Isolation and characterization

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Isolation and characterization

Chapter V Isolation and Characterization of Flavonoid Compound from Feronia Limonia

Dept. of Biotechnology, Gulbarga University, Kalaburagi

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Isolation and characterization 5.

ISOLATION

AND

CHARACTERIZATION

OF

FLAVONOID

COMPOUND FROM FERONIA LIMONIA 5.1 Introduction The correlation among the specific food, diet and disease expression has been indicated over the past three decades by the epidemiological survey. An evolution in the new diet-health pattern has resulted drastically in the discovery of a number of biologically active phytocompounds drastically which relatively emphasizes more on the useful features of the diet than the adverse effects. As a consequence of this development several terms such as functional food, nutraceutical and phytochemical have been introduced to describe various aspects of the diet. There are various databases available to provide details of the reviews on the different bioactive compounds present in plants and also their quantity (Bidlack et al., 2000). A wide variety of bioactive compounds exist in plant are omega-3-fatty acids, carotenoids, sterols, phenols and benzopyrroles (indoles) which differ in their chemical structures. Robards (2003) has illustrated in his review article that the number, diversity and determination by analytical approach of various bioactive compounds with specific reference to the plant phenols. Plant produces a diverse range of bioactive molecules making them rich source for preparation of different types of medicines. There are a number of plants with powerful therapeutic effects, and as a result, an extensive investigation to study their content is necessary. The natural extracts of the plants are an important source for the identification of new biologically active compounds with possible applications in the pharmaceutical field. Phytotherapy embraces especially the isolation of drugs from herbs which are considered to be pharmacologically active compounds each with unique chemical structures. There are many classes of compounds that can be found in natural Dept. of Biotechnology, Gulbarga University, Kalaburagi

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Isolation and characterization extracts including amino acids, phenols, polyphenols, saponins, flavonoids and sugars. Phyto-compounds of great interest which show important pharmacological activities are the phenols and flavonoids as reported by Middleton and Kandaswami (1993). Most of the plant secondary metabolites, in particular the phenols are synthesized by the shikimate pathway using the carbohydrates as the precursor molecule. This biosynthetic pathway is also the route for the synthesis of the aromatic amino acids but it is restricted only in the plants and microorganisms. Phenolic compounds found in all fruits and vegetables are ubiquitous in the plant kingdom but their distributions by quantitative means are vary within the populations of the same species and also among different tissues of the same plant (Van, 1970). The plant phenolic compounds are made up of a complex mixture but only countable plants are investigated thoroughly for the qualitative and quantitative estimation of the phenolic content and have been summarized by various reviewers (Clifford, 2001; Robards et al., 1999; Tomas and Clifford, 2001; Tomas et al., 2000; Ryan, 2002). However, the quantitative data is not reliable because of the presence of different quantification and extraction methods. 5.1.1

Flavonoids Flavonoids are the low molecular weight plant secondary metabolites having an

essential function in the photosynthesis of cell (Fernandez et al., 2006; Heim et al., 2002; Hollman and Katan, 1999; Cushine and Lamb, 2005). These are classified under the subclass of the polyphenols, which are characterized as containing two or more aromatic rings, each ring bears at least one aromatic hydroxyl group connected with a carbon bridge. Flavonoids are among the most ubiquitous groups of plant secondary metabolites distributed in various foods and medicinal plants. Flavonoid family is divided into

