Chapter 5. Isolation, Purification & Characterization. Phytochemical and Pharmacological Profiling of Ficus glomerata Roxb

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1 Chapter 5 Isolation, Purification & Characterization Department of Biotechnology, Gulbarga University, Kalaburagi Page 119

2 5. ISOLATION, PURIFICATION AND CHARACTERIZATION OF FLAVONOID FROM FRUITS OF FICUS GLOMERATA ROXB INTRODUCTION Identification of bioactive natural compounds for their anti-bacterial, antifungal and anti-cancer capacity by using bioprospecting is one of the important assignment (Hiil, 2010; Demain and Sanche, 2009). The process of bio prospecting can be compared to challenge of Needle in the Haystack in many facets. As we are expecting a single successful compound as an outcome from large number of test extracts (Ebada, 2008). Repeated research on less effective compounds or earlier identified ones results in wastage of resources and time. The development of rational screening methodologies helps to avoid wastage of resources. (Snowden and Green, 2008; Klinkenberg, 2011). Influence of different extraction techniques, extraction solvents on the natural antioxidant compounds was reported by many researchers. Plant materials selected used for extraction majorly decides the efficiency of solvents used for extraction and method of extraction employed. Extraction of phenolic compounds from fresh plant material can be commonly achieved by using solvents, such as methanol, ethanol, acetone, propanol and ethyl acetate (Grigonisa et al., 2005; Durling et al., 2007; Alothman etal., 2009). Variation in total phenolic content (±25% variation) and total antioxidant capacity (up to 30% variation) of fresh fruits and vegetables can be observed with varying properties of extracting solvent system. Polarity of solvent plays an important role in the solubility of phenolics in extraction by using solvent system. Generally phenolics are found to be with better soluble nature in high polarity solvents. Polar Department of Biotechnology, Gulbarga University, Kalaburagi Page 120

3 alcohol based solvents have shown extractive yield up to 22.8%, which is highest among other solvents. Extraction rate of ethanol can be improved by addition of water to it. Solvents with more water content results in the lower concentrations of phenolics and increase yield of other contaminant compounds(spigno et al., 2009; Lolita et al., 2012). Since very long period, plants have become the most important therapeutic sources available to mankind. From a phytochemical and pharmacological view point only a relative less percentage of plant species are studied. The plant kingdom is still remain as unexploited treasure of new molecules with promising remedial concern (Hostettmann, 1998). Pharmacognosy research has confirmed that plants can provide a effective bioactive products. Submitting plant extracts to hyphenated chemical inspection techniques such as liquid chromatography and mass spectroscopy. These techniques rapidly provides a number of detailed structural information in order to discover new bioactive compounds present in plant sources which could become new leads or new drugs, this shows the way to a partial or a complete on-line structural identification of interested natural products (Wolfender, 2013; Payal et al., 2013). Herbs, vegetable and fruits are commonly contain the phenolics as universally distributed component in them. Phenolics are one of the most diverse groups of phytochemicals. Approximately 8000 phenolics are have been isolated from natural sources. These compounds contain at least one phenol moiety (an aromatic ring with one or more hydroxyl substituent) (Robbins, 2003). The epidemiological, clinical and laboratory studies assumes that the risk of chronic diseases such as coronary heart disease and cancer may be reduced by the consumption of fruits, vegetables and herbs (Middleton et al., 2000; Hertog, 1995). Department of Biotechnology, Gulbarga University, Kalaburagi Page 121

