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Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Vaishnavi Chandrasekar Entitled Optimizing the microwave-assisted extraction of phenolic antioxidants from apple pomace and microencapsulation in cyclodextrins For the degree of Master of Science Is approved by the final examining committee: Tameshia Ballard Chair Fernanda San-Martin Gonzalez Peter Hirst To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Tameshia Ballard Approved by Major Professor(s): ____________________________________ ____________________________________ Approved by: Suzanne Nielsen June 30, 2010 Head of the Graduate Program Date Graduate School Form 20 (Revised 1/10) PURDUE UNIVERSITY GRADUATE SCHOOL Research Integrity and Copyright Disclaimer Title of Thesis/Dissertation: Optimizing the microwave-assisted extraction of phenolic antioxidants from apple pomace and microencapsulation in cyclodextrins Master of Science For the degree of ________________________________________________________________ I certify that in the preparation of this thesis, I have observed the provisions of Purdue University Teaching, Research, and Outreach Policy on Research Misconduct (VIII.3.1), October 1, 2008.* Further, I certify that this work is free of plagiarism and all materials appearing in this thesis/dissertation have been properly quoted and attributed. I certify that all copyrighted material incorporated into this thesis/dissertation is in compliance with the United States’ copyright law and that I have received written permission from the copyright owners for my use of their work, which is beyond the scope of the law. I agree to indemnify and save harmless Purdue University from any and all claims that may be asserted or that may arise from any copyright violation. Vaishnavi Chandrasekar ______________________________________ Printed Name and Signature of Candidate 07/01/2010 ______________________________________ Date (month/day/year) *Located at http://www.purdue.edu/policies/pages/teach_res_outreach/viii_3_1.html OPTIMIZING THE MICROWAVE-ASSISTED EXTRACTION OF PHENOLIC ANTIOXIDANTS FROM APPLE POMACE AND MICROENCAPSULATION IN CYCLODEXTRINS A Thesis Submitted to the Faculty of Purdue University by Vaishnavi Chandrasekar In Partial Fulfillment of the Requirements for the Degree of Master of Science August 2010 Purdue University West Lafayette, Indiana UMI Number: 1489654 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI 1489654 Copyright 2011 by ProQuest LLC. All rights reserved. This edition of the work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106-1346 ii For Amma and Appa iii ACKNOWLEDGMENTS I would like to thank my advisor Dr. Tameshia Ballard for being my mentor and friend, and guiding me through the graduate program. I enjoyed working with her and learnt a lot in the process! I would also like to thank Dr. Fernanda San-Martin and Dr. Peter Hirst for serving on my committee, and for all their help and advice. I am grateful to all the faculty and staff in Food Science for their help over the past two years. A special thanks to Dr. Nivens for his guidance in various analytical techniques. Also to Dr. Liceaga for permitting me to use of her lab equipment whenever I needed to. I am also thankful to Dr. Campanella and Bhavesh Patel for helping me with the DSC. I thank my office mates Rei and Mandar for their support and friendship during these last two years. I am also eternally grateful to my ‘mafia’ friends in Food Science… Preetha, Deepak, Shivangi and Azalenah for all their support and help, and the endless cups of coffee that provided motivation during my day! Finally, I am forever grateful to my family for their support and strength in all my endeavors. My parents, for the love that keeps me positive everyday; my older sister Preethi for her spontaneity and generosity; and my twin sister Vaidehi for being there always. iv TABLE OF CONTENTS Page LIST OF TABLES……………………………………………………………………..…vi LIST OF FIGURES…………………………………………………………………..….vii ABSTRACT .................................................................................................................. ix CHAPTER 1 INTRODUCTION ................................................................................1 Hypothesis ...................................................................................................................3 Objectives....................................................................................................................3 Significance and Rationale...........................................................................................4 Thesis organization ......................................................................................................4 References ...................................................................................................................6 CHAPTER 2 LITERATURE REVIEW .....................................................................8 2.1 Antioxidants ..........................................................................................................8 2.2 Apple polyphenols ...............................................................................................11 2.3 Extraction techniques ...........................................................................................13 2.3.1 Solvent extraction..........................................................................................13 2.3.2 Supercritical fluid extraction (SFE) ...............................................................14 2.3.3 Accelerated solvent extraction (ASE) ............................................................16 2.3.4 Microwave assisted extraction (MAE) ...........................................................17 2.4 Quantification of phenolic compounds .................................................................21 2.5 Estimation of antioxidant activity.........................................................................22 2.5.1 ORAC assay ..................................................................................................23 2.5.2 DPPH Assay .................................................................................................24 2.5.3 FRAP Assay ..................................................................................................25 2.5.4 TEAC Assay .................................................................................................26 2.6 Encapsulation of polyphenols...............................................................................26 2.6.1 Wall materials ...............................................................................................27 2.6.2 Analysis of inclusion complexes....................................................................32 2.7 Summary .............................................................................................................34 References .................................................................................................................35 v Page CHAPTER 3 OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION OF PHENOLIC ANTIOXIDANTS FROM APPLE POMACE ........................................... 41 Abstract .....................................................................................................................42 Introduction ...............................................................................................................43 Methods and Materials...............................................................................................46 Results and Discussion ..............................................................................................50 Conclusion ................................................................................................................58 References .................................................................................................................75 CHAPTER 4 ENCAPSULATION OF POLYPHENOL EXTRACTS FROM APPLE POMACE IN CYCLODEXTRINS ................................................................................77 Abstract .....................................................................................................................78 Introduction ...............................................................................................................79 Methods and materials ...............................................................................................81 Results and discussion ...............................................................................................85 Conclusion ................................................................................................................89 References .................................................................................................................96 CHAPTER 5 CONCLUSIONS ................................................................................98 vi LIST OF TABLES Table Page Table 3.1 Coded values and corresponding actual values of the optimization parameters ......................................................................................................................68 Table 3.2 Experimental design for extraction of phenolic compounds by MAE in terms of coded values ..................................................................................................69 Table 3.3 Response surface models fitted based on TPC for different apple cultivars and solvent systems ..............................................................................................70 Table 3.4 Response surface models fitted based on DPPH activity for different apple cultivars and solvent systems .........................................................................71 Table 3.5 Optimum conditions for the extraction of phenolic antioxidant compounds by MAE based on both TPC and DPPH activity .................................................72 Table 3.6 Predicted and experimental values of TPC and DPPH activity for Red Delicious apple pomace extracted under optimal conditions for 60% ethanol .73 Table 3.7 HPLC quantification of major apple polyphenols from optimized MAE extracts for each apple cultivar and solvent system ..................................................... 74 Table 4.1 HPLC quantification of apple polyphenols from Red Delicious apple pomace extract............................................................................................................95 vii LIST OF FIGURES Figure Page Figure 2.1 Common polyphenols in apple ...................................................................... 12 Figure 2.2 Structure of Cyclodextrin molecule ............................................................... 30 Figure 2.3 Phase solubility diagram (adapted from Higuchi & Connors, 1965) .............. 32 Figure 3.1 Response surface of Red Delicious apple pomace extracted with 70% acetone - Effect of extraction time and microwave power on TPC ............................. 59 Figure 3.2 Response surface of apple pomace extracts with 70% acetone - Effect of solvent volume and power on TPC ............................................................... 60 Figure 3.3 Effect of solvent volume on TPC of Winesap apple pomace extracted with 70% acetone ................................................................................................. 61 Figure 3.4 Effect of solvent volume on DPPH inhibition percentage of Jonathan apple pomace extracted with 70% acetone ............................................................. 62 Figure 3.5 Interaction plot of Red Delicious apple pomace extracted with 60% ethanol Effect of microwave power and time on TPC ............................................... 63 Figure 3.6 Response surface of Jonathan apple pomace extracted with 60% ethanol Effect of solvent volume and power on DPPH inhibition percentage ............ 