorption, but typically this is determined by the other absorptions, in particular the C-Ostretching frequency. Absorption of lower importance, but often characteristic is assigned to another form of bending vibration, the out-of-plane bend or wagging vibration of the O-H. The OH bendingvibrations are broadened by hydrogen bonding as is the stretching absorption, but often to a lesser extent. The figures (3a-3f) of the compound were identified and determined by using LC-MS, FTIP, NMR and DSC methods. The obtained compound was off white powder with a melting point of C. Analysis by mass spectroscopy (ESI) gave molecular mass at m/z. The IR, NMR, melting point and the chemical test of compound 3 can be correlated to the available literature data of the flavonoid apigenin. Figure 2 e- Compound-2 e 13C NMR The 1H- and 13C-NMR spectra of compound 2 exhibited resonances due to aromatic systems. In the 1H-NMR spectrum of 2, the aromatic region exhibited an ABX system at δ 7.6 (1H, d, J = 2.0 Hz, H-2 ), 7.62 (1H, dd, J = 2.2 and 2.2 Hz, H-6 ), and 6.8 (1H, d, J = 8.48 Hz, H-5 ) due to a 3, 4 disubstitution of ring B and a typical metacoupled pattern for H-6 and H-8 protons (δ 6.1and 6.3, d, J = 2.0 Hz). The 13C NMR spectrum showed the presence of 15 aromatic carbon signals. Based on the NMR data and comparison of the data given in the literature, the structure of compound 2 was identified as quercetin.the melting point and other recorded properties of the isolated constituents were presented.melting point of quercetin as 325 C. The infrared spectrum revealed two absorption bands in 3409and 3324 cm-1 that typically result from the OH link axial deformation. Moreover, the existence of an aromatic ring may be observed, evidenced by a set of bands that Figure 2f - Compound-2 f 1H NMR Figure 3 a -Compound-3 a LC-MS In the 1H NMR spectrum of 3, the aromatic proton signals of two m-coupled doublets at δ H 6.4 and 6.1(each,J =

6 Hz) showing HMQC correlations to the carbon resonances at δ C (d) and C (d),and 7.9 (3 and5 ) and 6.9(2 and 6 )showing doublets(each j=12.1hz). Phenolic Compounds from Scutellaria pontica, T. ERS OZ, et al., respectively, were attributed to the H-6 and H-8 of the A- ring. Two vicinally coupled doublets at δ H 7.9 and 6.9 (each, 2H, J = 12.1 Hz) showed long-range couplings with the 13C NMR signal at δ C (C-4 ) and, therefore, were assigned to H-2 /6 and H-3 /5, respectively, of the B-ring. Additionally, a singlet at δ H 6.7 was ascribed to H- 3. The assignment of H-3 was confirmed by its heteronuclear long-range correlations to C-2 (δ C 121.6) and C-1 (δ C 121.6). The 13C NMR resonance at δ C which showed HMBC correlations with H-6 and H-8 was attributed to C-7. Based on the above-mentioned data and comparison of 1H and 13C NMR data with those given in the literature, the structure was identified as apigenin. For both flavones the vicinal CH couplings in Ring B obey the following rules: ca. 8-9 Hz (Cq-CH-CH pathway); ca. 7 Hz (CH-Cq-CH or Cq-COH-CH); ca. 3-5 (CH-COH-CH)( Tayfun ERS OZ et al., 2002, Wild.TIAN Ying et al.,2009, T.J. Mabryet et al., 1970, K.R. Markham 1982, J.B. Harborne and T.J. Mabry 1982). Table: 1 Compound name HPLC peak Meting point by Peak purity by Mass Uv-spectrum purity% DSC ºC DSC % m\z Nm Apigenin 95% ,266,336 Qucertin 96% ,254,230,228 keampferol 97% ,264,266 Figure 3 b- Compound-3 c FTIR Figure 3c -Compound-3 DSC Thermogram melting point and DSC by purity Figure 3d -Compound-3 c HPLC Chromatogram Figure 3e -Compound-3 d 1H NMR Figure 3f- Compound-3 e 13C NMR The other most important set of bands are the aromatic ring vibrations centered around 1600 and 1500 cm1, which usually appear as a pair of band structures, often with some splitting. The appearance and ratio of these band structures is strongly dependent on the position and nature of substituents on the ring. In the spectrum of νoh vibrations one bands (stretchbonded broad strong OH alcohol) are found, one at approx cm- 1.the intensive band at cm is probably the result of c-h vibration of aryl group from the aromatic ring, c-h band at occurs in approx.3095 cm. The intensive band at 1609 cm-1 is most