4 Antioxidant nature of phenolics supports the beneficial properties of them (Rice- Evans et al., 1996; Hollman et al., 1998). It is critical to estimate the exact levels of phenolic compounds present in plants. Diverse nature among structure of phenolics and their solubility, structural diversity are important challenges in the extraction and analytical procedures of phenolics. Preparation of sample in analytical measurement contributes around 30% of the total errors (Majors, 1995). Extraction of desired product from target plant material is the first step in any analytical studies. A variety of parameters such as plant part, particle size, solvent polarity, extraction procedure and extraction conditions will influence the extraction of phenolic compounds from plant material. The impact of the extraction of phenolic compounds on the analysis has often been overlooked as substantial variations found in the extraction procedures and solvents are documented in the recent literature (Naczk and Shahidi, 2006). Several solvents such as methanol, ethanol, acetone, water, ethyl acetate and, to a lesser extent, propanol, dimethyl formamide, dimethyl sulfoxide and their combinations have been used for the extraction of different classes of phenolic compounds (Antolovich et al., 2000; Parejo et al., 2004). The reasoning for the selection of a particular extraction solvent or solvent mixture in addition to the extraction procedure is frequently not well studied or not clearly documented. (Luthria et al., 2006). Plants produce a diverse range of bioactive molecules making them as rich source of different types of medicines. Various techniques are employed for their investigation, which includes bioassays for chemical screening and their evaluation for proving the biological activities. Department of Biotechnology, Gulbarga University, Kalaburagi Page 122

5 Isolation of pure pharmaceutically active constituents from plants remains a long tedious process. Chemical screening is performed to target isolation of new or useful type of constituents having potential activities. This procedure enables recognition of known metabolites present in extracts in the earliest stages of separation and thus there are economically very important. To characterize the bioactive compounds several techniques are employed among which chromatographic techniques were extensively applied. Phenolic compounds are large and diverse group of molecules, which includes different families of secondary aromatic compounds in plants (Dai and Mumper, 2011). Around 8000 chemical structures of plant phenolics are studied until now. These compounds are simple, having low molecular weight, consisting of single aromatic ring to large and complex-polyphenols (Carlo et al., 1999). Plant phenolic compounds are primarily derived from phenyl - propanoid and acetate pathway and commonly found in conjugation with sugars and organic acids. Phenolics are classified into two groups: Flavonoids and Non- Flavonoids (Mabry et al., 1970). Flavonoids Flavonoids are the largest groups of phenols. More than 5000 chemical structures of flavonoids have been identified till date.(middleton and Kandaswami, 1986). The basic structure of flavonoid is comprised of C6-C3-C6 structure forming the A, B and C rings respectively. These are group of structurally related compounds with a chromane-type skeleton having phenyl substituent in C2-C3 position. The basic structural feature of flavonoid is 2-phenyl-benzo-γ-pyrane nucleus consisting of two benzene rings (A and B) linked through a heterocyclic pyran ring (C) (Sandha et al., Department of Biotechnology, Gulbarga University, Kalaburagi Page 123

6 2011). Based on the backbone structure flavonoids are classified into subgroups: flavanone, flavone, isoflavone, flavanol and flavonol (Ren et al., 2003). Biosynthesis of Flavonoids Two biochemical pathways shikimic acid pathway and acylpolymalonate pathway together results the biosynthesis of flavonoids. Phenylpropane a derivative of cinnamic acid, synthesized from shikimic acid, acts as the precursor in a polyketide synthesis, to this cinnamic acid extra three acetate residues are added into its structure followed by ring closure. Plants are capable to synthesize various classes of flavonoids through successive hydroxylations and reductions (Sameulsson, 1993; Giulia and Nicola, 1999). Pharmacokinetics of Flavonoids Due to the limited data available about the extent of absorption of flavonoids it remains a clearly unsolved question particularly in humans. Flavonoids present in food are assumed to be non- absorbable as they are attached with sugar molecules as P-glycosides except catechin. Aglycones which are the flavonoids free from sugar molecules are thought to be capable of passing through the gut wall. Hydrolysis of glycone results in the Aglycones i.e. hydrolysis detaches the sugar moiety from flavonoid and makes it as sugar free flavonoids. 13-glycosidic bonds of flavonoids are degraded only by microorganism of colon at the same time of degradation of dietary flavonoids. This is because no enzymes are capable of splitting the bonds present in flavonoids or the phenolics secrets into the gut. After absorption, the subsequent metabolism of flavonoids are quite well known from animal studies (Hacket, 1986). Metabolisms of absorbed flavonoids largely occur in liver which is responsible for this function. Kidney and intestinal wall are the secondary sites of the flavonoid metabolism. However, this depends on the various sources of flavonoids: those found Department of Biotechnology, Gulbarga University, Kalaburagi Page 124

