Chapter III Optimization of extraction method and profiling of plant phenolic compounds through RP-HPLC 1. INTRODUCTION Phenolics compounds are naturally present antioxidants, found in a variety of plant based food and vegetables. These compounds are attracting a great deal of attention due to increasing evidence suggesting that they may prevent oxidative diseases such as cancer and other neurological diseases (Catherine et al., 1997). Generally flavonoids and phenolic compounds are widely distributed in plants have been exhibiting multiple biological effects such as, diuretics, antinephrities, antiaging, antiinflammatory, anticarcinogenic and antidiabetic etc (Wu et al., 2009). Most well known common polyphenolics are gallic acid, ellagic acid, catachin, quercetin, flavon, rosmeric acid, chlorogenic acid, ferulic acid, coumaric acid, quinic acid, vanilic acid, syringic acid, catechuic acid and kaempferol etc (Dai and Mumper, 2010). Green leafy vegetables are major part of constitute in balanced diet and are well balanced nutrition sources of minerals, vitamins and other secondary metabolites. Green leafy vegetables are the most important sources of polyphenols (Drewnowski and Carneros, 2000). For extraction of phenolic compounds, previously several methods have been reported namely supercritical extraction, solid-liquid extraction, Microwave Assisted extraction (MAE), Conventional Assistant Extraction (CAE) and Ultrasound-Assisted Extraction (UAE) etc. (Naczka and Shahidi, 2004). In UAE, ultrasonic frequency sound is very high (>20 khz) that is greater than the upper limit of human detection. Ultrasound-Assisted Extraction (UAE) involves passing ultrasonic energy in the form of waves through a liquid solvent containing the solid particles. As the waves hit the surface of the material, a force that is either perpendicular or parallel to the surface is 52
generated. If the force is perpendicular to the surface, a compressive wave is formed whereas if the force is parallel to the surface, a shear wave is produced. This constant production of compressive and shearing forces gives rise to a phenomenon known as cavitations. Microwaves are a form of non-ionizing electromagnetic energy. In contrast, MAE uses microwave energy to directly heat the molecules within the material often in a matter of seconds. This thermal energy was responsible for phenolic compounds extraction from plant material. Interest in Conventional-Assisted Extraction (CAE) has increased significantly over the past few years as a result of its inherent advantages (reduction in extraction solvent volume and well percolation) over more traditional extraction techniques. In CAE, the solvent and sample are kept at boiling temperature, all phenolic compounds percolated from plant cells to solvent. In addition, the direct heating that occurs with CAE, typically allows for a reduction in the volume of solvent needed for extraction. The majority of the work had focused on the extraction of phenolic compounds by CAE method only, rather than by other extraction methods such as MAE and UAE. Lianfu and Zelong, (2008) reported combination of Ultrasound/Microwave Assistant Extraction (UMAE) as a optimized procedure for lycopene extraction from tomato and its efficacy in the extraction of phenolic compounds from plant material was investigated. To our knowledge, there are no previous reports on the extraction of polyphenols from M. emarginata, the green leafy vegetable using the combination of Ultrasound and Conventional - Assistant Extraction (UCAE) method. The spectrometric method is used traditionally for quantification of phenolic compounds in plant extracts. However, due to complexity of the phenolic compounds, quantification of individual phenolic compound cannot be determined by spectrometric method. Modern chromatographic techniques are successfully used for the individual 53
phenolic compound quantification. Chromatographic methods combined with instrumentation analysis used for the profiling and quantification of phenolic compounds. Gas chromatography (GC) is widely used for phenolic and flavonoid compounds identification based on volatile and non volatile nature of compounds (Stalikas, 2007). The most successful instrumentation method is using HPLC, by which phenolic compounds including anthocyanins, tannins, flavonols, flavan-3-ols, flavanones, flavones, and phenolic acids in different plant extract and food samples (McCallum et al., 2007; Mertz et al., 2007; Pawlowska et al., 2008) could be qualitatively and quantitatively analysed. The reversed-phase (RP) columns have considerably enhanced HPLC separation of different classes of phenolic compounds and RP C 18 columns are almost exclusively employed. It was found that column temperature may affect the separation of phenolics such as individual anthocyanin. Acetonitrile and methanol are the most commonly used organic solvents as mobile phase. In many cases, the mobile phase was acidified with a modifier such as acetic, formic and phosphoric acid for