probably the result of νc=o vibrations of C=O group from the central heterocyclic ring, while the νc-o vibration should occur at approx cm-1(strong). The other most important set of bands are the aromatic ring vibrations centered around 1600 and 1500 cm1, which usually appear as a pair of band structures, often with some splitting. The C=C band vibrations of C=C group from the aromatic ring, the C=C vibration should occurs at approxy.1502, 1444 and 1400(stretch) cm (for confirming aromatic C-H peaks slightly above 3000 cm. its occurring in spectrum rang3095 cm). The appearance and ratio of these band structures is strongly dependent on the position and nature of substituents on the ring. The above spectral characteristics indicate with high probability that the compound is apigenin(jordan Siniša Đorđević et al., 2000, Charlie Corredor a etal., 2009, Mabry, T. J., et al., 19 18

7 Conclusion For this article were studied and identified flavonoids and phenolic acid etc., compare to this leaves, flower are also having same compounds,there is no any significant changes from this plant. Methanolic extract of coriander flower Out of ten fractions have been isolated instead of these only three and purified and achieved above 95% purity. And moreover, HPLC peak purity were compare to DSC peak purity, it s more or less counterpart to HPLC peak purity.a significant phenolic content in agreement with good radical Scavenging activity and therapeutically value for health. References 1. Mahesh Chand Meena and Vidya Patni Isolation and Identification of Flavonoid "Quercetin" from Citrullus colocynthis (Linn.) Schrad. Asian J. Exp. Sci., Vol. 22, No. 1; Susanne Schmidt1, Michaela Zietz, Monika Schreiner1, Sascha Rohn, Lothar W. Kroh and Angelika Krumbein Identification of complex, naturally occurring flavonoid glycosides in kale (Brassica oleracea var. sabellica) by high-performance liquid chromatography diode-array detection/ electrospray ionization multi-stage mass spectrometry. 3. Shang-Tse Ho, Yu-Tang Tung, Kai-Chung Cheng, Jyh-Horng Wu Screening, determination and quantification of major antioxidants from Balanophora laxiflora flowers, Food Chemistry, 122; Pietta, P. G Flavonoids as antioxidants. Journal of Natural Products, 63, Haslam E Practical Polyphenolics: From Structure to Molecular Recognition and Physiological Action. Cambridge: Cambridge University Press. 6. Harborne JB The Flavonoids: Advances in Research Since London: Chapman & Hall. 7. PLTM Karin Janssen, Ronald P Mensink, Frank JJ Cox, Jan L Harryvan, Robert Hovenier, Peter CH Hollman, and Martijn B Katan Effects of the flavonoids quercetin and apigenin on hemostasis in healthy volunteers: results from an in vitro and a dietary supplement study Kühnau J The flavonoids. A class of semiessential food components: their role in human nutrition. World Rev Nutr Diet; 24: Markham.KR Flavones, flavonols and their glycosides. Methods Plant Biochem; 1: Herrmann K On the occurrence of flavonol and flavone glycosides in vegetables. Z Lebensm Unters Forsch; 186: Herrmann K Flavonols and flavones in food plants: a review. J Food Technol; 11: Hertog MGL, Hollman PCH, Katan MB, Kromhout D Intake of potentially anticarcinogenic flavonoids and their determinants in adults in The Netherlands. Nutr Cancer; 20: Keli SO, Hertog MGL, Feskens EJM, Kromhout D Dietary flavonoids, antioxidant vitamins, and incidence of stroke. Arch Intern Med; 156: Knekt P, Reunanen RJ, Maatela J. Flavonoid intake and coronary mortality in Finland: a cohort study. Br Med J 1996; 312: Hertog MGL, Kromhout D, Aravanis C, et.al Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries. Arch Intern Med; 155: Hertog MGL, Feskens EJ, Hollman PCH, Katan MB, Kromhout D Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet; 342: Rimm ER, Katan MB, Ascherio A, Stampfer MJ, Willett WC Relation between intake of flavonoids and risk for coronary heart disease in male health professionals. Arch Intern Med; 125: Hertog MGL, Sweetnam PM, Fehily AM, Elwood PC, Kromhout D Antioxidant flavonols and ischemic heart disease in a Welsh population of men: the Caerphilly Study. Am J Clin Nutr; 65: Hollman