7 in citrus fruits are poorly metabolized by the intestinal microflora, quercetin is not absorbed in humans, rutin is poorly absorbed, whereas procyanidolignanes are readily absorbed in mice. Flavonoids metabolized by intestinal bacteria are converted to hormone-like compounds with a weak estrogenic and antifecondative activities. Hydroxy groups are conjugated with glucuronic acid or sulphate and, in addition, methylation may occur. The glucuronides and sulphates are excreted in the bile (Hacket, 1986). Flavonoids, once absorbed may influence various biological functions including protein synthesis, cell proliferation, differentiation and angiogenesis, making them beneficial in a variety of human disorders (Giulia et al., 1999). Biological activities of Flavonoids Flavonoids have been reported to exert wide range of biological activities. These include anti-inflammatory, antibacterial, antiviral, antiallergic, cytotoxic antitumor and vasodilatory actions (Harborne and Williams, 2000) In addition, flavonoids are known to inhibit lipid-peroxidation, platelet aggregation, capillary permeability and fragility, cyclo-oxygenase and lipoxygenase enzyme activities (Li et al., 2007) Flavonoids are capable of modulating the activity of enzymes and affect the behavior of many cell systems and exerting beneficial effects on body(gomes et al., 2008). Department of Biotechnology, Gulbarga University, Kalaburagi Page 125

8 MATERIALS AND METHODS Plant material Fresh ripe fruits of Ficus glomerata Roxb (Family Moraceae) are collected from the Gulbarga University Campus. Fruits are washed thoroughly, dried in shade and grounded to powder and sieved. The fruit powder was stored at room temperature in airtight container until it is used for extraction. Extraction Fruit powder was subjected to hot extraction by using soxhlet extractor. Fruit powder was defatted with petroleum ether for three times. Further, extracted with methanol, and resulted methanolic extract was centrifuged at 10,000 rpm for 15minutes; supernatant was collected, dried and used for the purification (Thangavel and Gupta, 2010). Qualitative examination of Flavonoids Shinoda Test: Three pieces of magnesium chips was added to the filtrate followed by few drops of concentrated hydrochloric acid. A pink, orange, or red to purple colouration indicates the presence of flavonoids. Ferric chloride test: To 2 ml of the filtrate, few drops of 10% ferric chloride solution were added. A green-blue or violet colouration developed indicating the presence of a phenolic hydroxyl group. Separation of flavonoids by thin layer chromatography Sample: Dried methanol extract dissolved in distil water was used for thin layer chromatographic study. Loading: Approximately 20µl of the extract was loaded on the activated pre coated chromatographic plate (Merck, India) with help of capillary tube. Department of Biotechnology, Gulbarga University, Kalaburagi Page 126