64 Figure 3.7 Response surface of the effect of solvent volume and power on both TPC and DPPH inhibition percentage (desirability) for Red Delicious apple pomace extracted with 60% ethanol .......................................................................... 65 Figure 3.8 Response surface of the effect of solvent volume and power on both TPC and DPPH inhibition percentage (desirability) for Red Delicious apple pomace extracted with 70% acetone .......................................................................... 66 Figure 3.9 HPLC chromatogram at 280 nm of Red Delicious apple pomace extracted with 60% ethanol under optimum conditions ........................................................ 67 viii Figure Page Figure 4.1 Complex formation study of apple extract with (A) α-CD (B) β-CD and (C) 2hydroxypropyl-β-CD .................................................................................... 90 Figure 4.2 DSC thermograms of pure apple pomace extract and inclusion complexes under oxidative conditions ............................................................................ 93 Figure 4.3 Phase solubility study of apple pomace extract with α-, β, and γ-CD at 30 °C (n=2) ............................................................................................................94 ix ABSTRACT Chandrasekar, Vaishnavi, M.S. Purdue University, August, 2010. Optimizing the microwave-assisted extraction of phenolic antioxidants from apple pomace and microencapsulation in cyclodextrins. Major Professor: Tameshia Ballard. Apple pomace is the skin, seeds and flesh leftover after juice extraction, and is rich in polyphenols that have demonstrated high antioxidant activity both in vivo and in vitro. Despite this fact, apple pomace remains an underutilized agricultural waste product. To add value and increase the commercial applicability of the pomace, efficient methods for the extraction of phenolics from apple pomace are needed. Traditional solvent extraction is both solvent and time intensive, leading to the consideration of alternate extraction techniques such as microwave-assisted extraction (MAE). MAE of phenolic compounds from pomace of four apple cultivars (Red Delicious, Golden Delicious, Winesap and Jonathan) was optimized for various extraction parameters (microwave power, extraction time, solvent volume to sample ratio and solvent type) using response surface methodology. Optimum conditions were based on maximizing two responses: total polyphenol content (TPC) and antioxidant activity as measured by the percent inhibition (%IP) of the DPPH free radical. Response surface models were developed by regression, taking into consideration significant model parameters (p<0.05). Each solvent system showed similar optimum extraction conditions independent of apple cultivar. Red Delicious pomace variety had the highest TPC (15.8 mg GAE/g DW) and DPPH radical x scavenging activity (94.4 % inhibition) obtained under the optimum conditions of 735 W and 149 s, with 10.4 mL of 60% ethanol and 5.65 mL of 70% acetone, respectively. Catechin, phloridzin, caffeic acid, chlorogenic acid and quercetrin were some of the major phenolic compounds identified by HPLC in the extracts. Stabilization of the extracted phenolics obtained under optimized conditions was carried out by encapsulation in α-, β-, and 2-hydroxypropyl-β-cyclodextrin. Inclusion complex formation was confirmed by differential scanning calorimetry for all the three CD types. Complexes were also found to be stable to oxidation up to temperatures of 250 °C. Phase solubility studies indicated an A L type diagram and established the formation of a 1:1 complex of the extract with β-CD, with a moderate binding constant (K c = 1.02 mg GAE/g extract -1). The overall results of this research indicate that MAE is an efficient method for extraction of valuable antioxidant compounds from apple pomace. Further, their encapsulation in β-CD results in the formation of stable inclusion complexes with enhanced solubility for potential use as functional food ingredients. Keywords: apple pomace, microwave-assisted extraction, encapsulation, cyclodextrin, inclusion complex 1 CHAPTER 1. INTRODUCTION Polyphenols are a diverse group of compounds that are present in plants, and are synthesized as secondary metabolites. They are known to possess very good antioxidant activity (1). Currently the industry makes use of a number of synthetic antioxidants such as BHA, BHT and TBHQ to combat the problem of lipid oxidation, and enhance the shelf-life of lipids and lipid-containing foods. Due to increasing consumer demand for healthier, all natural products, industry’s reliance on these synthetic compounds must be minimized. Thus, the challenge for the food industry moving forward is to create foods containing more natural ingredients while maintaining sensory quality. The use of these naturally occurring antioxidant polyphenols in place of synthetic antioxidants in foods is a potential area worth investigating. Several million tons of apples are processed as juice or cider, generating hundreds of tons of apple pomace as a waste stream globally (2). Polyphenols from apple pomace have been extensively studied, and are known to possess high antioxidant and antiproliferative activities and effectively inhibit lipid oxidation (3). The major phenolic compounds present in apple include catechin, epicatechin, quercetin glycosides and phloridzin (4). The type and amount of phenolic compounds present varies widely 2 depending on the apple cultivar, maturity and environmental growth conditions (3). Polyphenols have conventionally been extracted from the apple matrix by solid-liquid extraction using various organic solvents including ethanol, methanol, acetone, or mixtures of these with water (5-6). However, conventional extraction techniques consume large amounts of solvent, and are also time intensive. As a result, alternate extraction techniques such as ultrasound-assisted extraction (7), sub-critical carbondioxide extraction (8) and pressurized liquid extraction (9) have been studied to improve the extraction of phenolic compounds from apples. Microwave-assisted extraction (MAE) is a relatively new extraction technique that utilizes microwaves for heating up the sample and solvent system, rapidly and homogenously. The principle of heating using microwaves is based on the direct action of the electromagnetic radiation on the molecules through ionic conduction and dipole rotation, resulting in heating (10). Polar solvents with high dielectric constants tend to heat up more rapidly with microwaves than non-polar solvents with lower dielectric constants (11). Thus, polar solvents or non-polar solvents diluted with water are frequently employed solvent systems with MAE. MAE has been increasingly used for the rapid extraction of bioactive compounds from plant sources (12-14). Compared to solvent extraction, it has been shown to reduce solvent requirements and extraction time (12, 1415). However, the extraction of phenolic compounds from apple pomace using MAE has not been studied and optimized. As phenolic compounds are antioxidants, they are highly prone to oxidation, and degrade in the presence of heat and light (16). This makes them difficult to store and 3 transport. Furthermore, many of these compounds are hydrophobic in nature, and therefore, demonstrate limited solubility in water. Encapsulation of phenolic antioxidants in suitable wall materials may enhance their functionality. Cyclodextrins, or modified starch molecules, are commonly used wall materials for the encapsulation of hydrophobic compounds (17). They are cyclic molecules that consist of a relatively hydrophobic cavity which may form inclusion complexes with the phenolic compounds. Several hydroxyl groups are present on the outer surface, which enhances the water solubility of the inclusion complex. Modified cyclodextrins with higher water solubility have also been developed by the addition of polar side chains. Therefore, the stability and functionality of the extracted polyphenols may be improved by their encapsulation in native or modified cyclodextrins. Hypothesis This work is based on the hypothesis that microwave-assisted extraction may be used as a rapid and efficient technique to increase the yield of valuable natural antioxidant compounds from apple pomace. Additionally, the water solubility and oxidative stability of phenolic compounds present in the pomace extracts will be increased by encapsulation in cyclodextrins. Objectives 1. Optimize the microwave-assisted extraction of phenolic antioxidants from apple pomace using response surface methodology. 4 2. Encapsulate apple pomace extract in cyclodextrins and characterize their complex formation, oxidative stability and solubility using DSC and phase solubility studies. Significance and Rationale Several million tons of apple pomace is generated globally as an industrial waste stream from apple cider or juice production. The extraction of phenolic antioxidant compounds from this waste stream could prove to be beneficial in terms of generating value-added food ingredients. Polyphenols have traditionally been extracted by solidliquid extraction, and conditions for maximizing yields have been optimized. However, conventional solvent extraction is both time and solvent intensive. The use of alternative extraction techniques, in this case MAE, may prove to be more practical. Once removed from their native plant matrix, polyphenols are unstable and prone to oxidation in the presence of heat and light. In addition, several of these phenolics have very low solubility in water, which limits their applications in the food industry. Encapsulation of these compounds in cyclodextrins may enhance their stability and improve their solubility, thereby increasing overall commercial applicability. Thesis organization This thesis consists of five chapters. Following the introduction is the second chapter consisting of a concise literature review. The third and fourth chapters outline the work carried out under the two objectives stated above, and have been written in manuscript format to be submitted to peer-reviewed journals. The third chapter focuses on optimizing MAE of phenolic compounds from apple pomace. The fourth chapter 5 details the encapsulation of the polyphenol extracts in cyclodextrins that were obtained in the MAE study. The final chapter summarizes the overall conclusions, and outlines some possible future work that may be carried out. 6 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Dapkevicius, A.; Venskutonis, R.; van Beek, T. A.; Linssen, J. P. H., Antioxidant activity of extracts obtained by different isolation procedures from some aromatic herbs grown in Lithuania. J Sci Food Agr 1998, 77, (1), 140-146. Vendruscolo, F.; Albuquerque, P. M.; Streit, F.; Esposito, E.; Ninow, J. L., Apple Pomace: A Versatile Substrate for Biotechnological Applications. Critical Reviews in Biotechnology 2008, 28, (1), 1-12. Boyer, J.; Liu, R. H., Apple phytochemicals and their health benefits. Nutr J 2004, 3, 5. Lu, Y.; Foo, L. Y., Identification and quantification of major polyphenols in apple pomace. Food Chemistry 1997, 59, (2), 187-194. Foo, L. Y.; Lu, Y., Isolation and identification of procyanidins in apple pomace. Food chemistry 1999, 64, (4), 511-514. Garcia, Y. D.; Valles, B. S.; Lobo, A. P., Phenolic and antioxidant composition of by-products from the cider industry: Apple pomace. Food Chemistry 2009, 117, (4), 731-738. Lee, K. W.; Kim, Y. J.; Kim, D. O.; Lee, H. J.; Lee, C. Y., Major phenolics in apple and their contribution to the total antioxidant capacity. Journal of Agricultural and Food Chemistry 2003, 51, (22), 6516-6520. Adil, I. H.; Çetin, H. I.; Yener, M. E.; BayIndIrlI, A., Subcritical (carbon dioxide + ethanol) extraction of polyphenols from apple and peach pomaces, and determination of the antioxidant activities of the extracts. The Journal of Supercritical Fluids 2007, 43, (1), 55-63. Alonso-Salces, R. M.; Korta, E.; Barranco, A.; Berrueta, L. A.; Gallo, B.; Vicente, F., Pressurized liquid extraction for the determination of polyphenols in apple. Journal of Chromatography A 2001, 933, (1-2), 37-43. Eskilsson, C. S.; Bjorklund, E., Analytical-scale microwave-assisted extraction. Journal of Chromatography A 2000, 902, (1), 227-250. Wang, L.; Weller, C. L., Recent advances in extraction of nutraceuticals from plants. Trends in Food Science & Technology 2006, 17, (6), 300-312. Hong, N.; Yaylayan, V.; Raghavan, G.; Paré, J.; Bélanger, J., Microwaveassisted extraction of phenolic compounds from grape seed. Natural Product Research 2001, 15, (3), 197-204. Pan, X.; Niu, G.; Liu, H., Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves. Chemical Engineering and Processing 2003, 42, (2), 129-133. Kaufmann, B.; Christen, P., Recent extraction techniques for natural products: microwave-assisted extraction and pressurised solvent extraction. Phytochemical analysis 2002, 13, (2), 105-113. Proestos, C.; Komaitis, M., Application of microwave-assisted extraction to the fast extraction of plant phenolic compounds. LWT-Food Science and Technology 2008, 41, (4), 652-659. 