6 CHAPTER-6 TOTAL PHENOLIC AND FLAVONOID CONTENT DETERMINATION
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1 6 CHAPTER-6 TOTAL PHENOLIC AND FLAVONOID CONTENT DETERMINATION 6.1 PHENOLIC COMPOUNDS Phenolic compounds are a group of chemical compounds that are widely distributed in nature. They are simple compounds present in most fresh fruits and vegetables, or complex compounds present in bark, roots and leaves of plants. A group of polyphenols, responsible for the color of many fruits, vegetables, and flowers, are known as anthocyanins. Phenolic compounds, ubiquitous in plants are an essential part of the human diet, and are of considerable interest due to their antioxidant properties. These compounds possess an aromatic ring bearing one or more hydroxyl groups and their structures may range from that of a simple phenolic molecule to that of a complex high-molecular weight polymer (Waterman et al., 1994). Natural phenolic compounds play an important role in cancer prevention and treatment. Phenolic compounds from medicinal herbs and dietary plants include phenolic acids, flavonoids, tannins, stilbenes, curcuminoids, coumarins, lignans, quinones, and others. Plant foods are rich sources of phenolics, which are molecules that can act as antioxidants to prevent heart disease (Jin et al., 2010; Wijngaard et al., 2009; Hoye et al., 2008), reduce inflammation (Mohanlal et al., 2012; Zhang et al., 2011; Jin et al., 2006), lower the incidence of cancers (Sawadogo et al., 2012; Pieme et al., 2010; Ramos, 2008; Slivova et al., 2005) and diabetes (Kusirisin et al., 2009; Scalbert et al., 2005), as well as reduce rates of mutagenesis in human cells (Sawadogo et al., 2012; Pedreschi et al., 2006; Gomez-Cordoves et al., 2001). The protection afforded by the consumption of plant products such as fruits, vegetables and legumes is mostly associated with the presence of phenolic compounds. Phenolic compounds are synthesized in plants partly as a response to ecological and physiological pressures such as pathogen and insect attack, UV radiation and wounding (Diaz et al., 2010; Kennedy et al., 2011; Zulak et al., 2006; 106
2 Chung et al., 2003). The basic structural feature of phenolic compounds is an aromatic ring bearing one or more hydroxyl groups (Figure 1) (Chirinos et al., 2009). Plant phenolic compounds are classified as simple phenols or polyphenols based on the number of phenol units in the molecule. Thus, plant phenolics comprise simple phenols, coumarins, lignins, lignans, condensed and hydrolysable tannins, phenolic acids and flavonoids (Soto-Vaca et al., 2012). Fig. 6.1: Basic structures of phenolic acids and flavonoids. Flavonoids are some of the most common phenolics, widely distributed in plant tissues, and often responsible alongside the carotenoids and chlorophylls for their blue, purple, yellow, orange and red colors. The flavonoid family includes flavones, flavonols, iso-flavonols, anthocyanins, anthocyanidins, proanthocyanidins and catechins (Ferreira et al., 2012; Rong, 2010). All flavonoids are derived from the aromatic amino acids, phenyalanine and tyrosine, and have three-ringed structures (Routray et al., 2012). Variation in flavonoid structure arises from the scale and pattern of hydroxylation, prenylation, alkalinization and glycosylation reactions that alter the basic molecule (Stalikas, 2007). Phenolic acids are one of the other main phenolic classes within the Plant Kingdom and occur in the form of esters, glycosides or amides, but rarely in free form. Variation in phenolic acids is in the number and location of hydroxyl groups on the aromatic ring (Pereira et al., 2009). Phenolic acids have two parent structures: hydroxycinnamic and hydroxybenzoic acid. Hydroxycinnamic acid derivatives include ferulic, caffeic, p-coumaric and sinapic acids, while hydroxybenzoic acid derivatives consist of gallic, vanillic, syringic and protocatechuic acids. Another major class of phenolic compounds is the cell wall phenolics. They are insoluble and found in complexes with other types of cell components. The two main groups of cell wall phenolics are lignins and hydroxycinnamic acids (Vanholme et al., 2010; Baucher et al., 1998). These compounds play a critical role in the cell wall during plant growth by protecting against stresses such as infection, wounding and UV radiation (Naczk et al., 2004). Tannins can be divided into two groups, hydrolysable tannins and condensed tannins, and have great potential to form oxidative linkages to other plant molecules. 107