9 Development: The loaded plate was kept in a saturated chromatographic chamber containing Toluene Chloroform Acetic acid mixture as mobile phase with ratio equal to (4: 5: 1). After running of about 15 cm the plate was removed from the chromatographic chamber and dried. Detection: The developed chromatographic plate was observed under (UV 254 nm) chamber. Thus, the colour and Rf values of fluorescent bands obtained recorded. The values of rate of flow (Rf) are calculated for separated flavonoid. Separation of flavonoids by silica gel column chromatography Preparation of column A clean and dry 500 ml capacity column of about 60 cm length was filled with the slurry of Silica gel-h of mesh size µ (Himedia, Mumbia) to 45 cm portion using hexane. Due care was taken to avoid air bubbles while packing the cloumn with a stationary phase. Then the column was run through twice with the solvent system containing hexane to make the column air tight and compact one. Loading Column chromatography technique was employed for purification of methanolic extract. A glass column (Merck, India) with 50cm X 2.5cm dimensions was cleanly washed with distill water and followed by eluting solvent. Column was packed thoroughly with the Silica gel (60-120; Merck, India). 5g of methanol extract of Ficus glomerata was ground well with a small amount of silica gel and loaded on to the top of the column. Mixture of methanol-water in various ratios were used as eluting solvent. Collection of fraction Totally 35 fractions with each 100ml were collected, as they came off the column in a series of conical flasks (100 ml). Thin layer chromatography was done Department of Biotechnology, Gulbarga University, Kalaburagi Page 127

10 with collected fractions. Based on the results similar fractions are pooled together and concentrated in vacuo to isolate the active principle. Characterization Characterization of compound was carried out by following techniques. FTIR spectroscopy Fourrier transformation (FT) IR spectrum of flavonoid compound isolated was recorded by using (FT IR Bruker, Japan) spectrophotometer for studying the functional groups. The sample compound was fixed in potassium bromide (KBr) disc then the spectrum was measured at wave number ranged from ( cm-1). The various bands are identified related to their wave numbers. NMR Spectroscopy 5mg-purified compound was dissolved in di methyl sulphoxide and used for Nuclear magnetic resonance spectroscopic analysis. Sophisticated multinuclear FTNMR Spectrometer model Avance II (Bruker) is used for the study, with a cryomagnet of field strength 9.4 T and 1H frequency 400 MHz. The chemical shifts expressed in ppm are recorded. MS spectrometry LC MS instrument (Waters Micromass Q-Tof Micro ) having quadra pole time of flight, mass equipped with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APcI) sources having mass Range of 4000 amu in quadruple and amu in ToF is used to detect the molecular mass and mass by charge ratio of compound dissolved in DMSO. Department of Biotechnology, Gulbarga University, Kalaburagi Page 128

11 RESULTS Qualitative examination of flavonoid (Table 5.1) Two preliminary qualitative tests Shinoda test and Ferric chloride test are performed to screen the presence of flavonoids in methanol extract of Ficus glomerata fruits. Appearance of pink colour in Shinoda test confirms the presence of flavonoids. Phenolic hydroxyl group present in compound was confirmed by the formation of violet red colour in ferric chloride test. Thin layer chromatography of Flavonoid (Table 5.2, Fig. 5.1) Developed thin layer chromatography plates were subjected for iodine vapour test and appearance of brown colour spots on thin layer chromatography plates when placed in iodine chamber confirms the presence of flavonoids, and in ferric chloride test appearance of yellow colour spot after spraying (1%) ethanol ferric chloride solution also confirms the presence of flavonoids. FTIR Spectroscopy (Fig 5.2) Furrier transformation infra red (FT IR) spectrum result of isolated flavonoid is shown in Fig.5.2. Absorption peaks belonging to functional and / or structural groups were recorded. Appearance of broad band at cm-1 represents presence of stretching vibration of phenolic groups containing hydrogen bonding. The weak band at cm-1 indicates the stretching vibration of aromatic (C-H) group and also the medium band at cm-1 represents the bending vibration of aliphatic (C-H) group. NMR Spectroscopy (Fig 5.3, Table 5.3) The 1H NMR spectrum of the compound as in Fig. 5.3 demonstrated the signals of aromatic, pyron ring and prenyl group. The 1H NMR spectrum of the compound displayed signals at d 6.53 assignable to a phenolic hydroxyl group at C- Department of Biotechnology, Gulbarga University, Kalaburagi Page 129