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Isolation and characterization different sub families namely flavones, isoflavones, flavonols, flavanols, anthocyanidins and flavanones (Clifford, 2001). However, exact mechanism of the actions attributed by the flavonoids have not yet been established, but the relationship between their activity and the presence of specific functional groups in the molecules is undeniable. The fundamental research on flavonoids commenced in the year 1936 by Albert Szent-Gyorgi, an Hungarian scientist who worked on the synergy between vitamin C obtained in pure form and the unidentified co-factors in the lemon peel, where he named it first as citrin and later on as vitamin P (Murray, 1998). Looking into the fundamental structure of the flavonoid, they have two benzene rings (A and B) consisting of nucleus 2-phenyl-benzo-γ-pyrane which is linked by a heterocyclic pyran ring (C). Characterization of the flavonoids is done based upon the C6-C8-C6 carbon-skeleton and the flavan nucleus (Heim et al., 2002; Peterson et al., 1998; Tsuchiya, 2010). Flavonoids are generally classified into different groups depending on the chromane-type skelton with phenyl substituent in the C2-C3 position which yield the structurally related compounds (Rijke et al., 2006). The previous studies on traditional medicines data reveals that the widely distributed phytocompounds are the flavonoids that are non-toxic, and usually present in beverages of all edible plants (Bimlesh et al., 2011; Cushnie and Lamb, 2005; Cook and Samman, 1996). They hold a diverse range of functions in the mammalian cells; yet, to be used as a drug in the field of modern medicine. However, the in-vivo evaluation is necessary to confirm their side effects. The selectivity and metabolism of flavonoids by the enzymes in eukaryotic cell differs and thus systematic study of toxicity is essential. Flavonoids are less toxic to the normal cells but possess toxicity towards immortalized

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Isolation and characterization cancer cells (Middleton et al., 2000). They are non-toxic and non-allergic for humans because of the properties such as low coefficient of absorption, low solubility of aglycone flavonoids in water and short duration of stay of flavonoids in the intestine. Thus there is increase in usage of flavonoids for the therapeutic use in human health than all other phytocompounds and drugs (Havsteen, 2002). The fresh or non-dried plant material results in the degradation of flavonoids particularly glycosides by enzyme action; thus usage of dry, frozen and lyophilized samples are usually preferred. For extraction of dry and powdered plant material, the solvent is selected based on the type of flavonoid which is targeted for isolation. Polarity plays an important role in the process where the extraction of less polar flavonoids including flavonols, flavanones, isoflavones and methylated is achieved by the use of solvents such as diethyl ether, dichloromethane, chloroform and ethyl acetate, while the extraction of more polar aglycones and flavonoid glycosides is achieved by the use of alcohols and aqueous-alcohol. Due to the increased water solubility of glycosides usually solutions of aqueous alcohol are preferred for its extraction (Andersen et al., 2006). A number of extraction methods are followed to isolate pure flavonoids. Simple direct solvent extraction method is employed when the powdered plant material is bulk. It is also extracted by the use of Soxhlet apparatus where lipids are eliminated by using hexane first and further extracted using ethyl acetate or ethanol which yields the phenolics; but this method is not suitable if heat-sensitive compounds are present. Thus most widely and convenient method employed is the sequential solvent soxhlet extraction method where extraction of flavonoid aglycones and less polar material is the first step achieved usually with dichloromethane. The next is extraction of flavonoid glycosides

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Isolation and characterization and polar constituents using alcohol. There are few important chalcone glycosides and flavanone which does not dissolve easily in alcohol–water mixtures, ethanol and methanol whose solubility depends on the pH of water-containing solutions (Andersen et al., 2006). There are other flavonoids such as catechins, proanthocyanidins, and condensed tannins belonging to class of flavan-3-ols which are extracted directly using water only (Takuya et al., 2013). Thus in general, the extraction process and composition of the extract varies with the type of solvent used. Hussein et al., (1990) have reported that for extraction of catechins methanol is the best solvent and for procyanidins, 70% acetone is good. For the separation and isolation of flavonoids, there is no single isolation procedure and thus successive isolation steps are necessary. Therefore, there are two prerequisites as described by Hostettmann et al., (1998) namely the quantity of sample available and the polarity of the compounds on which the choice of method depends. The process of structure elucidation of a natural product involves the determination of many physico-chemical properties including melting point, optical rotation, solubility, absorption, and resonance. The chemistry of natural products consists of three main areas i.e. isolation, characterization and synthetic methods. The isolation step is considered to be a part of structure elucidation and therefore analysis by using UV-Vis and infrared spectroscopy, mass spectrometry and various chromatographic techniques all are important tools for proper identification of the compounds present in an extract (Busquet et al., 2005). The organic solvent extracts contain various bioactive compounds which is determined by means of several separation techniques such as extraction & isolation of bioactive constituents by using thin-layer chromatography, column chromatography,