7 16. 17. Cheynier, V., Polyphenols in foods are more complex than often thought. Am J Clin Nutr 2005, 81, (1), 223S-229. Szejtli, J., Introduction and General Overview of Cyclodextrin Chemistry. Chemical Reviews 1998, 98, (5), 1743-1754. 8 CHAPTER 2. LITERATURE REVIEW 2.1 Antioxidants Lipids are a major component of foods, and are classified in very basic terms as fats (lipids that are solid at room temperature) and oils (lipids that are liquid at room temperature). They contribute to the sensory and flavor profile of various foods, and may also serve certain functional properties within the food system such as emulsification. Lipid oxidation is a major problem in the food industry, resulting in loss of nutritional and sensory qualities and producing undesirable flavors, color and toxins (1). Food manufacturers currently incorporate synthetic antioxidant compounds such as butylated hydroxy toluene (BHT), butylated hydroxy anisole (BHA), propyl gallate (PG) and tertiary-butyl hydroxy quinone (TBHQ) into food products to slow the rate of lipid oxidation and enhance shelf life. These synthetic antioxidants have proven to be effective in inhibiting lipid oxidation, but have shown toxicological effects, particularly in higher concentrations (2). Structurally, all these compounds closely resemble naturally occurring polyphenols. They all contain one or more hydroxyl groups with hydrogen donating potential, attached to a modified benzene ring which provides resonance stabilization to the radical form. As today’s consumers are demanding more all natural food products, 9 the addition of naturally derived polyphenols from plant sources may be a good alternative to replace their synthetic counterparts. Polyphenols are a wide group of compounds that are found in a variety of plant tissues. They are secondary plant metabolites and are synthesized during normal plant growth as well as during conditions of stress (3). These phenolic compounds contribute to the organoleptic and sensory properties of plant foods in terms of enhancing their astringency and bitterness. Polyphenols are a ubiquitous group of compounds that can be classified into several types based on their chemical structure. Flavonoids are the largest class of polyphenolic compounds with greater than 5000 compounds identified to date, and they can be further classified into several subclasses such as flavones, flavonols, flavanols and anthocyanidins. Other important groups of polyphenolic compounds include phenolic acids, hydroxycinnamic acids, coumarins, stilbenes and lignans. Bravo et al. (4) summarizes the major classes of naturally occurring polyphenolic compounds based on their chemical structure. Interest in phenolic compounds has been rapidly increasing owing to their roles as effective antioxidants and scavengers of free radicals (5-6). Rice-Evans et al. (7) investigated the structure of several naturally occurring phenolic compounds and how they function as terminators of free radicals and chelators of metal ions that are capable of catalyzing lipid peroxidation. Phenolics have several hydroxyl groups which are capable of donating a hydrogen atom to free radicals to form phenoxy radical intermediates. These intermediate radicals are relatively stable due to resonance. As a result, a new chain reaction is not easily initiated. Due to their antioxidant properties, 10 there is good potential to extract and utilize these phenolic compounds as replacers of synthetic antioxidants. The advantage of this approach would be to produce food products with a clean label with no synthetic preservatives. Consumer demand for products containing natural ingredients has been rapidly increasing, and nearly 76% of consumers would choose a product containing ‘all natural’ ingredients rather than synthetic compounds (Mintel, 2009). A large amount of these natural antioxidant compounds have been found in agricultural and industrial by-products. Grape pomace has been studied extensively and is known to be rich in catechin, epicatechin and quercetin glycosides (8-9). Other agricultural by-products that have been studied include citrus seeds and peels (10), carrot pulp waste (11), spent tea leaves (12), cacao by-products (13) and soy-bean molasses (14). All these studies showed that as a general rule, the natural phenolics extracted from the plant matrix had comparable antioxidant activity to that of synthetic antioxidants, and could be used as an alternative to the synthetic ones in foods. In some cases, the natural extracts showed a reduced antioxidant potential when compared to synthetic antioxidants. However, they could still be used advantageously as maximum levels established for synthetic food additives need not be applicable to naturally occurring compounds (15). A substantial amount of waste is generated from apple processing. Nearly 25 to 30% of the total world production of apples is estimated to be industrially processed into products such as cider and juice, and this results in generation of several million tons of apple pomace globally (9). Apple pomace is the skin, seeds and flesh leftover after juice 11 extraction, and is rich in several phenolic compounds (8). This waste stream thus has excellent potential to be used as a source of valuable antioxidant byproducts. 2.2 Apple polyphenols Consumption of apples has been related to several health benefits. A large number of epidemiological and animal studies have shown a link between consumption of apples and decreased risk of cancer and cardiovascular disease (16). Apple polyphenols have been extensively studied and are found to contain high antioxidant and anti-proliferative activity and effectively inhibit lipid oxidation (16). These health benefits have mainly been attributed to phytochemicals including phenolic antioxidants present in apples. The major polyphenols identified in apples are catechin, epicatechin, quercetin and quercetin glycosides, phloridzin, caffeic acid and chlorogenic acid (Fig 2.1). Lee et al. (17) quantified some of the major polyphenols in six cultivars of apples. The average concentrations in mg/100 g fruit were: epicatechin 8.65; phloretin glycosides 5.59; chlorogenic acid 9.02; procyanidin B 9.35; quercetin glycosides 13.2 and vitamin C 12.8. The total phenols content varies widely depending on the apple cultivar, state of maturation, availability of growth nutrients, etc. 12 Figure 2.1 Common polyphenols in apple Several studies indicate a higher concentration of polyphenols in the apple peel compared to the rest of the fruit (16). Quercetin glycosides are known to be exclusively present in the peel. Some of the other polyphenols typically present in the peel include procyanidins, catechin, epicatechin, phloridzin and chlorogenic acid. However, higher concentrations of chlorogenic acid have been reported in the apple flesh compared to the peel (18). Several animal studies were conducted to study and compare the beneficial health effects of apple peel and flesh phenolic compounds. In vivo studies indicate that apple peels were better able to inhibit cancer cell proliferation, had greater plasma antioxidant capacity and showed greater inhibition of lipid peroxidation in rats (19). A number of studies have reported the beneficial effects of individual apple polyphenols in relation to reducing the risk of cancer and cardiovascular disease. Boyer and Liu (16) have reviewed these studies comprehensively. However, the synergistic effect of all the polyphenols present in apples should be considered rather than the effect 13 of individual compounds. Thus, there is very good potential to use phenolic extracts from apples as a bioactive ingredient in foods. 2.3 Extraction techniques Studies on extraction of these compounds from plant matrices have traditionally involved solvent extraction. These classical extraction methods are time consuming, require large volumes of solvent and often generate heat, and consequently cannot be used for the extraction of thermolabile compounds, rendering these techniques impractical for commercial large scale extraction. Therefore, other more rapid and efficient extraction techniques are being explored such as supercritical fluid extraction, accelerated solvent extraction and microwave assisted extraction. Descriptions of these extraction methods will be given in the following sections as a means of comparison. 2.3.1 Solvent extraction Solvent extraction involves addition of a solvent or a mixture of solvents to the plant matrix for subsequent extraction of the desired analyte. The solvent diffuses into the plant matrix and the analyte is then solubilized in the solvent. The solvent along with the analyte can then be physically separated from the plant matrix using various filtration and centrifugation methods. The particle size of the plant matrix is thus an important factor. The smaller the particles, the easier it is for the solvent to diffuse into the matrix and extract the analyte. The important solvent characteristics that govern the solubility of phenolic compounds are polarity, degree of polymerization, interaction of phenolics with other food components and formation of insoluble complexes (4). Common solvents 14 employed for the extraction of polyphenols are methanol, ethanol, acetone, water, ethyl acetate, and to a lesser extent propanol, dimethylformamide, and their combinations (20). Existing classical techniques used for extracting phytochemicals from plants include soxhlet, hydrodistillation and maceration with an alcohol-water mixture or hot fat (21). Traditionally, solvent extraction coupled with the use of heat and/or agitation has been performed using a soxhlet extraction unit. Extraction of phenolics from apple pomace has widely been carried out using aqueous 70% acetone (9, 22-23). Foo and Loo (9) characterized the procyanidins in apple pomace after extraction of about 200 g freeze-dried pomace using 70% aqueous acetone (3 x 500 ml) at room temperature. The total phenols content of their extract was 7.24 mg/g DW. Quercetin glycosides accounted for more than half (4.24 mg/g DW) of the total phenols present followed by phloridzin and its oxidative products. 