A Study On The Bioactive Compounds From Agro Industrial Waste Products Of Citrus Fruits And Enhancement Of Their Antioxidant Activity

Foods from plant origin usually contain natural antioxidants that can scavenge free radicals and may contribute to reduce human diseases. Therefore, in recent years many studies have been undertaken to find the new sources of antioxidant compounds. The growing interest to find the cheap, renewable and abundant sources of antioxidant compounds has fostered research on vegetable sources of residual origin from agricultural industries.Citrus is an important crop mainly used in food industries for fresh juice production and peel and pomace are the main byproducts during its processing. Pakistan is one of the major citrus producing countries in the world. Kinnow(Citrus reticulata Blanco cv Kinnow) and Feutrell's Early (C. reticulata Blanco cv Feutrell's Early) are two main citrus mandarin cultivars grown in Pakistan. Citrus peel which represents roughly half of the fruit mass is a rich source of bioactive compounds. It is assumed that many antioxidative phenolic compounds in plants are usually presented in covalently bound forms. Therefore, in order to enhance the antioxidant activity of plant materials, reliable and practical methods for liberation, extraction, identification and quantitative analysis of phenolic compounds are of considerable interest. The objective of this research was to (1) analyze, compare and document the antioxidant potential of the peels and pomaces of Pakistani citrus mandarin cultivars, (2) find the suitable methods for the enhancement of their antioxidant capacity by liberating bound phenolic compounds and (3) to explore their bioactive compounds.To begin with, the phenolic compounds (phenolic acids), total flavonoid and polyphenol contents and antioxidant activities of 70% ethanolic extracts of the Kinnow and Feutrell's Early peels were investigated. Results were compared with synthetic antioxidants and also with Chinese Ponkan mandarin peel to see the varietal and geographical effects on phenolic content and antioxidant activity. The cultivars showed the significant differences in total polyphenols, flavonoids and phenolic acids. Ferulic acid ranging from 1193.84 to 1640.87μg/g dry weight was the dominant phenolic acid in all cultivars. The antioxidant activity of extracts as determined byβ-carotene bleaching assay ranged from 28.40 to 43.78% and was in the following order:Kinnow< Feutrell's Early< Ponkan. A strong positive correlation was observed among the analyzed phenolic compounds and the antioxidant capacities. Results revealed that citrus peels under study might be used as an economical source of natural antioxidants and both, cultivars and geographical factors affect the bioactive composition in citrus mandarin peel.In the further step, the optimum processing conditions were investigated for the extraction of phenolic acids from the peels of citrus mandarin cultivars using microwave-assisted extraction (MAE). The operating parameters were optimized using central composite design combined with response surface methodology. The optimum extraction conditions were found as follows:microwave power,152 W; extraction time,49 s; liquid to solid ratio,16; and methanol concentration,66%. After method development, the extraction yield of phenolic acids (divided into four fractions:free, ester-bound, glycoside-bound and insoluble-bound), FCs (flavanol, flavanone and flavonols) and the antioxidant activity of the extracts were compared with those of the extracts obtained from the conventional methods including ultrasonic extraction (USE) and rotary extraction. Though in some cases the yields of MAE and USE were not significantly different but MAE showed obvious advantages in terms of high extraction efficiency and antioxidant activity of extract within shortest extraction time. The results demonstrated that MAE could be a fast and reliable method for the quantitative analysis of phenolic compounds in citrus mandarin peels.Based on the above results that microwave can enhance the yield of phenolic compounds from citrus peels, further insights into the potential of microwaves to liberate bound phenolic compounds and to enhance the antioxidant activity of citrus peels were carried out. The citrus peel powder was heated in a house-hold microwave oven at different power level and time intervals. Samples were heated at 250 W for 5,10 and 15 min and at 125 and 500 W for 5 min. Non-heated powder was used as a control. After treatment, methanol extracts of citrus peels were prepared and the contents of phenolic acids (free and bound) and flavanol, flavanone and flavonol compounds were determined by high performance liquid chromatography. Antioxidant capacity of peel extracts was measured using DPPH radical scavenging assay, hydroxyl radical scavenging assay and reducing power. After microwave treatment, the free fraction of phenolic acids increased, whereas the bound fractions decreased and the total phenolic acids content was declined. The antioxidant activity in all the systems was increased with microwave power and treatment time; however, a slight decline was observed in the antioxidant activity of citrus peel powder treated at 250 W for 15 min. But statistically there was no difference in the antioxidant activity of the peel powder treated for 10 and 15 min at 250 W. The highest antioxidant activity of the citrus peel powder was found when heated at 250 W for 10 min. The content of total FCs increased with power but at longer irradiation time it was declined which meant that some FCs might be degraded on microwave heating of citrus peels. The correlation analysis among sum of individual phenolic acids content (SPC) in free fraction and different antioxidant assays showed a high correlation coefficient in each case, which meant that the increase in the antioxidant capacities of citrus peels was due to at least in a part to the increase of SPC in the free fraction. The results indicated that appropriate microwave treatment could be an efficient process to liberate and activate the bound phenolic compounds and to enhance the antioxidant activity of citrus mandarin peels.Effect of microwave heating was also investigated on citrus pomace and almost similar results were observed as for the citrus peels except that the antioxidant activity of pomace was increased both with microwave power and treatment time and the highest activity was found when pomace powder was heated at 250 W for 15 min.After the enhancement of antioxidant activity of citrus peels, the effect of microwave heating was also investigated on their nutrient value and volatile compounds. The results showed a great variation in the nutrient and volatiles composition of citrus mandarin peels on microwave processing. With few exceptions, the mineral content was increased after microwave heating of citrus peels. However, in general, the amino acids, vitamin, and sugar contents were decreased in microwave treated samples. D-limonene, acetic acid, furfural and 2-methoxy-4-vinylphenol were the major aroma compound to be afftcted by mirowaving process. On microwave treatmnet of citrus peels, some new volatile compounds were also formed which were not detected in untreated samples.Finally, antioxidative compounds were explored from citrus peels by isolation, purification and identification process. A flavonoid compound was isolated from Feutrell's Early peels and its chemical structure was identified by nuclear magnetic resonance spectra. The compound was identified as a polymethoxy flavone,2-(3,4- dimethoxyphenyl)-5,6,7,8-tetramethoxychromen-4-one (Nobiletin) with a molecular weight of 402.1 Da.This is a pioneer study on the antioxidant potential of Kinnow and Feutrell's Early citrus mandarin cultivars and the use of microwave for the enhancement of antioxidant activity, which will serve as a basis for the future work in this area of research.