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Isolation and characterization HPTLC, HPLC and gas chromatography. NMR, FTIR, UV-Vis and mass spectroscopy techniques are used in prediction of the chemical nature and structure of the isolated compound. The study of flavonoid research by using chromatography has been reported by a number of investigators (Mabry et al., 1970; Markham, 1975; Hostettmann and Hostettmann, 1982; Harborne, 1980, 1989; Santos et al., 2003). To prevent the rediscovery of a chemical drug that is previously identified for its chemical activity and structure, elucidation of the chemical structure plays a significant role. Mass spectrometry is used to determine the chemical structure where an individual compound is identified after ionization based on their mass/charge ratio. Since compounds occur as mixtures in nature, it is essential to separate the individual chemicals by the use of both liquid chromatography and mass spectrometry (LC-MS) which is further compared with the databases of available mass spectra of known compounds. To determine the chemical structures of natural products, NMR spectroscopy is employed which allows detailed reconstruction of the molecules architecture by generating the information about individual carbon and hydrogen atoms present in the structure (Jiang et al., 2012). Column chromatography (CC) has been widely employed in food, aromatic and medicinal plants to fractionate the phenolic compounds. Some common column chromatography methods such as RP-18, sephadex-LH20, repeated silica gel, RP-8 and MCI-gel are used to fractionate simple flavonoids, phenolics and tannins from various samples including fruits such as Morus nigra, apples and Punica granatum (Pawlowska et al, 2008; Lee et al, 2010); olive oil (Khanal et al, 2011); kernels and nuts (Karamac, 2009; Zhang et al, 2009); seeds such as lentils (Amarowicz & Karamac 2003); tea (Liu et

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Isolation and characterization al, 2009; Gao et al, 2010); aromatic plants such as mint and sage (Wang et al, 1998; She et al, 2010) and medicinal plants such as Tridax procumbens and Ulmus davidiana (Agrawal and Talele, 2011). Therefore the present research work is aimed to describe the isolation and structural elucidation of the flavone compound from the methanolic extract of Feronia limonia fruit pulp by FTIR, NMR, LC-MS spectroscopy methods.

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Isolation and characterization 5.2 Materials and Methods 5.2.1 Preliminary screening of Feronia limonia methanol extract for flavonoids Methanolic extract was used for preliminary screening of flavonoids using Shinoda test and ferric chloride test (Trease and Evans, 2002). 5.2.2 Separation and purification of flavonoids In the first step, purification of the flavonoids was done using Silica gel column chromatography technique (Lin and Harnly, 2007). Borosil glass column with standard laboratory size was washed and dried. Column was tightly packed with silica gel of mesh size 60. Using the eluting buffer column it was washed and checked for presence of air bubbles and cracks. 5gm of methanolic extract was loaded onto the column. Elution buffer (methanol: water:: 70: 30) was used for the elution. Fractions were collected at a regular interval in 2ml tubes. These fractions are subjected further for flavonoids separation using the preparative thin layer chromatography technique (Hu et al., 2006). The analysis was performed on pre-coated 20×20 cm (0.25mm thick) TLC plates K6Fsilica gel 60 A which was purchased from Whatman, USA. Collected fractions are run on the TLC plate; further fractions with similar retention factor (Rf) values are pooled together and concentrated to dryness by evaporation. The sample from each tube was applied as spot onto the TLC sheets. Five different mobile phases were used to optimize the TLC conditions as shown in table 5.1. The plates are developed at room temperature in a previously saturated vertical separating chamber. After drying, visualization was performed in two ways: First in short UV light (254 nm) and next in spraying with 1 % ferric chloride solution.

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Isolation and characterization 5.2.3 Characterization of flavonoid FTIR, NMR and LC MS spectroscopic techniques are used for the characterization of the flavonoids. The active functional group present in the compound was studied by using Fourier–Transformation–Infra Red spectroscopy ((FT–IR Bruker, Japan). Sample was prepared by fixing in potassium bromide discs and 600-4000 cm-1 spectrum range was scanned (Linder et al., 2005). Chemical shifts of purified compound was dissolved in Dimethyl sulphoxide and observed by using the sophisticated multinuclear FT-NMR Spectrometer model Advance II (Bruker) with 400 MHz frequency, with a cryo magnetic strength 9.4 Tesla (Holzgrabe et al., 1998). Molecular mass and mass by charge ratio of purified compound was calculated using Liquid Chromatography Mass Spectroscopy instrument (Waters Micromass Q-Tof Micro) equipped with electrospray ionization consisting of mass ranging from 4000– 20000 atomic mass units was used (Grayer et al., 2000).