better phenolic compounds separation and to minimize peak tailing. Both isocratic and gradient elution are applied to separate phenolic compounds. The choice depends on the number and type of the analyte and the nature of the matrix. Previously several reviews have been published on the application of HPLC methodologies for the analysis of phenolics (Naczka and Shahidi, 2004; Stalikas, 2007; Dai and Mumper, 2010). After choosing the appropriate column (i.e. stationary phase) and mobile phase (i.e. organic solvents or buffer), the next important step in RP-HPLC method development and choosing the elution mode, that is isocratic elution (constant elution) or gradient elution (The mobile phase composition does not have to remain constant). The gradient provides an adequate separation within an acceptable analysis time (Robards, 2003). Moreover, HPLC with mass spectroscopy (MS) techniques is the most 54
powerful analytical technique for identification of mass of the molecule; it offers an unique chance to analyze simultaneously all components of interest together with their possible derivatives or degradation products (Downey and Rochfort, 2008). This chapter evaluates the optimization of phenolic compound extractions from M. emarginata using MAE, UAE, CAE and UCAE methods. Further optimization of RP-HPLC conditions like mobile phase, ph, gradient programme and absorbance maximum for phenolic compounds separation was carried out. 2. MATERIALS AND METHODS 2.1. Plant material M. emarginata leaves were used for phenolic compound extraction and identification. Plant collection and sample storage were carried out as previously discussed in chapter I. 2.1. Optimization of phenolic Extraction 2.2. Microwave-Assisted Extraction (MAE) 500 mg of powdered plant material was extracted with 40 ml of 70 % methanol (with 10 ml 5 M HCl) in a microwave for 10 40 min. On mass yield basis, an extraction time of 20 min at 650 W microwave powers was found to be optimum. The extracts were filtered and concentrated to dryness under vacuum (temperature, 40 ± 3 C) (Lianfu and Zelong, 2008). 2.3. Ultrasound-Assisted Extraction (UAE) 500 mg of powdered plant leaves was sonicated with 40 ml of 70 % methanol (10 ml 5 M HCl) in an ultrasonic bath at a frequency of 40 KHz (Elma Ultrasonic, Germany) and at a controlled temperature of 30 ± 5 C for 40 80 min. An extraction time of 60 min was taken as optimum on mass yield basis. The extracts were filtered 55
and concentrated to dryness under vacuum (temperature, 40 45 C) and the yield obtained used for further analysis (Lianfu and Zelong, 2008). 2.4. Conventional-Assistant Extraction (CAE) 500 mg of powdered plant leaves with 40 ml of 70 % aqueous methanol was taken. Then 10 ml of 5 M HCl was added. The mixture was stirred carefully. The extraction mixture was then refluxed in a water bath at 100 C for 145 min. After cooling, it was filtered with Whatman No.1 filter paper). Then filtrate was centrifuged at 5000 rpm. The supernatant was filtered with 0.22 µm membrane syringe filter (Pall, Biosciences, USA) prior to injection in RP-HPLC (Proestos et al., 2005). 2.5. Ultrasound/Conventional-Assistant Extraction (UCAE) 500 mg of powdered plant leaves with 40 ml of 70 % aqueous methanol was taken. Then 10 ml of 5 M HCl was added. The mixture was stirred carefully. The extraction mixture was then refluxed in a water bath at 100 C for 145 min. After cooling, it was filtered with Whatman No.1 filter paper) and filtrate was sonicated at a frequency of 40 KHz in ultra sonic bath for 10 min. Then the filtrate was centrifuged at 5000 rpm and the supernatant was filtered with 0.22 µm membrane syringe filter (Pall Biosciences, USA) prior to injection. 2.6. Optimization of RP-HPLC Method The analytical HPLC system employed consists of High Performance Liquid Chromatography (Waters, USA) coupled with a photodiode array detector (PDA-2998), USA. C 18 reverse phase column of 4.6 250 mm, 5µm particle size (SYMMETRY) was used. The mobile phase used was water with 0.1% formic acid as solvent A and 100% methanol as Solvent B. The different isocratic and gradient programmes were followed for phenolic compound separation. The optimized gradient program of 0 10% B (5 min), 10 15% B (5 min), 15 20% B (5 min), 20 30% B (5 min), 30-40% B (10 56