3 6.2 TOTAL PHENOLIC CONTENT OF LEAVES EXTRACT OF KIGELIA AFRICANA AND ROTULA AQUATICA LOUR Procedure for the determination of total phenolic content The concentration of phenolic in plant extracts was determined using spectrophotometric method. Methanolic solution of the extract of Kigelia africana and Rotula aquatica lour in the concentration of 1 mg/ml was used in the analysis. The reaction mixture was prepared by mixing 0.5 ml of methanolic solution of extract, 2.5 ml of 10% Folin-Ciocalteau s reagent dissolved in water and 2.5 ml 7.5% NaHCO3. Blank was concomitantly prepared, containing 0.5 ml methanol, 2.5 ml 10% Folin- Ciocalteau s reagent dissolved in water and 2.5 ml of 7.5% of NaHCO3. The samples were thereafter incubated in a thermostat at 45 o C for 45 min. The absorbance was determined using spectrophotometer at λmax = 765 nm. The samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained. The same procedure was repeated for the standard solution of gallic acid and the calibration line was constructed. Based on the measured absorbance, the concentration of phenolic was read (mg/ml) from the calibration line; then the content of phenolic in extracts was expressed in terms of gallic acid equivalent (mg/g of GA/g of extract). All the tests were performed in triplicate (Milan et al., 2011) Results and Discussion Kigelia africana Total content of phenolic compound was calculated as Gallic acid equivalent. The obtained observations were mentioned in table and plotted graph absorbance vs. concentration. Total Phenolic content was found to be 3.52 g gallic acid equivalent per 100 g methanolic by using the equation (y = x ). Table 6.1: Phenolic content of methanolic extract of Kigelia africana S. No. Sample Concentration (mg/ml) Absorbance 1. Gallic Acid Kigelia africana Methanolic extract
4 Fig. 6.2: Graph of absorbance against concentration for total phenolic content of methanolic extract of Kigelia africana Rotula aquatica lour Total content of phenolic compound was calculated as Gallic acid equivalent. The obtained observations were mentioned in table and plotted graph absorbance vs. concentration. Total Phenolic content was found to be 6.80 g & 5.90 g gallic acid equivalent per 100 g methanolic & aqueous extract respectively by using the equation (y = x ). Table 6.2: Phenolic content of methanolic and aqueous extract of stems of Rotula aquatica lour S. No.. Sample Concentration (mg/ml) Absorbance 1. Gallic Acid Rotula aquatica lour Methanolic extract 3. Rotula aquatica lour aqueous extract
5 Absorbance Phenolic content determination graph y = x R² = Absorbance of standard Linear (Absorbance of standard) Concentration (mg/ml) Fig 6.3: Graph of absorbance against concentration for total phenolic content. 6.3 TOTAL FLAVONOID CONTENT OF LEAVES EXTRACT OF KIGELIA AFRICANA AND ROTULA AQUATICA LOUR Procedure for the determination of total flavonoids content Total flavonoids content was determined according to the procedure by Aluminium chloride colorimetric method. Extract solution (1 ml, 0.1 mg/ml) was placed in a 10-ml volumetric flask, and then 5 ml of distilled water was added followed by NaNO2 solution (0.3 ml, 5%). After 5 min, AlCl3 solution (0.6 ml, 10%) was added. After another 5 min, NaOH solution (2 ml, 1 M) was added and volume was made up with distilled water. The solution was mixed thoroughly and absorbance was measured at 510 nm. Total flavonoid contents were expressed as milligrams of quercetin equivalent per milligram of dry weight. All the tests were performed in triplicate (Pongtip et al., 2005) Results and Discussion Kigelia africana In this method, flavonoid content was determined as quercetin equivalent. The obtained observations were mentioned in table and the graph plotted absorbance vs. concentration. Total flavonoids content was found to be 5.89 g & 4.00 g quercetin 110
6 absorbance equivalent per 100 g methanolic & aqueous extract respectively by using the equation (y = 9.837x ). Table 6.3: Flavonoid content of methanolic extract of leaves of Kigelia africana Sr. No. Sample Concentration (mg/ml) Absorbance 1 Standard Standard Standard Standard Standard Standard Test (alc. Extract) Test (water Extract) flavonoid content graph y = 9.837x R² = Absorbance concertration (mg/ml) Fig 6.4: Graph of absorbance against concentration for total Flavonoid content Rotula aquatica lour In this method flavonoids content was determined as quercetin equivalent. The obtained observations were mentioned in table and the graph plotted absorbance vs. concentration. Total flavonoids content was found to be 2.60 g & 1.79 g quercetin equivalent per 100 g methanolic & aqueous extract respectively by using the equation (y = x ). 111
7 Absorbance Table 6.4: Flavonoid content of methanolic and aqueous extract of stems of Rotula aquatica lour S. No. Sample Concentration (mg/ml) Absorbance 1. Quercetin Rotula aquatica lour Methanolic extract 3. Rotula aquatica lour aqueous extract Flavonoid content graph y = x R² = Concentration (mg/ml) Absorbance of standard Linear (Absorbance of standard) Fig 6.5: Graph of absorbance against concentration for total Flavonoid content. 112
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