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Isolation and characterization 5.3 RESULTS 5.3.1 Separation and Purification (Table 5.1, Figure 5.1) In the present study five different solvent systems are used for the separation of flavonoids from methanolic extract of Feronia limonia (Table 5.1). The solvent system (Chloroform: Ethyl acetate: Formic acid) in the ratio of 5:4:1 (v/v/v) has supported a clear separation and with good resolution as shown in figure 5.1. Purified fractions are screened for their Rf values which have yielded five different bands clearly in samples 3, 4, 5, 6 and 7 whereas samples 1 and 2 have yielded visibility for band 4 only. Band 1 was obtained at Rf value of 0.57, band 2 with Rf value of 3.9, band 3 with Rf value of 5, band 4 with Rf value of 6.7 and band 5 with Rf value of 7.5 respectively. Bands with similar Rf values are pooled together and used for characterization studies. 5.3.2 Preliminary screening for flavonoids (Table 5.2) The result of the preliminary qualitative screening of flavonoids is tabulated in table 5. 2. Both the tests have indicated a positive result by formation of yellow colored precipitation for ferric chloride test and formation of pink color in shinoda test. 5.3.3 Characterization of flavonoid compound luteolin 5.3.3.1 FTIR Spectroscopy of flavonoid compound luteolin (Figure 5.2) Fourier transformation–infrared (FT–IR) spectrum result of the isolated flavonoid is shown in Figure 5.2. Several absorption peaks were recorded belonging to functional and structural groups. Appearance of broad band at 3396.3 cm-1 represents the presence of stretching vibration of phenolic groups which contains hydrogen bonding. The weak band at 2936.15 cm-1 indicates stretching vibration of aromatic (C-H) group and also the medium band at 2038.65 cm-1 represents bending vibration of aliphatic (C-H) group.

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Isolation and characterization Presence of weak peak at 2864.09 cm-1 indicates stretching aliphatic (C-H) group whereas the sharp and strong band at 1639.26 cm-1 represents stretching vibration of aromatic(C=O) group. Appearance of medium band at 1517.55 cm-1 indicates stretching vibration of aromatic (C=C) group. 5.3.3.2 NMR Spectroscopy of flavonoid compound luteolin (Figure 5.3) The NMR spectrum result of the isolated flavonoid luteolin is shown in Figure 5.3. The1 H-NMR spectrum exhibited two peaks at δ 4.21 (1H, d, J =1.8 Hz) and 4.14 ppm (1H, d, J =1.8 Hz) consistent with the meta protons H-6 and H-8 on A-ring and an AA’BB’ system at 4.54 (2H, d, J =8.9 Hz, H-2’, 6’) and 3.89 (2H, d, J =8.9 Hz, H-3’, 5’) corresponding to the protons on B-ring. The compound has presented the aglycone signal patterns. By comparison of proton field shift values with the available literature data, the 1

H NMR data of isolated compound was compatible with the literature libraries of

standard flavonoids and the peak values of isolated compound matched with the standard Luteolin. 5.3.3.3 Mass spectrometry study of flavonoid compound luteolin (Figure 5.4) The ESI-MS study of flavonoid yielded a quasi-molecular mass ion peak at m/z 293.05 has shown in Figure 5.4, was found to be 293.35 g/mol which is the nearest value to standard luteolin having molecular mass of 286.24 g/mol. 5.3.3.4 Structural elucidation of pure compound luteolin (Figure 5.5) The spectroscopic studies such as FTIR, NMR and LC-MS results reveals the functional group, chemical shifts and mass of the compound. Using these spectral details, the compound was identified as luteolin. Molecular formula and elemental analysis of isolated compound is C15H10O6 and C, 62.94; H, 3.52; O, 33.54 respectively. Molecular

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Isolation and characterization weight is 286.24 with m/z of 286.05 (100.0%), 287.05 (16.6%), 288.05 (2.5%). The two dimensional structure of the isolated flavonoid was drawn using Chem Draw UltraVersion 12 software. The structure of the isolated compound is depicted in Figure 5.5.