min) was followed, flow rate was 1.0 ml/min, and the injection volume was 20 µl. The wavelength was monitored between 210 to 400 nm. The analytical data was evaluated using EMPOWER 2 data processing software from Waters, USA. 3. RESULTS AND DISCUSSION 3.1. Optimization of UCAE for phenolic compounds extraction The phenolics extraction efficiency of CAE, MAE and UAE methods were assessed by comparing their RP-HPLC chromatogram (Figure 4.1, 4.2 and 4.3). Among these extraction methods, the highest recovery of phenolic compounds in terms of number and yield was obtained in CAE when compared with MAE and UAE. The CAE is traditionally successful extraction method because in this method, maceration or percolation mechanism accounts for better extraction efficiency and little amount of water in the extracting solvent can penetrate easily into the cells of the plant matrix and facilitate better heating of the plant matrix. This in twist increases the mass transfer of the active constituents into the extracting solvent (Wenkui Li et al., 2003). Further, the chromatographic profile of the combination of both UAE and CAE method (UCAE) was better in terms of number of phenolics eluted and resolution of their individual peak when compared with chromatographic profile of other extraction methods (Figure 4.4). Since, the conventional extraction gives better percolation of sample to solvents under boiling temperature and the ultrasound extraction technology, the sonication gives individual phenolic compounds fractions and also offers a mechanical effect to give high penetration of solvent into the sample surfaces, thereby increasing the contact surface area between the solid and liquid phase and result in quick diffusion of sample solute from the solid phase to the extraction solvent (Ghafoor et al., 2009). 57
Figure 4.1. Microwave-Assisted Extraction (MAE) RP-HPLC chromatogram at 280 nm Figure 4.2. Ultrasound-Assisted Extraction (UAE) RP-HPLC chromatogram at 280 nm 58
Figure 4.3. Conventional Assistant extraction (CAE) RP-HPLC chromatogram at 280 nm Figure 4.4. Ultrasound Conventional-Assistant extraction (UCAE) RP-HPLC chromatogram at 280 nm 59
3.2. Optimization of RP-HPLC condition A separation of mobile phase composition with a constant flow throughout the procedure is termed isocratic (meaning constant composition). In gradient programme, which the mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution. Both isocratic and gradient program was used for optimization of RP-HPLC condition for ME phenolic compound identification. The Figure 4.5 shows phenolic compound elution by isocratic program. Figure 4.6, 4.7 and 4.8 shows the different gradient programs such as 1, 2 and 3 respectively. Among this gradient elution, the gradient program no.3 was chosen since it gave more phenolic compounds interms of yield and number compared to isocratic and other gradient programs. Many of the previous reports shown gradient programs were used for better separation phenolic compounds from plant materials (Fang et al., 2002; Min Ye et al., 2005; Navindra et al., 2006). Recently Beer and Joubert, (2010) reported development of HPLC method for phenolic compound analysis of Cyclopia subternata. Kokotkiewicz et al, (2012) have reported isolation and structural elucidation of phenolic compounds from Cyclopia subternata using gradient program. Generally phenolic compounds absorbance shows maximum peaks in the UV region. So in this study, the multiwavelength absorbance between of 210-500 nm was used. Phenolic compounds were monitored in the range between 280, 300 to 320 nm (Figure 4.9). Among these monitoring wavelengths, at 280 nm, most of the abundant phenolic compounds were detected. It is because most of the phenolic compounds absorbance maximum is near to 280 nm. Escarpa and Gonzalez, (1999) have reported phenolic compounds identification from apples and pears was carried out using highperformance liquid chromatography with diode-array detector and was monitored at 280 nm. 60
Figure 4.5. Isocratic program RP-HPLC chromatogram at 280 nm Mobile phase Solvent A : Water with 0.1% formic acid Solvent B : 100% Methanol Isocratic program Solvent A - 50 % Solvent B - 50 % 61
Figure 4.6. Gradient program 1 RP-HPLC chromatogram at 280 nm Mobile phase Solvent A : Water with 0.1% formic acid Solvent B : 100% Methanol Program 1 0 5% B (5 min), 10 10% B (5 min), 15 15% B (5 min), 20 20% B (5 min), 30 25% B (10 min), 25 40% B (20 min). 62
Figure 4.7. Gradient program 2 RP-HPLC chromatogram at 280 nm Mobile phase Solvent A : Water with 0.1% formic acid Solvent B : 100% Methanol Program 2 0 5% B (5 min), 10 10% B (5 min), 15 20% B (5 min), 20 25% B (5 min), 30 35% B (10 min), 35 40% B (30 min). 63
Figure 4.8. Gradient program 3 RP-HPLC chromatogram at 280 nm Mobile phase Solvent A : Water with 0.1% formic acid Solvent B : 100% Methanol Program 3 0 10% B (5 min), 10 15% B (5 min), 15 20% B (5 min), 20 30% B (5 min), 30 40% B (10 min), 40 40% B (20 min). 64
a b c Figure 4.9. Different nm absorbance of RP-HPLC chromatogram detection a. Ultrasound Conventional-Assistant extraction (UCAE) RP-HPLC chromatogram at 280 nm b. Ultrasound Conventional-Assistant Extraction (UCAE) RP-HPLC chromatogram at 320 nm c. Ultrasound Conventional-Assistant Extraction (UCAE) RP-HPLC chromatogram at 340 nm 65
4. CONCLUSION In this chapter, we successfully investigated the optimization of phenolic compounds extraction using CAE, MAE, UAE and UCAE methods. The optimized phenolic compound extraction method was named as ultrasound-conventional assistant extraction (UCAE). Further, the optimized RP-HPLC condition for phenolic compound detection was chosen as gradient programme No.3 which gave more number of phenolic compounds yield at 280 nm wavelength. 66