2.3.2 Supercritical fluid extraction (SFE) SFE with carbon dioxide is a non-thermal approach to extracting thermolabile compounds, and has therefore been successfully used in extraction of phenolics. SFE makes use of a supercritical fluid to extract the analyte of interest from the plant matrix. A supercritical fluid is any substance which is held above its critical temperature and pressure, and exhibits the characteristics of both a liquid and a gas. Such a fluid has the advantage of lower viscosity and higher diffusivity. This increases the rate of mass transfer and diffusion, thereby decreasing the extraction time from hours (for typical solvent extraction) to a few minutes. The solvation power depends on the temperature and pressure. Hence, selectivity for particular analytes can be increased by manipulating 15 these parameters. The extracted solutes, now dissolved in the supercritical gas, can then be separated by depressurization. There is no residual solvent in the extract, as in the case with other extraction techniques. Further, only small volumes of sample and solvents are required. Supercritical fluid extracts have reported higher antioxidant power compared to extracts obtained by solid-liquid or soxhlet extraction (24). Carbon dioxide is often used in SFE because of its low critical values (T c = 31.1°C, P c = 72.8 atm). Carbon dioxide is also readily available in a highly pure form and is inexpensive and non-toxic. Pure CO 2 , however, is not suitable for the extraction of the more polar phenolic compounds. Therefore, a modifier such as EtOH or MeOH must be used to increase solubility of these compounds in the supercritical fluid. The important parameters involved in the extraction of polyphenols by this technique include supercritical fluid carbon dioxide density, type of organic modifier used, modifier percentage, extraction temperature and trap type. SFE is widely used for extraction of plant phenolics owing to the absence of both heat and light during the extraction process, which can reduce the incidence of degenerative reactions that may occur when using other extraction techniques. A recent review on the applications of SFE in food analysis and natural products analysis has been given by Mendiola et al. (25). Subcritical (carbon dioxide and ethanol) extraction of polyphenols from apple pomace was optimized by Adil et al. (26) using various pressures, temperatures, ethanol concentrations and extraction times. Maximum extraction yields were obtained at 20% ethanol concentration, 60 °C at 60 MPa for 40 minutes. The total polyphenols content and DPPH free radical scavenging of the extracts under these conditions was 0.47 mg gallic acid equivalents (GAE)/g fresh 16 weight of sample (1.566 mg GAE/g DW) and 3.3 mg DPPH/mg sample. The total phenols content of this extract was low compared to extracts obtained previously from solvent extraction techniques, probably because of limited solubility of the specific apple polyphenols in carbon dioxide. Therefore, other extractions techniques need to be considered for this purpose. 2.3.3 Accelerated solvent extraction (ASE) ASE (also known as Pressurized Solvent Extraction or Pressurized Fluid Extraction) is a solid-liquid extraction process which is performed at elevated temperatures and pressures. The elevated pressure increases the boiling point such that the solvent remains in a liquid state at elevated temperatures. The increased pressures also tend to “push” the solvent into the matrix pores, thereby making the analyte more accessible. This is a type of pressurized solvent extraction similar to SFE, and is usually considered as an alternative to SFE for the extraction of polar compounds (27). This technique is rapid (compared to classical extraction methods) and can be used with any kind of solvent or solvent mixtures (with the exception of solvents which have flash points at low temperatures and corrosive acids and bases). Most of the applications for ASE are in the area of environmental research for the extraction of pollutants from environmental matrices. Extensive work has been done in the extraction of polycyclic aromatic hydrocarbons (PAH) and polychlorinated biphenyls (PCB) as well as other pollutants. These have been summarized by GiergielewiczMoajska et al. (28) . Kaufmann and Christen (29) reviewed recent developments in 17 accelerated solvent extraction of natural products. Some of these include xanthones and flavonones, curcuminoids, flavonolignans, terpenes and saponins. The use of ASE in the extraction of bioactive compounds from apple pomace is limited. Alonso-Salces et al. (30) optimized a pressurized liquid extraction of apple polyphenols from Golden Delicious apple pomace by varying the temperature, solvent composition, pressure and extraction time. The optimum extraction efficiency was obtained at 5 minutes extraction time, 40 °C using 100% methanol at 1000 psi. The total phenols obtained under these conditions as measured by reverse phase HPLC was 7.566 mg/g DW, which is comparable to yields obtained using solvent extraction techniques. However, considering the capital cost of the instrumentation and relatively high solvent volumes required, consideration of other extraction techniques may prove to be valuable. 2.3.4 Microwave assisted extraction (MAE) Principle and Mechanism: Microwaves are electromagnetic radiation with frequencies ranging from 0.3 to 300 GHz. Microwave assisted extraction has been used increasingly for extraction of phenolics and other compounds. It has a number of advantages compared to other extraction techniques, such as increased yield, shorter extraction time and use of less solvent. MAE delivers energy rapidly to a total volume of solvent and solid plant matrix with subsequent heating of the solvent and solid matrix, efficiently and homogenously. The principle of heating using microwaves is based on the direct effect of the waves on molecules by ionic conduction and dipole rotation. The ionic conduction generates heat due to the resistance of the medium to ion flow. Dipole rotation also causes heat 18 generation as the polar molecules try to line up with the electric field and collide with each other. MAE involves first choosing a solvent in which the target analyte is soluble. The polarity of the solvent is important as solvents with high dielectric constants (example water) can absorb more microwave energy. Usually polar solvents are considered better than non-polar ones (31) due to their high dielectric constants which results in rapid heating on application of microwaves. On the other hand, several papers have described the ‘broken cell-wall theory’ according to which microwave-transparent solvents with lower dielectric constants, and thus lower microwave absorbing capacity, are better than microwave-absorbing ones. This is because in a microwave-transparent solvent (e.g. hexane), the microwaves are absorbed by the plant matrix alone. This matrix may have some amount of water which gets heated up, disrupts the cellular structure and releases the desired components into the surrounding solvent medium (32). This type of MAE is also called a ‘cold-extraction’. Parameters influencing MAE There are a number of parameters that influence the microwave extraction process. The commonly studied ones include choice of solvent, solvent volume, extraction temperature and time, and matrix characteristics. Eskilsson et al. (33) discusses each of these parameters in detail and are summarized below: Choice of Solvent: The important characteristics that must be considered while choosing a solvent are its microwave-absorbing properties, interaction of the solvent with the matrix, 19 and analyte solubility (34). The solvent must also be capable of selective solute extraction. Common solvents employed are acetone, methanol, ethanol, hexane and water, or various mixtures of these (35). Solvent Volume: The volume of the solvent required varies depending on the sample, but is usually within 10 – 30 ml (33). Very low volumes are required in some cases. For example, phenol and methyl phenol extracted from soils required very small amounts of solvent, with an optimum volume of 10 ml for a 5 gm sample size (36). The solids/solvent ratio and solvent concentration are also important parameters that must be taken into account. Shahidi and Naczk (37) found that changing the sample/solvent ratio from 1:5 to 1:10 significantly increased the extraction yields of condensed tannins and total phenolics from commercial canola meals. However, other studies have shown that increasing the solvent volume may reduce the extraction yield after a certain value. Chee et al. (38) reported this effect while extracting polycyclic aromatic hydrocarbons (PAH’s) from sediment. In that study, it was found that 30 ml of solvent gave higher recoveries than 45 ml. Kovaks et al. (39) also found on extracting free amino-acids from various foodstuffs, that higher yield was obtained when the extraction was performed at lower solvent volumes. The solids/solvent ratio must thus be obtained experimentally and varies with each sample matrix. Temperature: The influence of temperature seems to depend on the matrix. Some studies have shown that the temperature is not a very significant factor (40) while others have shown significant variation in extraction yields at different temperatures (41). Higher temperatures, when used for long periods of time, result in degradation of thermolabile 20 phenolic compounds. Kiss et al. (42) studied the extraction of carotenoids from paprika using MAE. The extractions were optimized to below 60 °C, as higher temperatures lead to degradation and reduced yield. Extraction Time: Extraction times are relatively short (typically 30 seconds to 20 minutes) in MAE as compared to other conventional extraction techniques (43). About four minutes of irradiation was sufficient to extract the polyphenols from various aromatic plants of Greek origin (44). Matrix Characteristics: Some studies report that MAE is not matrix dependent, and similar extraction yields are obtained irrespective of the nature of the matrix (45). However, many other papers report that the matrix can have a profound effect on the extraction yield. Lopez-Avila et al. (41) performed PAH (Polycyclic Aromatic Hydrocarbon) extraction of 6 different matrices using the same procedure and obtained recovery rates ranging from 57% to 99% for different matrices. The matrix moisture content is an important parameter that can affect the extraction efficiencies, depending on the nature of the solvent used. Samples with high water content can be easily extracted using relatively non-polar solvents; while dry sample matrices are preferred for extraction with more polar solvents. Microwaves have been used increasingly for the fast extraction of a variety of compounds. A number of scientific publications report the use of MAE for persistant organic pollutants (POPs) (41, 46-49). A large number of pesticides have also been extracted using MAE such as organochlorine pesticides (OCP) and OPP’s (36, 50-51). In 21 addition, microwaves have been used for the extraction of several biological compounds such as essential oils from the leaves of rosemary and peppermint (52), taxanes from taxus biomass (53) and extraction of ergosterol and total fatty acids from fungal hyphae and spores, mushrooms, filtered air, artificially contaminated corn, naturally contaminated grain dust and soil (54). Extraction of phenolic compounds from tea leaves have been studied recently (55). Phenolic compounds have also been extracted from grape seed (56) using MAE. MAE is increasingly being used as an alternative to traditional extraction methods for the removal of phenolics from plant tissues, as it significantly reduces extraction time and solvent consumption while generating higher extraction yields (43, 55). Limited studies have investigated the potential of MAE in the extraction of bioactive compounds from plant waste streams such as apple pomace. The extraction of pectin from dried apple pomace powder was optimized by Wang et al. (57), using HCl buffer (pH 1.01) at 499.4 W microwave power, 20.8 minutes extraction time and 0.069 solid/liquid ratio. However, the extraction of polyphenols from apple pomace using MAE has not been previously reported, and could be a potential tool for rapid and efficient extraction. 