12 40 and a singlet for three protons at d 3.83 was ascribed to a methoxy group at C-7. The 1H NMR spectrum further displayed a one proton singlet at d 5.35 was attributed to H-8 proton. The spectral details of the purified compound is tabulated in Table 5.3. At base 2.00 a chemical shift of 2.0 corresponding to OH group indicates the presence of alcohol group. Chemical shift of 3.75 at base 3.00 corresponding to OH group shows the presence of C-OH group. Presence of 1-benzene moiety was confirmed by a representing node with a chemical shift of 3.36 at base 4.00 Department of Biotechnology, Gulbarga University, Kalaburagi Page 130

13 Mass spectrometry (Fig 5.4) The mass spectrometry data revealed that the mass of purified compound is (Fig.5.4), which is the closest value to the flavonoid myricetin (350.03). The molecular formula is C 15 H 10 O 10. Elemental analysis of purified compound found to be: C H O From the entire above chemical and spectral test evidences and previous studies on Ficus species by Li (1998) used to identify the isolated compound as a flavonol, dihydroxy myricetin [3, 5 dihydroperoxy-7- hydroxyl-2-(3,4,5- trihydroxyphenyl)-4h-chromen-4-one]. Department of Biotechnology, Gulbarga University, Kalaburagi Page 131

14 DISCUSSION When a plant has been identified as a source for new products, the bioactive compounds present in the relevant fractions in the so called Bioassay guided isolation. In this process the plants are successively extracted with solvents of increasing polarity and tested by a range of bioassays relevant to the activity of the compounds (Gedara and Zaghloul, 2005). It s a long and tedious process to isolate pure, pharmacologically active constituents from plants. Thus, it is necessary to have the good methods available which eliminate unnecessary separation procedures. Chemical screening is thus performed to allow localization and targeted isolation of new or useful constituents with potential activities. This procedure enables in recognition of known metabolites present in extracts or at the earliest stages of separation and is thus economically very important (Kumara, 2001). Sample preparation and removal of unwanted substances for quantification of phenolics is an important method. The extraction procedure is the primary determinant for the separation and recovery of phenolics. Extraction is generally influenced by the sample nature, particle size, solvent type as well as extraction techniques employed. Soxhlet, heated reflux extraction and maceration are the conventional procedures frequently applied to recover phenolics from solid samples. The Soxhlet and heated reflux methods are normally performed at 90 C for several hours while maceration is performed over days at ambient temperature. These methods are simple, require relatively cheap apparatus and result in adequately high phenolic extraction rates. Castro-Vargasa et al., (2010) reported that the highest total phenolic content of Guava seed extract was achieved by using Soxhlet extraction Department of Biotechnology, Gulbarga University, Kalaburagi Page 132

15 techniques. In another study, the phenolic compounds present in seeds of three wild grapevines were successfully extracted by using Soxhlet technique. Flavonoids are highly bioactive compounds found in both edible and nonedible plants. They are often extracted with methanol, ethanol, acetone, water or mixtures of these solvents using heated reflux extraction methods (Routray and Orsat, 2012). Biesaga (2011) has extracted flavonoids in maize samples using heated reflux, microwave-assisted extraction, ultrasonic-assisted extraction and maceration and compared to the stability of the extracted compounds. The highest stability of the extracted flavonoids in methanol-water (60:40 v/v) was found in compounds extracted with traditional heated reflux in a water bath. In our study we have used a combinatorial solvent methanol water in a proportion of 70:30 for extraction of phenolics from fruits of Ficus glomerata. High extractive value was achieved using this solvent system. Paper chromatography and thin-layer chromatography are the two partitioning techniques employed to separate phenolics in foods (Naczk and Shahidi, 2006) paper chromatography is a simple method and less utilized compared to HPLC and gas chromatography. In other studies, flavonoids, phenolic acids and glycoflavones have been separated from three green leafy vegetables using paper chromatography (Nambiar, 2010). Thin layer chromatography is more powerful technique than Paper chromatography to analyze phenolics, especially in crude plant extracts. Phenolics in crude plant extracts can be separated by a number of thin layer chromatography techniques, which are cheap and provide a multiple detection on the same thin layer chromatography plate in a short analysis time (Ignat, 2011). Sajewicz et al (2012) Department of Biotechnology, Gulbarga University, Kalaburagi Page 133