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Isolation and characterization 5.4 DISCUSSION The fundamental step in any drug discovery process is the analysis of preliminary phytochemical compound (Plazonic et al., 2009; Lin et al., 2008). Only if the samples respond positively for the preliminary tests, then they are used further for the separation and purification of the compounds. The protective role of the flavonoids and phenolic acids have been found in treating a variety of diseases such as atherosclerosis, carcinogenesis, thrombosis, antioxidant and inflammation. Flavonoids are also been reported to have role in treatment of diabetes where it acts as inhibitor to block the function of the aldose reductase in sorbitol pathway (Tapiero et al., 2002; Harborne and Williams, 1992; Hung et al., 2006; Yonathan et al., 2006; Bonita et al., 2007). Flavonoids also possess potential activity to inhibit various enzymatic systems such as lipooxygenase and cyclooxygenase that finally results in the development of defense against cardiovascular disorders, decrease in the platelet activation and aggregation, antiinflammatory activity and potential cancer chemopreventive functions (Yao et al., 2004; Sadhu et al., 2006; Kong et al., 2001; Sadik et al., 2003; Al-Fayez et al., 2006). They also possess

various

other

bioactivities

including

antihepatotoxic,

antimicrobial,

antiosteoporotic, antiviral, antiulcer, antiproliferative, immunomodulatory, and apoptosis (Li et al., 2005; Sousa et al., 2006; Kim et al., 2007; Oh et al., 2004; Innocenti et al., 2007; Ha et al., 2006; Chiang et al., 2006). Methanolic extract of Feronia limonia was screened for the flavonoids using two preliminary tests. With regard to detection, brief exposure of the TLC plate to iodine vapor produces yellow-brown spots against a white background. Several studies have indicated that the therapeutic properties of the extract depends on the type of the

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Isolation and characterization extraction methods used and thus various innovative extraction methods are being developed since the past few decades which are used for the isolation of bioactive compounds from numerous medicinal plants (Hayouni et al., 2007; Mujeeb et al., 2014). As a result, based on the physical and chemical properties of the sample matrix it is essential to choose the suitable solvent and extraction method which is not adversely affects in the presence of any interfering substances. Thin layer chromatographic (TLC) is the simplest technique used to separate and identify the natural products of interest. This method readily provides qualitative information and only little of the quantitative data. In order to achieve the bioassay separation of any plant metabolite, the optimization conditions like concentration, solvent system and visualizing methods are followed. In the present study, TLC procedure was optimized with a view to quantify a potential activity of methanol extract of Feronia limonia. The mobile phase was (Chloroform: Ethyl acetate: Formic acid) in the ratio of 5:4:1 yielded better, sharp and well‑defined resolution. The derivatization of the well defined spots are obtained when the chamber was saturated with a mobile phase for 20 minutes at room temperature. The TLC plate was further visualized at 254 nm after derivatization. A photograph of a TLC plate after chromatography was compared with the retention factor of standards using databases which have revealed the spot at Rf = 0.57 and the compound was identified as luteolin. The peak corresponding to luteolin from the isolated sample solution had retention factor (Rf = 0.57) near to that of luteolin standard (Rf = 0.55). There are different solvent systems available for the separation of flavonoids by using thin layer chromatography (TLC). Markham (1975) has extensively reported on the