2.4 Quantification of phenolic compounds Once the phenolic compounds are extracted from their plant source, they are typically characterized and quantified by spectrophotometric and chromatographic methods. The Folin-ciocalteu assay is the most widely used analytical method to estimate the total polyphenol content in a sample. The phosphomolybdic acid in the Folin reagent 22 reacts with the phenols in alkaline solution to give a blue colored complex which is then read spectrophotometrically at 760 nm. The phenolic content is commonly expressed as percent gallic acid equivalents (GAE) per gram of sample. However, reducing substances like sugars and ascorbic acid may also react with the Folin reagent and cause interference and inaccurate estimation, which is one of the main drawbacks of the assay. HPLC is the most common quantitative analytical tool used to study polyphenols, such as anthocyanins, procyanidins, flavonoids, phenolic acids, etc. Several reviews on the application of HPLC for the study of plant phenolics have been published. A typical separation system involves a reverse phase HPLC (RP-HPLC) comprising a C-18 or C-30 stationary phase. Isocratic elution has been proved sufficient in some separations. However, gradient elution is more commonly applied and results in better separation. A number of different mobile phases have been employed, but a typical separation system uses a binary solvent system comprising an aqueous component and a less polar organic solvent such as acetonitrile. The common detectors used for food phenolics include UVVis, photodiode array (DAD) and UV-fluorescence detectors, although the use of various other detectors has also been reported. Structural characterization of phenolics is usually done using HPLC coupled with mass spectrometry (58). 2.5 Estimation of antioxidant activity Polyphenols are known to be effective antioxidants and scavengers of free radicals (7). There are a number of methods to estimate antioxidant capacity of phenolics. Based on the mechanism of the reaction involved, assays are often classified as HAT 23 (hydrogen atom transfer) or SET (single electron transfer). HAT based assays measure the ability of an antioxidant to quench free radicals by the donation of a hydrogen atom, and some examples of this type include oxygen radical absorbance capacity (ORAC) and total radical trapping antioxidant parameter (TRAP). SET based assays measure the ability of the antioxidant molecule to reduce free radicals, metals or carbonyls by the transfer of an electron. Examples of the SET type of antioxidant assays include diphenyl picryl-hydrazyl (DPPH), trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP) as well as the Folin-ciocalteu total phenols assay (59). Unfortunately, these different assays used to evaluate antioxidant potential vary depending on the testing conditions, medium, type of substrate and reaction period. This complicates our ability to effectively compare values of antioxidant effectiveness of plant extracts from literature. Further, most of these assays occur under controlled conditions which do not simulate physiological conditions, so understanding the antioxidant potential of the test substrate in food and biological systems becomes complicated. However, despite these drawbacks, these methods are still used for reporting antioxidant potential because they are rapid, inexpensive and easy to use. Further, simple comparisons of plant extracts obtained between different experimental treatments can be successfully carried out with these types of assays due to their good within-run and between-day reproducibility (59). 2.5.1 ORAC assay The ORAC measures the antioxidant inhibition of peroxyl radical induced oxidations by hydrogen atom transfer (60). The peroxyl radical reacts with a fluorescent 24 probe to form a non-fluorescent product, which is then quantified by fluorescence (59). Antioxidants delay degradation of the probe by intercepting free radicals generated by AAPH (2,2’-azobis(2-aminopropane) dihydrochloride), thereby causing a delay in fluorescence decay. The reduced rate and amount of product formed over time can then be monitored to estimate the antioxidant capacity, which is reported as micromoles of Trolox equivalents per liter or gram of sample. Trolox is a water-soluble form of vitamin E and is used as the antioxidant standard. The values are determined by measuring the area under the curve. ORAC is generally preferred over the other antioxidant estimation methods because it models the reaction of antioxidants with peroxyl radicals as occurs in the human body. Many food companies now include ORAC values on their product labels. This assay may be easily automated and several improvements in the design of the instrumentation and fluorescent probe (commonly fluorescein) have been reported (6061). The fluorescent markers used in the assay are very sensitive. However, their use requires fluorometers for detection, which are rather expensive and not routinely available in laboratories. Another disadvantage of this technique is the relatively long analysis time (approximately 1 hour). Further, the ORAC reaction is temperature sensitive and small temperature differences in the external wells of the microplate can decrease the reproducibility of the assay (60). 2.5.2 DPPH Assay DPPH is a stable nitrogen-centered free radical that can be used to estimate antioxidant activity. This assay is based mainly on electron transfer reaction, rather than 25 hydrogen atom abstraction (59). The odd electron in DPPH gives a strong absorption maximum at 517 nm and is purple in color. When the odd electron of the DPPH radical becomes paired with a hydrogen from a free radical scavenging antioxidant to form reduced DPPH –H, the molar absorptivity of the DPPH radical reduces from 9660 to 1640 and its color changes from purple to yellow (62). The antioxidant assay is based on spectroscopic measurement of the loss of color at 517 nm after reaction with the test compounds. This assay is extremely rapid and simple, requiring only a UV-Vis spectrophotometer. However, there are a number of drawbacks with the DPPH that may result in inaccurate interpretation of the antioxidant capacity. Certain compounds may interfere by absorbing at the wavelength of measurement. Carotenoids, for example, have been found to interfere (63). Further, DPPH is a very stable free radical unlike the highly reactive radicals which are responsible for lipid oxidation in foods. Many antioxidants show different activities with DPPH and with highly reactive radical species, leading to either over- or under-estimation of the radical scavenging potential of the extract. Despite these limitations, DPPH is one of the most widely used methods for reporting the antioxidant potential of plant extracts because it is simple, rapid and relatively inexpensive. 2.5.3 FRAP Assay The FRAP assay is another assay which is based on singlet electron transfer. This method has been widely used to evaluate the antioxidant capacity of several botanical extracts (64-65). The FRAP assay measures the reduction of ferric 2,4,6-tripyridyl-striazine (TPTZ) to a colored (blue) product and this can be monitored 26 spectrophotometrically at 595 nm. The FRAP assay is simple, rapid and inexpensive and does not require any special equipment. However, the results may fluctuate widely depending on the analysis time. Fast reacting phenolics must be analyzed with short reaction times while polyphenols that react slowly require longer reaction times for detection (59). 2.5.4 TEAC Assay The TEAC assay utilizes both electron transfer and hydrogen atom transfer to estimate the antioxidant capacity. This method measures a long life radical cation ABTS+ which are formed by the oxidation of ABTS by oxidants. There is a decrease in color which is measured spectrophotometrically for a fixed time to determine the antioxidant activity of the test compound. This assay is rapid and simple. ABTS reacts over a wide range of pH and is soluble in both aqueous and organic solvents. 2.6 Encapsulation of polyphenols Phenolic compounds may easily be destroyed by light, oxygen and other factors once removed from the plant matrix. This makes them difficult to store and transport. In addition, several of these compounds are highly hydrophobic and insoluble in aqueous systems. Encapsulation of these compounds using some wall material with high stability and good solubility characteristics would help in overcoming these problems. This would enable the use of these systems in oil-in-water emulsions, etc. 27 Encapsulation is a technique by which a material or a mixture of materials is entrapped within another material or system. It is commonly used to stabilize the entrapped compound, and to provide protection from environmental or subsequent harsh processing conditions. In several cases, encapsulation enables the incorporation of the compound in systems where it is insoluble in the native state. Encapsulation has traditionally been used extensively in the pharmaceutical industry for the incorporation of various drugs and other ingredients within a capsule. This was later extended to food processing and has several applications. A number of components have been encapsulated in the food industry, such as vitamins, minerals, amino-acids, flavors, colorants, enzymes, sweeteners and antioxidants (66). The substance that is encapsulated is often referred to as the core material, while the coating material is called the shell, wall, carrier or encapsulant (67). The process of encapsulation consists of two essential steps: the first is the formation of an emulsion between the core material and the wall material. The second is drying or cooling of the emulsion to obtain microcapsules. These microparticles may be obtained by chemical processes, such as coacervation, co-crystallization, molecular inclusion and interfacial polymerization, or they may be obtained by mechanical processes such as spray-drying (most common), spray chilling/cooling, extrusion and fluidized bed. Madene et al. (67) has reviewed the theory of each of these techniques in detail. 2.6.1 Wall materials There are a number of wall materials available for use in encapsulation. A suitable wall material must protect the core material from factors that may cause its degradation, 28 prevent premature interaction between the core material and other ingredients, limit volatile losses and allow a controlled release of the core material under desired conditions (66). There are several sources of wall materials permitted for use in food ingredients such as proteins (milk or whey proteins, gelatin, etc.), natural gums (gum arabic, alginates, carragenans, etc.), maltodextrins with different dextrose equivalence, waxes and their blends. The right choice of a wall material can be determined experimentally by forming and evaluating the microcapsules for encapsulation efficiency, stability under different storage conditions, degree of protection provided to the core material and by surface observation using scanning microscopy (68). Gums Acacia gum or gum Arabic has good emulsification properties and has been used extensively for food emulsification (69). However, it is very expensive compared to other available wall materials. Bixin (a carotenoid obtained from the seed of Bixa orellano) was encapsulated with gum arabic and maltodextrin. The core material was found to be more stable when trapped in gum Arabic, compared to maltodextrin + Tween 80 (70). Zhang et al. (71) studied various microencapsulation methods of procyanidins extracted from grape seeds using gum Arabic and maltodextrin as the wall material. Spray-drying was used to prepare the microcapsules, with an efficiency of 88.84%. Proteins Proteins such as milk or whey protein and gelatin also have good functional properties and possess good binding properties for flavor compounds. Whey proteins 29 have been successfully used for encapsulating anhydrous milk fat by spray drying, with an encapsulation yield greater than 90% (72). Gelatin is a water-soluble wall forming material that has wide applications in the food industry (73). Carbohydrate based wall materials Several carbohydrate compounds have been used for incorporation of ingredients. Maltodextrins were found to be effective in protecting the carotenoids of paprika oleoresin (74). Modified starches in which the starch molecule has been chemically treated with