16 have indicated that the silica gel thin layer chromatography -based video imaging method is a valuable complementary fingerprint technique to identify phenolic acids and flavonoids fractions of different sage species. Oliveira et al., (2012) also utilized silica gel thin layer chromatography to identify phenolic compounds from Baccharis trimera extract. The purified compound was screened to check the presence of flavonoids. Two generally accepted tests such as Shinoda and Ferric chloride tests are conducted. The pure compound has responded positively to Shinoda test and Ferric chloride test confirming the presence of flavonoid. FTIR has proven to be a valuable tool for the characterization and identification of compounds or functional groups (chemical bonds) present in an unknown mixture of plant extracts (Rao,1963). In our present study the of FTIR spectrum, broad band at cm-1 represents the presence of stretching vibration of phenolic groups containing hydrogen bonding. Weak band at cm-1 has indicated stretching vibration of aromatic (C-H) group and also the medium band at cm-1 represents bending vibration of aliphatic (C-H) group. NMR is one the most powerful research technique followed to investigate the structure and some properties of molecules. One of the main applications of NMR in flavonoid research is the structural elucidation of novel compounds, for which nothing is known. The proton Nuclear magnetic resonance of purified compound was analyzed by referring the standard flavonoid NMR spectra (Yamaguchi, 2005) In the present study, the 1 H NMR spectrum of the isolated compound has displayed signals at d 6.53 assignable to a phenolic hydroxyl group at C-40 and a singlet for three protons at d 3.83 was ascribed to a methoxy group at C-7. The 1H Department of Biotechnology, Gulbarga University, Kalaburagi Page 134

17 NMR spectrum further displayed a one proton singlet at d 5.35 was attributed to H-8 proton. Mass Spectrometry (MS) has proved to be one of the most effective technique in biomedical research. The main advantage of MS is its high sensitivity, which allows analysis of compounds present in the microgram scale, and high specificity, as it is able to separate molecules of the same molecular weight but different atom composition (Cuyckens and Claeys, 2005). The mass spectrometry data of isolated compound in the present study revealed the mass of which is the closest value to the flavonoid myricetin (350.03). The molecular formula is C 15 H 10 O 10.Elemental analysis of purified compound was found to be: C , H and O Myricetin is flavonol, consisting of 3-hydroxyflavone backbone and 6 hydroxyl groups. It is found in several foods such as walnuts, onions, berries, herbs and red grapes. Myricetin exerts a wide variety of biological effects, including antioxidant and free radical-scavenging activities (Li and Kuo 1998). Cereal, vegetable and fruit consumption contributes to improve human health and lowers the risk of disease. These benefits may depend significantly on the presence of phenolic content in these foods. The therapeutic benefits of phenolics have made researchers to discover, modify and utilize techniques for the extraction, separation and quantification of these compounds from natural sources. The methods need to be simple, rapid, environmentally friendly and comprehensive. In our study, we have followed a simple and conventional soxhlet extraction method for extraction of phenolics using methanol and water mixture as solvent. Thin layer chromatography was made for separation of compound and silica gel column chromatography used for purification of flavonoids. Three spectral techniques FT-IR, Department of Biotechnology, Gulbarga University, Kalaburagi Page 135

18 NMR and LC-MS techniques applied for characterization and identification of isolated compound. Hence, our study relies on basic and simple techniques for isolation of flavonoids from plant source and succeeded in it. The further biological activity of the isolated flavonoid is needed to screen its specific therapeutic properties. Department of Biotechnology, Gulbarga University, Kalaburagi Page 136