PCH, Hertog MGL, Katan MB Role of dietary flavonoids in protection against cancer and coronary heart disease. Biochem Soc Trans; 24: Hertog MGL, Hollman PCH Potential health effects of the dietary flavonol quercetin. Eur J Clin Nutr; 50: Gordon M.C. and David J.N Natural product drug discovery in the next millennium. Pharm. Biol., 39, Lamson D.W. and Brignale M.S Antioxidants and cancer III: quercetin. Alt.Med. Rev., 5, Middleton E. and Kandaswami C The impact of plant flavonoids on mammalian 24. Biology: Implications for immunity 25. Wink M Introduction Biochemistry, role and biotechnology of secondary products. In: M Wink, Ed, and Biochemistry of Secondary Product Metabolism. CRC Press, Boca Ratom, FL, pp Winkel-Shirley, B Biosynthesis of flavonoids and effects of stress. Curr. Opin. 27. Plant Biol., 5, Springob K. and Saito K Metabolic engineering of plant secondary metabolism: Promising approach to the production of pharmaceuticals. Sci. Cul., 68, Long-Ze Lin, James M. Harnly Identification of the phenolic components of chrysanthemum flower (Chrysanthemum morifolium Ramat) Food Chemistry 120; Hao Liu, Yan Mou, Jianglin Zhao, Jihua Wang, Ligang Zhou, Mingan Wang Daoquan Wang, Jianguo Han, Zhu Yu and Fuyu Yang Flavonoids from Halostachys caspica and Their Antimicrobialand Antioxidant Activities Molecules, 15, ; doi: /molecules Fathy M. Soliman, Afaf H. Shehata, Amal E. Khaleel and Shahera M. Ezzat An Acylated Kaempferol Glycoside from Flowers of Foeniculumvulgare and F. DulceMolecules, 7, François N. Muanda, Rachid Soulimani, Amadou Dicko Study on biological activities and chemical composition of extracts from Desmodium adscendens leaves Journal of Natural Products, Vol. 4,

8 33. Naira Nayeem, Karvekar MD Isolation of phenolic compounds from the methanolic extract of Tectona Grandis April June, RJPBCS Volume 1 Issue Kamran G, Yosef G,Mohammed Ali. Pak J Pharm Sci 2009; 22(3) Sarvanakumar A,Venkateshwaran K,Vanitha J,Ganesh M et al. Pak J Pharm Sci 2009; 22(3): Marian N,Fereidoon S. J Chroma A 2004;1054: Majumdar M, Nayeem N, Kamath JV, Asad M. Pak J Pharm Sci 2007; 20(2): Nayeem N and Karverkar MD. Internet J Pharmacol 2010; 8 (1). 39. Fatma A.Ahmed Inas M.Abd Ei-wahab khamis and samar y.desoukey flavonoid of neotorularia aculeolata plant jouranal of pharmacy and nutrition science. 2011, 1, Rutinoside and vicianoside of quercetin,apigenin and luteolin from symphorcarpus albus l. blanke (caprifoliaceae)wieslawa bylaka and irena matlawskadrug research vol 56 no.3 pp Muhammad Khalid Saeed, Yulin Deng, Zahida Perveen, Waqar Ahmad, Rongji Dai And Yuhong Yu Optimal recovery of Apigenin from Torreya grandis by extraction, fractionation and structure elucidation Proceedings of the 2006 WSEAS Int. Conf. on Cellular & Molecular Biology, Biophysics & Bioengineering, Athens, Greece, July 14-16, pp Nawal, H. Mohamed and Atta, E. M Cytotoxic and Antioxidant Activity of Marrubium vulgare and its Flavonoid Constituents 2 nd International Conference on Chemical, Environmental and Biological Sciences (ICCEBS'2013) March 17-18, 2013 Dubai (UAE) 43. antiinflamatory and antioxidant flavonoid and phenols from cardiospermum halicacabum.hui-ling cheng et al., journal of traditional and complementary medicine vol.3 no.1 pp Medicinal benefits of coriander (Coriandrum Sativum L) Kişnişin (Coriandrum Sativum L) Faydaları sapuula Ullagaddi Rajeshwari, Bondada AndalluTıbbi dd (1): encyclopedia.com [Access date: ]. 46. Sanjeev Shukla and Sanjay Gupta Apigenin: A Promising Molecule for Cancer Prevention Pharm Res June ; 27(6): Doi: /s McKay DL, Blumberg JB. A review of the bioactivity and potential health benefits of chamomile tea (Matricaria recutita L.). Phytother Res 2006; 20: [PubMed: ] 48. Cheung ZH, Leung MC, Yip HK, Wu W, Siu FK, So KF A neuroprotective herbal mixture inhibits caspase-3-independent apoptosis in retinal ganglion cells. Cell Mol Neurobiol; 28: The Isolation of Carboxylic Acids from the Flowers of Delphinium formosum Received Nedime D UR UST_ et al Susanne Schmidt, Michaela Zietz, Monika Schreiner1, Sascha Rohn, Lothar W. Kroh and Angelika Krumbein Identification of complex, naturally occurring flavonoid glycosides in kale (Brassica oleracea var. sabellica) by high-performance liquid chromatography diode-array detection/electrospray ionization multi-stage mass spectrometry. 