hydrophobic groups such as octenyl side chains to increase their affinity for hydrophobic flavor compounds, have widely been used as encapsulating materials for flavor compounds (75). Pectin is another polymer capable of producing stable emulsions at low concentrations of around 1-2% (75). Alginates are gum like material obtained from brown seaweed, and contains a number of free carboxylic acid groups which react with divalent cations, mainly calcium to form stable gels (76). They are widely employed in drug delivery systems. Deladino et al. (77) studied the encapsulation of natural antioxidant compounds extracted from yerba mate (Ilex paraguariensis) using two different systems, calcium alginate and calcium alginate-chitosan. Cyclodextrins Modified starches have very good functionality to be used as wall materials. Cyclodextrins (CD) are enzymatically modified starch molecules obtained by the action of cyclodextrin glucosyltransferase on starch. The starch molecule is cleaved by the 30 enzyme and the ends join together to form a circular molecule with six, seven or eight glucose residues linked by α 1,4 glucosidic bonds, to form α-cyclodextrin, βcyclodextrin and γ-cyclodextrin, respectively (Fig 2.2 a). CDs can form inclusion complexes with a variety of hydrophobic compounds, by entrapping the entire compound, or at least part of the compound, in the apolar cavity. The complex formation relates to its structure (Fig 2.2 b). The hydroxyl groups of the CD molecule are located on the wider edge of the ring, resulting in a hydrophilic outer shell suitable for solubility in aqueous food systems. The apolar carbon and hydrogen atoms and the ether-like oxygen atoms are present in the inner surface to form the relatively hydrophobic cavity (78). (a) (b) Figure 2.2 Structure of Cyclodextrin molecule (a) top view of α, β and γ-CD (b) 3 -d surface view of β-CD 31 The important factors associated with complex formation with a guest molecule are steric and thermodynamic effects (78). The size of the hydrophobic cavity must be suitable with the guest molecule. In addition, the reaction must be driven forward by the displacement of water molecules from the cavity, and formation of hydrophobic interactions between the guest and the CD. This results in a thermodynamically favorable reaction. Studies have shown that addition of excess water to the system tends to shift the equilibrium to the left, resulting in dissociation of the guest-CD complex, and release of the guest molecule (78). β-CD is the most widely used CD for encapsulation purposes as it is more available and lower priced compared to the other two parent CDs. However, it has lower water solubility (1.85% w/v at 25°C) compared to α-CD (14.5% w/v) and γ-CD (23.2% w/v) (78). However, the number of glucopyranosyl residues, and thus the cavity diameter of β-CD is more suitable for the entrapment of a variety of molecules. Beta-cyclodextrins have been commonly used for the encapsulation of phenolic compounds such as resveratrol (79). Lucas-Abellan et al. (79) studied the encapsulation of quercetin and myricetin flavonols in beta-cyclodextrins in acidic medium. Derivatives of β-CD have been produced by chemical modification of the parent molecule by aminations, etherifications and esterifications in order to produce wall materials with improved solubility and stability characteristics (78). 2-hydroxypropyl-βCD is an example of a modified CD with improved solubility characteristics, and has been used as an encapsulant for a variety of food ingredients. 32 2.6.2 Analysis of inclusion complexes Phase solubility study Enhancing the water solubility of hydrophobic compounds is one of the major advantages of encapsulation. The most widely applied analytical method used to study the aqueous solubility of inclusion complexes formed by cyclodextrins is the phase solubility study developed by Higuchi and Connors (80). This method has been applied to analyze the solubility of several ligand-drug complexes, and is widely used in the pharmaceutical industry. Usually, an excess amount of the drug molecule is added to aqueous solutions containing the ligand (cyclodextrin) at increasing ligand concentrations. Inclusion complexes are allowed to form by stirring at constant temperature. This may take anywhere between 48 hours to 1 week (78). Following this, samples are filtered and the water soluble inclusion complexes are analyzed for quantification of the drug molecule. Phase solubility diagrams are then constructed by plotting the ligand (cyclodextrin) concentration on the x-axis and the drug concentration on the y-axis (80). A typical phase-solubility curve is shown in Fig 2.3. Figure 2.3 Phase solubility diagram (adapted from Higuchi & Connors, 1965) 33 Curves obtained are classified mainly into 2 categories, the formation of inclusion complexes with good water solubility (A) and the formation of inclusion complexes with poor water solubility (B). The A L type curve indicates a linear increase of drug solubility with cyclodextrin concentration, and is indicative of a 1:1 binding of the drugcyclodextrin complex (78). AP and A N are representative of positively deviating and negatively deviating isotherms respectively. B S and B I are representative of poorly soluble and insoluble complexes. This method allows a qualitative assessment of complex formation, and may also be used to derive stability constants between the drug and ligand. Differential Scanning Calorimetry (DSC) DSC is a thermal analysis tool that measures the amount of energy required to increase the temperature of the sample and reference, as a function of temperature. DSC has been widely used for the verification of complex formation, by comparing the thermograms of typically four types of samples: pure drug molecule, pure ligand (cyclodextrin), the inclusion complex and a physical mixture of the ligand and drug, prepared at the same ratio as for the inclusion complex. The disappearance of thermal peaks associated with the pure drug or ligand, in the thermogram of the inclusion complex is usually indicative of successful complex formation (81-82). DSC studies carried out under an oxygen atmosphere have also been used for checking the oxidative stability of the complex. The complex and pure drug compound are placed in DSC pans with a hole on their lid, and the experiment is carried out under an oxygen atmosphere. Degradation peaks are usually visible at low temperatures for the 34 pure drug molecule, while they are absent or shifted to the right in the thermogram of the inclusion complex (81, 83). 2.7 Summary Polyphenols are a ubiquitous group of plant compounds that have high antioxidant activity, and have been shown to inhibit lipid oxidation. They have been extracted from their native plant matrix traditionally by solvent extraction. However, research has shown that conventional solid-liquid extraction consumes large amounts of solvent and is also time intensive. Alternate extraction techniques have been discussed, with emphasis on microwave-assisted extraction. Furthermore, studies indicate the poor functionality of polyphenols in their native state due to low oxidative stability and poor water solubility. Encapsulation of these compounds may improve their stability and solubility for use in various food systems. 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Kalogeropoulos, N.; Konteles, S.; Mourtzinos, I.; Troullidou, E.; Chiou, A.; Karathanos, V., Encapsulation of complex extracts in -cyclodextrin: An application to propolis ethanolic extract. Journal of microencapsulation 2009, 99999, (1), 1-11. Kalogeropoulos, N.; Yannakopoulou, K.; Gioxari, A.; Chiou, A.; Makris, D., Polyphenol characterization and encapsulation in [beta]-cyclodextrin of a flavonoid-rich Hypericum perforatum (St John's wort) extract. LWT-Food Science and Technology 2010. Pralhad, T.; Rajendrakumar, K., Study of freeze-dried quercetin-cyclodextrin binary systems by DSC, FT-IR, X-ray diffraction and SEM analysis. Journal of pharmaceutical and biomedical analysis 2004, 34, (2), 333-339. 41 CHAPTER 3. OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION OF PHENOLIC ANTIOXIDANTS FROM APPLE POMACE† Vaishnavi Chandrasekar1, Tameshia S. Ballard1*, Fernanda San-Martín1 and Peter Hirst2 1 Department of Food Science, Purdue University, West Lafayette, IN 47907 2 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907 * Correspondence should be sent to: Tameshia S. Ballard, 745 Agriculture Mall Drive, Purdue University, West Lafayette, IN 47907 Phone: (765) 496-6545 Fax: (765) 494-7953 Email: firstname.lastname@example.org † Submitted to J. Agricultural and Food Chemistry 42 Abstract Microwave-assisted extraction (MAE) was used to extract phenolic antioxidants from apple pomace of four cider apple varieties (Red Delicious, Golden Delicious, Winesap and Jonathan). Response surface methodology was used to optimize solvent type (70% acetone, 60% methanol and 60% ethanol), microwave power (100-900 W), solvent volume to sample ratio (4-12 ml/g dry pomace) and extraction time (30-180 s) for each pomace variety. Optimum conditions were based on total polyphenol content (TPC) and antioxidant activity as measured by inhibition percentage (IP) of the 2,2-diphenyl-1picrylhydrazyl (DPPH) free-radical. Red Delicious pomace had the highest TPC (15.8 mg GAE/g DW) and DPPH radical scavenging activity (94.4%) obtained under the optimum extraction conditions. Catechin, phloridzin, caffeic acid, chlorogenic acid and quercetrin were some of the major phenolic compounds identified in the extracts. MAE was found to be an effective method of extracting valuable antioxidant compounds from apple pomace. Keywords: polyphenols, apple pomace, microwave-assisted extraction, total polyphenol content, DPPH, response surface methodology 43 Introduction Waste generated from apple processing is substantial. It is estimated that about 25 to 30% of the total world production of apples is processed into cider, juice and other products, which results in several million tons of apple pomace being generated globally (1). Apple pomace consists of the peels, seeds, stems and flesh left over from juice or cider processing and is primarily being treated as an agricultural waste material, with no real value to processors. The pomace is currently being composted or used as animal feed, although this application is limited due to its low protein content (2). However, apple pomace is rich in phenolic antioxidants including epicatechin, caffeic acid, phloridzin, quercetin glycosides and quercetrin (3). Phenolic extracts from apples have shown high in vitro and in vivo antioxidant and antiproliferative activity (4). Consumption of apples has been associated with reduced risk of cardiovascular disease (5), lung cancer (6) and stroke (7), and this has largely been attributed to the presence of polyphenols in the fruit, many of which remain in the pomace after juice extraction. The relative quantities and types of phenolics present vary widely depending mainly on the apple cultivar and other factors such as state of maturation and availability of growth nutrients (8). Extraction of phenolics from apple pomace has been carried out typically by solvent extraction. Foo and Lu (1) obtained a phenolic extract with 70% aqueous acetone (1500 ml/200 g) that contained 7.24 mg GAE/g dry apple pomace and mainly consisted of quercetrin glycosides. Adil et al. (9) optimized the subcritical (carbon dioxide and ethanol) extraction of phenolics from apple pomace at 20% ethanol concentration, 60°C 44 at 60 MPa for 40 min. The total phenols content and DPPH free radical scavenging activity of the extracts obtained was 1.57 mg GAE/g dry pomace and 3.3 mg DPPH/mg sample, respectively. The low values were attributed to limited solubility of apple phenolics in carbon dioxide. Alonso-Salces et al. (10) optimized the pressurized liquid extraction of apple polyphenols from Golden Delicious apple pomace. Total phenols content of the extracts was 7.57 mg/g dry sample