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Isolation and characterization solvent systems used in the separation of flavonoids which are still employed by many researchers today. Wagner and Bladt (1996) have described on the separation of rutin and vitexin-2’’-O-rhamnoside by the use of solvent system ethyl acetate: ethyl methyl ketone: formic acid: glacial acetic acid: water in the ratio of 50:30:7:3:10. According to Budzianowskin (1991) the careful selection of solvent system will allow the separation of flavonoid glucosides from their galactosidic analogs which is required for the distinction of C-glucosides from C-galactosides. The extraction of luteolin and apigenin from different parts of the celery plant was achieved by using a combination of ultrasonic pretreatment and enzymatic hydrolysis (Zhang et al., 2011). Tania et al., (2012) have reported that the silica gel chromatography technique is particularly suitable for separation of phenolic compounds, it mainly depends on the polarity in which the degree of adsorption is determined by number of hydroxyl groups present in the extract. The principle of separation on Sephadex LH-20 chromatography which is a cross linked dextran-based resin is governed by the molecular size of the phenolic compound that exists outside the range of both adsorption and partition methods. The other method for separation of compounds include silica gel flash and open column chromatography and Sephadex LH-20 elution with hexane-ethyl acetate (1:1); ethyl acetate-methanol (1:1), hexane and methanol which were used by Cespedes et al., (2010) to separate phenolic acids such as ferulic acid and gallic acid; flavonoids derivatives including flavonol, catechins and anthocyanidins of the plant Aristotelia chilensis fruits which is a type of wild black berry. Also phenolic acids and Pyrogallol are separated and purified by using the repeated column chromatography procedure on

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Isolation and characterization Sephadex LH-20 eluted with methanol in Juglans regia kernels defatted extract which was followed by silica gel column chromatography using 40:1 v/v of chloroform–acetone as elution buffer; further on recrystallization by using methanol ethyl gallate was obtained. Zhang (2009) has demonstrated the separation of protocatechuic acid with the use of 1:1 v/v CH2Cl2–EtOH for elution in Sephadex LH-20 column chromatography and was further purified with 20:1 of Chloroform–methanol using the silica gel column chromatography. Karamac (2009) and Zhang (2009) have used silica gel column chromatography with

a

mixture

of

chloroform–

methanol

for

eluting

the

3,4,8,9,10-

pentahydroxydibenzo[b,d]pyran-6-one and gallic acid. Also defatted hazelnut, walnut and almond kernels of high-molecular-weight were used to isolate tannins with 50% (v/v) acetone as elution buffer by Sephadex LH-20 gel column chromatography. The ethanol fraction of Dipteryx lacunifera kernels was found to exhibit high radical scavenging activity and was subjected to further fractionation. Separation of butein, (-)-butin, sulfuretin, (-)-eriodictyol and luteolin on silica gel column chromatography with gradient solutions of elution buffer using chloroform to methanol and later eluted with methanol on Sephadex LH-20 (Junior et al., 2008, Wu et al., 2011). All these reports indicate that the separation technique of various phenolics is mainly dependent on the type of sample used, solvent system, column chromatography type and buffer used for elution. Thus the successful separation of flavonoids from methanolic extract of Feronia limonia in the present research work was achieved by using the silica gel column chromatography with chloroform: methanol (7:3) as elution buffer; and solvent system Chloroform: Ethyl acetate: Formic acid (5:4:1) which has supported a clear separation of flavonoid luteolin.

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Isolation and characterization NMR and FTIR spectroscopy offers the most useful and valuable information about the structure of any natural product. The methods have the advantage of excellent reproducibility. Even though they are considered to be the expensive techniques, but are relatively fast, sensitive and easily used as a routine application for amino acid analysis. 1

In the present investigation, H-NMR spectrum has showed two peaks at δ 4.21 (1H, d, J =1.8 Hz) and 4.14 ppm (1H, d, J =1.8 Hz) and by comparison of proton up field shift values with the literature data, the NMR data was compatible with the literature data of standard luteolin. Several absorption peaks belonging to functional and / or structural groups were recorded in the FTIR spectra by our study which describes the functional groups present in the isolated compound is luteolin. Patra et al., (2010) have reported the chromatographic separation using 1 H NMR analysis and bioautography screening of methanol extract of Excoecaria agallocha which is analogous to our findings. Several other investigators also worked on the isolation and characterization of compounds using the NMR, FTIR and MS techniques to illustrate the spectral data (Bremner and Surya, 2001; Anand et al., 2011). Plazonic et al., (2009) have described that the information on the carbohydrate sequence and the aglycone can be obtained by the use of mass spectrometric methods. The structurally diverse group of natural products are the flavonoid aglycones which vary in their structures at the level of oxygenation by presence of hydroxyl or methoxyl groups and also at the position of ring B attachment particularly in isoflavonoids and flavonoids. Thus, the information on the molecular mass, structure of the aglycone by measuring the pattern of aglycone hydroxylation and the point of connection of ring B on ring C is obtained by using the mass spectra of flavonoid glycosides. Also the data on the number