51. Shang-Tse Ho, Yu-Tang Tung, Kai-Chung Cheng, Jyh-Horng Wu Screening, determination and quantification of major antioxidants Food Chemistry 122 (2010) from Balanophora laxiflora flowers 52. Siniša Đorđević1, Milorad Cakić1, Salameh Amr the Extraction of Apigenin and Luteolin from the Sage Salvia Officinalis L. FROM JORDAN Janmejai K Srivastava and Sanjay Gupta Extraction, Characterization, Stability and Biological Activity of Flavonoids Isolated from Chamomile Flowers Mol Cell Pharmacol. January 1; 1(3): María L. Tereschuk, Mario D. Baigorí, Lucia I. C. de Figueroa, and Lidia R. Abdala Flavonoids From Argentine Tagetes (Asteraceae) With Antimicrobial Activity Methods in Molecular Biology, vol Jolanta Nazaruk and Jan Gudej.2001.Qualitative and Quantitiescharomatographicinvestigation of flavonoids in bellis perennis l., durg reach vol.58 no.5 pp Triterpenoid, flavonoids and sterols from Lagenaria siceraria fruits Gangwal A., Parmar S. K., Sheth N. R. Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, India Der Pharmacia Lettre, 2010: 2 (1) ( archive.html) 58. Shafaghat, F. Salimi Extraction and Determining of Chemical Structure of Flavonoids in Tanacetum parthenium (L.) Schultz. Bip. from IranJ. Sci. I. A. U (JSIAU), Vol 18, No. 68, Summer Flavonol Glycosides from Asperula arvensis L. Z uhal G UVENALP, L. Om ur DEMIREZER Turk J Chem 29 (2005), 163 {169.C T UBITAK. 60. Aarti chourasiya, Anil Upadhayay, R.N. shukla. To Assess Isolation of quercetin- from the leaves of Azadirachta indica and antidiabetic study of The crude extracts. Journal of pharmaceutical and biomedical sciences (J Pharm Biomed Sci.) 2012, December; 25(25); (Article no 11). 61. K. Selvaraj, Ranjana Chowdhury, Chiranjib Bhattacharjee.Isolation and structural elucidation of flavonoids from aquatic fern azolla microphylla and evaluation of free radical scavenging activity International Journal of Pharmacy and Pharmaceutical Sciences ISSN Vol 5, Suppl 3, Phytochemical constituents and preliminary toxicity evaluation of leaves from Rourea induta Planch. (Connaraceae) Milena Kalegari, Marilis Dallarmi Miguel, Josiane de Fátima Gaspari Dias, Ana Luísa Lacava Lordello, Cristina Peitz de Lima, Cristina Mayumi SasakiMiyazaki, Sandra Maria Warumby Zanin1, Maria Christina dos Santos Verdam1, Obdulio Gomes MiguelBrazilian Journal of Pharmaceutical Sciences vol. 47, n. 3, jul./sep., Silverstein, R.M.; Bassler, F.X.; Morril, T.C. Identifi- 20

9 cação espectrométrica de compostos orgânicos. 5.ed. Rio de Janeiro: Guanabara Koogan, P.K. Agrawal, \Carbon-13 NMR of Flavonoids", Elsevier Science, New York, (1989). 65. J.B. Harborne, \The Flavonoids", Chapman & Hall, (1994). 66. Interpretation of Infrared Spectra, A Practical Approach John Coates John Wiley & Sons Ltd, Chichester, Phenolic Compounds from Scutellaria pontica Tayfun ERS OZ, U. Sebnem HARPUT, Iclal SARACOGLU, Ihsan CALIS Turk J Chem 26 (2002), Apigenin glycosides from Euphorbia humifusa Wild.TIAN Ying, LIU Xi-qiao, DONG Jun-xing Acta Pharm Sin, 2009, 44: T.J. Mabry, K.R. Markham and M.B. Thomas, \The Systematic Identi_cation of Flavonoids", Springer- Verlag, Berlin (1970). 70. K.R. Markham, \Techniques of Flavonoid Identi_cation", Academic Press, London (1982). 71. J.B. Harborne and T.J. Mabry, \The Flavonoids: Advances in Research", Chapman and Hall Ltd., London (1982). 72. The extraction of apigenin and luteolin from the sage salvia officinalis l. from jordan Siniša Đorđević, Milorad Cakić, Salameh Amr Working and Living Environmental Protection Vol.1,No5,2000, pp Raman and surface-enhanced Raman spectra of chrysin, apigenin and luteolin Charlie Corredor a, Tatyana Teslova a,1, Maria Vega Can amares a, Zhanguo Chen a, Jie Zhang a, John R. Lombardi a, Marco Leona Vibrational Spectroscopy 49 (2009) Mabry, T. J., K. R. Markham and M. B. Thomas, The Systematic Identification of Flavonoides, Springer Verlag, New York-Heidelberg-Berlin Source of support: Nil; Conflict of interest: None declared 21