obtained under the optimum conditions of 5 min extraction time, 40°C using 100% methanol at 1000 psi. Considering the capital cost of instrumentation needed for these extraction methods and the relatively high solvent volumes required, consideration of other extraction techniques may be helpful. Microwave-assisted extraction (MAE) is a relatively new extraction technique that delivers microwave energy rapidly to a total volume of solvent and solid plant matrix. This results in subsequent heating of the solvent and solid matrix, efficiently and homogenously (11). The principle of heating using microwaves is based on the direct effect of the waves on molecules by ionic conduction and dipole rotation. The plant matrix may contain some water which is rapidly heated by microwaves, disrupting the cellular structure and releasing the desired components into the surrounding medium (12). There are a number of parameters that influence the microwave extraction process. Those most commonly studied include choice of solvent, solvent volume, extraction temperature and time, and matrix characteristics (13). MAE is increasingly being used as an alternative to traditional extraction methods for the removal of phenolics from plant tissues, as it significantly reduces extraction time and solvent consumption while generating higher extraction yields. Ballard et al. (14) 45 found that MAE increased the extraction yield of phenolic antioxidants from peanut skins using less solvent and in a fraction of the time required for traditional solid-liquid extraction. Limited studies have investigated the potential of MAE in the extraction of bioactive compounds from plant waste streams such as apple pomace. The extraction of pectin from dried apple pomace powder was optimized by Wang et al. (15) using 499 W microwave power, 20.8 minutes extraction time and 0.069 solid:liquid ratio. However, the extraction of polyphenols from apple pomace using MAE has not been previously reported and could be a potential tool for rapid and efficient extraction. The influence of several factors on a desired response can be determined using response surface methodology (RSM) which was first described by Box and Wilson (16). This method requires fewer experimental runs while still producing statistically acceptable results (17). It is widely used to evaluate the effect of various factors and their interactions on the response variable, and to optimize multiple responses simultaneously. The central composite design (CCD) is the most commonly used experimental design in this method, and has been used in optimization studies for a number of applications including the extraction of bioactive compounds from plant sources (18-20). The objective of this work was to optimize and compare the extraction of phenolic antioxidants from pomace of four common apple cider varieties (Red Delicious, Golden Delicious, Winesap and Jonathan) using MAE. Response surface methodology was used to optimize solvent type, microwave power, solvent volume to sample ratio and extraction time for each pomace variety. Optimum conditions were based on total 46 polyphenol content (TPC) and demonstrated antioxidant activity as measured by inhibition percentage (IP) of the DPPH free-radical. Methods and Materials Chemicals Catechin, epicatechin, procyanidin B2, caffeic acid, chlorogenic acid, phloridzin, hydrocaffeic acid, hydrocoumaric acid, quercetin and quercetrin were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). 2,2-Diphenyl-1-Picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, sodium carbonate anhydrous and 3,4,5-trihydroxybenzoic acid (gallic acid) were obtained from VWR International (Seattle, WA). Analytical grade methanol, ethanol and acetone, and HPLC grade acetic acid and water were obtained from Mallinckrodt Baker Inc. (Phillipsburg, NJ). Preparation of samples Frozen pomace from four cider apple varieties (Red Delicious, Golden Delicious, Winesap and Jonathan) was provided by Beirsdorfer Orchard (Guilford, IN). Pomace samples were placed in Ziploc freezer bags, flushed with nitrogen gas and stored at -20 °C until use. Samples were lyophilized for 48 hr. in a freeze dryer (Virtis Co., Gardiner, NY), ground to less than 1 mm particle size using a bench top coffee grinder (Hamilton Beach brand, Washington, NC) and used for extraction. 47 Microwave-assisted extraction Extractions were carried out in a Mars Xpress microwave extraction system (CEM Corp., Matthews, NC). One gram of ground apple pomace was accurately weighed and quantitatively transferred to teflon-lined extraction vessels. The appropriate volume of solvent was added, a stir-bar was inserted and the vessel was closed and exposed to microwaves for the designated time period. The extraction vessels were allowed to cool to room temperature after extraction. Extracts were then transferred to 15 ml centrifuge tubes and centrifuged at 1050 rpm for 20 min. in a Dynac centrifuge (Clay Adams, Division of Becton Dickinson and Co., Parsippany, NJ). The supernatant was then transferred to glass centrifuge tubes, flushed with nitrogen gas and stored at -20°C until analysis. Extracted samples were stored no longer than 2 weeks prior to analysis. Two replications were carried out for each treatment. Total phenols determination The total polyphenols content (TPC) of the extracts was determined using the Folin-Ciocalteu assay as outlined by Spanos & Wrolstad (21). Gallic acid standards ranging from 0.1 to 0.5 g/L were prepared. 0.1 ml of the sample and standards were taken in 15 ml test tubes. 3 ml of 0.2 N Folin-Ciocalteu reagent (Sigma-Aldrich Co., St. Louis, MO) was added to each tube and mixed on a vortex mixer. 2 ml of 9% (w/v) sodium carbonate solution in water was added to each tube, and again mixed using a vortex mixer. Absorbance was read after 2 hr. at 765nm using DU720 spectrophotometer (Beckman Coulter Inc., Fullerton, CA). The phenolic content of the extracts were expressed in mg gallic acid equivalents (GAE)/g dry weight (DW) of apple pomace. 48 DPPH radical scavenging assay The free radical scavenging activity was determined spectrophotometrically using a modified version of the DPPH assay reported by Garcia et al. (22). Extracted samples were evaporated to dryness under vacuum, and reconstituted in same volumes of methanol prior to analysis. 40 µL of sample, appropriately diluted in methanol, was added to 1.45 mL DPPH solution (40 mg/L in methanol). The solution was mixed using a vortex mixer and allowed to stand in the dark for 30 minutes. Absorbance of the samples was read at 517 nm using a DU720 spectrophotometer (Beckman Coulter Inc., Fullerton, CA). The decrease in absorbance was calculated in comparison to a blank sample (40 µL methanol and 1.45 mL DPPH) and results were expressed as inhibition percentage (IP): IP (%) = 1 - (A e/A b ), where A e = absorbance of sample extract and A b = absorbance of blank. HPLC analysis The major phenolic compounds in the extracts were analyzed by reversed phase HPLC based on the method used by Suarez et al. (23). An Agilent 1200 Series HPLC (Santa Clara, CA) system with a diode array detector was used. An Xterra C-18 column (3.9 x 100 mm, I.D. 3.5 µm; Waters Corp.) was utilized and the column temperature was equilibrated to 25 °C. The binary mobile phase consisted of solvent A: 2% aqueous acetic acid and solvent B: 100% methanol. The gradient elution was performed as follows: a linear step from 0 to 45% of solvent B in 55 min, an isocratic step of 15 min and finally another linear step from 45 to 55% of solvent B in 10 min. Flow rate of the mobile phase 49 was set at 0.8 mL/min. Injection volume was 10 µL. Identification of phenolics in the extracts was achieved by comparing their spectra and retention times with those of externally injected standards, when available. Absorbance was measured at 313 nm for detection of hydroxycinnamic acids, 355 nm for flavonol glycosides and 280 nm for the rest of phenolic compounds (benzoic and 3-phenylpropionic acids, flavanols, procyanidins and dihydrochalcones). Experimental design and statistical analysis Response surface methodology with a central composite design was used to optimize the extraction of phenolic antioxidant compounds from each of the four apple pomace varieties and three solvent systems. Design Expert 7.1.6 (Stat-Ease Inc., Minneapolis, MN) was used for experimental design and statistical analysis. The effects of three independent variables: solvent volume to sample ratio (A), microwave power (B) and extraction time (C) on the two measured responses (TPC and IP) were studied. The coded values and corresponding actual values of these independent variables are shown in Table 3.1. The variables were studied at five levels: -1.7, -1.0, 0.0, 1.0, 1.7 (with α = 1.7), with four replicates at the center, giving rise to 18 experimental points (Table 3.2). The data was analyzed to fit the quadratic model given below: Y = β 0 + Σ β i X i + Σ β ii X i 2 + ΣΣ β ij X i X j whereY is the predicted response; X i and X j are independent variables; β 0 is a constant ; β i, β ii and β ij are the coefficients for the linear, quadratic and interaction terms respectively. Models were generated through least-squares regression analysis. Three-dimensional 50 response surface and contour plots were generated by the software by keeping one variable at the optimal value and varying the other two. The models were evaluated based on their F statistic, p value and R2 value generated by analysis of variance (ANOVA). Significance of model terms was established at the 5% probability level (p < 0.05). Significance of difference test was carried out using SAS statistical software package (SAS Version 9.2, SAS Institute Inc., NC). Results and Discussion The concentrations of solvents used and the range of extraction parameters chosen in this study were based on preliminary experiments (data not shown). Response surface models were fitted based on the TPC and DPPH radical scavenging activity for each apple cultivar and solvent system. Model fitting Response surface models were fitted using regression, taking into consideration only significant factors (p < 0.05). Model assumptions of normality, constant variance and independence of observations were checked. Transformation of the data was carried out in some cases to obtain a better model fit, where the response variable is related to the model parameters as a power function instead of the customary linear model. ANOVA of the regression equations were performed. The predicted models in terms of coded units, along with their F and p statistics and R2 values are shown in Table 3.3 (based on TPC) 51 and Table 3. 