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Isolation and characterization of sugar rings, possible methylation or sulphation of aglycone hydroxyl, acylation of sugar hydroxyl groups and the arrangement of the glycosidic bonds. Mass spectrometry represents one of most efficient techniques for structure elucidation of natural product. It functions by separation of the ions formed in the ionization source of the mass spectrometer, according to their mass-to-charge (m/z) ratios (Kitson et al., 1996). Over the past two decades, mass spectrometry study has proved to be one of the most effective techniques in biomedical research, particularly for the analysis of complex mixtures of biological samples. A particular advantage of liquid chromatography mass spectrometry (LC-MS) is having capability to determine both free and conjugated forms of flavonoids (Clifford et al., 2008). As stated by Markham (1975), flavonoids when observed in UV light (254 nm) are visible as the dark spots against a green fluorescent background on plates containing a UV-fluorescent indicator such as silica gel F254 matrix. Further, when it is observed in UV light at 365 nm, flavonoids appear as dark yellow, green or blue fluorescence depending on the structural type of flavonoid, which is again intensified by the use of spraying reagents. One of the widely used significant spraying reagents is the ‘natural products reagent’ which yields an intense fluorescence when sprayed with 1% solution of diphenylboric acid-b-ethylamino ester commonly called as diphenylboryloxyethylamine dissolved in methanol under UV light at 365 nm. The average range for TLC detection limit of the flavonoids to obtain good resolution is between 2.5-10 mg of sample when sprayed with 5% solution of polyethylene glycol-4000 (PEG) in ethanol. The colors for different flavonoids observed in UV light at 365 nm are as follows:

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Isolation and characterization 

Myricetin, quercetin and their 3- & 7-O-glycosides ranges from orange to yellow.



Isorhamnetin, kaempferol and their 3- and 7-O-glycosides ranges from yellow to green.



Luteolin and its 7-O-glycoside appears orange.



Apigenin and its 7-O-glycoside ranges from yellow to green

Flavonoids such as luteolin are ubiquitous plant secondary metabolites and have a variety of biological effects, including antioxidant, anti-inflammatory activities (Clifford et al., 2008; Dall et al., 2004; Hirano et al., 2006; Xagorari et al., 2001). Luteolin belongs to the flavone group of flavonoids which has a C6-C3-C6 structure and possesses two benzene rings, a third oxygen-containing ring and a 2−3 carbon double bond as shown in figure 5.5. Luteolin also possesses hydroxyl groups at carbons 5, 7, 3’, and 4’ positions. The hydroxyl moieties and 2−3 double bond are important structural features in luteolin that are associated with its biochemical and biological activities. As in other flavonoids, luteolin is often glycosylated in plants, and the glycoside is hydrolyzed to free luteolin during absorption. Present investigation on spectral data reveals the elemental, structural and molecular details of the isolated compound. To our best knowledge, it is the first report on the isolation and identification of flavonoid luteolin from the fruit pulp of F. limonia. Using preliminary phytochemical screening, preparative thin layer chromatography and silica gel column chromatography a pure flavonoid 2- (3,4- dihydroxyphenyl)- 5,7dihydroxy-4H-chromen-4-one was obtained from fruit pulp of Feronia limonia Linn. We have also developed a solvent extraction and TLC methods with optimized conditions to

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Isolation and characterization separate and identify the constituents present in the methanolic extract. The isolated compound is known to exert many medicinal properties. Luteolin is an important constituent of F. limonia. Chemically it is 3’,4’,5,7-tetrahydroxyflavone and possesses various therapeutic activities. Though many flavonoids are present in this medicinal plant but due to luteolin’s small content in the drug and high therapeutic efficacy, its quantitative analysis becomes indispensible. Hence further specific and in depth research is requisite to identify its biological activities.

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