4 (based on DPPH IP). The models obtained using the three different solvent systems are discussed in detail. Acetone extraction All models for acetone extracts based on TPC were found to be significant (p < 0.05) with R2 values of 0.90, 0.87, 0.92 and 0.85 for Red Delicious, Golden Delicious, Jonathan and Winesap apple pomace varieties, respectively. This indicated that the models could be used to predict the response variables. Solvent volume, microwave power and time were found to be significant model parameters. Fig. 3.1 shows the effect of microwave power and extraction time on the TPC of Red Delicious pomace. Increase in microwave power from 265 W to 735 W and extraction time from 61 s to 149 s produced higher yields of total phenols in the extract. However, it was found that further increases in microwave power and extraction time lead to superheating of the solvent, which exceeded pressure limits of the microwave extraction unit. Due to this constraint, a larger range of these parameters were not studied and optimizations were carried out within this range. This was true for all subsequent extractions. The effect of solvent volume and power on the TPC of pomace extracts can be seen in Fig. 3.2. In Fig. 3.2 (a), solvent volume of 5.6 ml/g DW and microwave power of 735 W yielded the maximum predicted TPC of 0.46 mg GAE/ml (2.6 mg GAE/g dry pomace) for Golden Delicious apple pomace variety. Similar patterns were observed for Winesap and Red Delicious pomace samples extracted with 70% acetone. In general, 52 smaller solvent volumes produced higher extraction yields. For the Winesap pomace extraction, it was found that as the solvent volume was reduced from 10.3 to 5.6 ml, the TPC increased by 54.2% from 0.59 to 0.91 mg GAE/g DW (Fig. 3.3). This is in contradiction to what is typically observed during conventional extraction methods where higher solvent volumes generally produce increased yields of phenolic compounds. However, there have been reports of the opposite trend occurring using MAE, where higher solvent volumes have been shown to decrease extraction yield in some instances. For example, it was found that lower solvent volumes lead to higher yields of pectin from apple pomace using MAE (15). The same phenomenon was observed when MAE was used to extract polycyclic aromatic hydrocarbons (PAHs) from sediment using acetone:hexane (1:1) as the extraction solvent (24) and for the MAE of felodipine tablets (25). It is not currently known why lower solvent volumes have lead to higher extraction yields when using MAE and this phenomenon warrants further investigation. Response surface models for acetonic extracts fitted based on the DPPH radical scavenging activity were also found to be significant (p < 0.05) with R2 values of 0.95, 0.93, 0.82 and 0.66, respectively for Red Delicious, Golden Delicious, Jonathan and Winesap pomace samples. The solvent volume to sample ratio significantly impacted the extraction of phenolics with high DPPH radical scavenging activity for all the apple pomace varieties. When extracting Jonathan pomace with 70% acetone, there was an 18.5% increase in the inhibition percentage from 73.09 to 86.62, as the solvent volume was decreased from 10.3 to 5.6 ml/g DW (Fig. 3.4). Thus, it appears that MAE of apple 53 pomace using 70% acetone requires lower solvent volumes for maximum TPC and DPPH activity. Ethanolic extraction The models for ethanolic extraction were also found to be significant (p < 0.05) and were adequate for predicting the response variables. The factors, microwave power and extraction time, had a significant effect on the models based on TPC. Higher microwave power and extraction time produced maximum TPC values in all the apple pomace samples. The interaction plot between microwave power and time for Red Delicious apple pomace extracted with 60% ethanol illustrates the effect of power and time on TPC (Fig. 3.5). Even though solvent volume did not seem to be significant, its interaction effects were fairly significant (p < 0.1), and as a result, higher solvent volumes were found to be more effective in obtaining extracts with higher total phenols. Solvent volume was the only parameter that had a significant effect on the DPPH inhibition percentage in Jonathan ethanolic extracts (Fig. 3.6). Decreasing the solvent volume resulted in higher DPPH radical scavenging activity in the extract. Methanolic extraction The methanolic models based on TPC were also found to be significant (p < 0.05). Inverse transformations of the data were required for ensuring the validity of model assumptions and obtaining better fit. In this case, the response was inversely related to the model parameters. Models based on the DPPH inhibition percentage were found to be significant for Red Delicious, Golden Delicious and Winesap pomace 54 extracts. Trends similar to those obtained for ethanolic extracts, where increasing microwave power and time lead to increased TPC were observed. In addition, a decrease in solvent volume caused an increase in DPPH activity for all pomace varieties extracted using 60% methanol. Extraction time was a significant factor in all the models. An increase in the extraction time within the range tested in this study yielded extracts with higher TPC and DPPH activity. Model optimization and verification The objective of the optimization was to achieve extraction conditions that yield extracts containing high amounts of phenolic antioxidants and demonstrate high DPPH radical scavenging activity. The TPC and DPPH inhibition percentage were closely correlated (R2 >0.80) for all the extraction conditions. However, the ethanolic and methanolic extracts that had high phenolic content exhibited lower radical scavenging activity when compared to acetone extracts having lower TPC. This indicates the specificity of certain polyphenols to be extracted under different conditions, and the varying degrees of antioxidant capacities of individual compounds. The Design Expert software was used to estimate the “sweet-spot” in which the optimization was based on maximizing both TPC and DPPH activity simultaneously. The predicted optimum conditions were referred to as “desirability” and are shown in Table 5.5. Each solvent system had identical extraction conditions independent of the apple cultivar. A microwave power of 735 W was observed as optimum for all samples and solvent systems, indicating that phenolic compounds are not degraded by high temperatures in this method as they are exposed for very short periods of time. 55 In general, Red Delicious pomace extracts contained higher amounts of total phenols compared to the other cultivars (Table 3.5). The optimum conditions were predicted at 10.3 ml solvent volume per gram dry sample extracted at 735 W power and 149 s extraction time. Due to equipment constraints that resulted in solvent superheating, higher microwave powers and extraction times were not tested. The maximum DPPH inhibition percentage of 94% was obtained using 5.65 ml of 70% acetone at 735 W and 149 s extraction time. The response surface plots optimized for both the TPC and DPPH IP (desirability) for Red Delicious samples using acetone and ethanol solvent systems are shown in Figs. 3.7 and 3.8. To determine the accuracy of the model, Red Delicious pomace was extracted under the predicted optimum conditions based on TPC and DPPH IP (Table 3.6). Extractions were performed in triplicate and analyzed for their TPC and DPPH IP. Extraction with 60% ethanol resulted in an experimental TPC value of 16.8 mg GAE/g DW compared to the predicted value of 15.8 mg GAE/g DW, representing a 6.5% increase over the predicted value. The radical scavenging activity was also higher by 4.1% (77.12) compared to the predicted value (74.1). A possible explanation for this deviation could be that the optimal design points were located near the edge of the model, which is typically less accurate than the model center. However, the experimental values are still satisfactorily close (< 10%) to the predicted values. When extracting with 70% acetone, smaller deviations from the predicted responses were observed, possibly due to the fact that these models had better fit and R2 values. 56 HPLC characterization The major phenolic compounds extracted under optimal conditions by MAE were quantified by HPLC (Table 3.7). Extracts from all apple pomace varieties were found to contain catechin (192.51-351.60 mg/kg), phloridzin (25.25-102.9 mg/kg), caffeic acid (9.32-44.92 mg/kg), chlorogenic acid (22.56-38.40 mg/kg) and quercetrin (44.69-211.34 mg/kg). However, each solvent system showed a slightly different phenolic profile, indicating the ability of different solvents to preferentially extract certain compounds. Catechin and chlorogenic acid were found to be present exclusively in the methanolic extracts, independent of pomace variety. Caffeic acid was also present in higher amounts in the methanolic extracts as compared to acetone and ethanol in the four tested apple cultivars. Procyanidin B2 was present in the methanolic extracts from Jonathan and Winesap varieties. This selectivity can be attributed to solvent polarity. Methanol is the most polar solvent and thus was capable of extracting the more polar phenolics and hydroxycinnamic acids. Acetonic extracts had higher amounts of phloridzin compared to the other solvent systems, independent of pomace variety. This may be responsible for the high antioxidant activity of these extracts. Lee et al. (4) found that phloretins were one of the major contributors of antioxidant activity in apple pomace extracts along with quercetin. The HPLC chromatogram of Red Delicious apple pomace extracted with 60% ethanol is shown in Fig. 8. A significant peak (retention time 25.38 min) was obtained in the chromatogram of all extracts, but remained unidentified due to lack of standards, and is 57 suspected to be a quercetin glycoside. High amounts of quercetin glycosides were reported in gala apple pomace analyzed by HPLC (26). Comparison with other extraction techniques MAE was compared to traditional solid-liquid extraction for the ability to extract polyphenols from apple pomace. Wijngaard and Brunton (27) optimized the solvent extraction of polyphenols from an industrial blend of cider varieties. Optimum conditions were obtained using 100 ml/g of 65% acetone extracted at 25 °C for 60 minutes, which resulted in an extract containing 15.1 mg GAE/g DW. Slightly higher, although not significantly different amounts of total phenols (15.8 mg GAE/g DW) were observed in the Red Delicious pomace extracts in the current work using MAE, and this was verified by experimental runs. It can be noted that though comparable amounts of total phenols were extracted by both techniques, significantly shorter extraction times and solvent volumes were required for MAE compared to the traditional solid-liquid extraction method employed by Wijngaard and Brunton (27). The optimal extraction time for MAE was predicted as 149 s versus 60 min for solid-liquid extraction, resulting in an approximate 95% reduction in extraction time. This is consistent with other studies (2829) that have shown reduced extraction time of MAE compared with conventional extraction methods as well as other relatively new extraction techniques (ultrasound assisted extraction, supercritical fluid extraction, etc). In addition, MAE required significantly lower solvent volumes (10.3 ml/g) compared to solvent extraction (100 ml/g). 58 Conclusion MAE was found to be a viable alternative to traditional solvent extraction techniques for the extraction of phenolic compounds from apple pomace. Phenolic antioxidants from a variety of apple pomace cultivars were extracted under conditions of varying extraction parameters (solvent volume to sample ratio, power and time) and solvent systems. Red Delicious pomace variety had the highest TPC (15.8 mg GAE/g DW) and DPPH radical scavenging activity (94.4%) compared to the other pomace varieties. The optimal extraction conditions for this cultivar based on TPC were 10.4 mL of 60% ethanol at 735 W and 149 s. Optimal conditions for DPPH radical scavenging activity were 5.65 mL of 70% acetone at 735 W and 149 s. The predicted values were verified experimentally and found to be satisfactory in predicting the response variables (TPC and DPPH). Catechin, phloridzin, caffeic acid, chlorogenic acid and quercetrin were some of the major polyphenols present in the extracts. Compared to traditional solvent extraction, MAE produces extracts with comparable yields and significantly shorter extraction times and lower solvent volumes. Thus, MAE can be used for the rapid extraction of valuable antioxidants from industrial plant waste streams such as apple pomace, thereby adding value to a waste material and increasing the potential development of these extracts into functional food ingredients and natural food preservatives. 59 Design-Expert® Software total phenols Design points below predicted value 0.928981 0.36367 0.69 Actual Factor A: solvent volume = 8.00 0.635 total phenols (mg GAE/g DW) X1 = B: power X2 = C: time 0.58 0.525 735.29 0.47 617.65 500.00 149.12 127.06 B: power (W) 382.35 105.00 82.94 60.88 264.71 C: time (s) Figure 3.1. Response surface of Red Delicious apple pomace extracted with 70% acetone - Effect of extraction time and microwave power on TPC 60 Design-Expert® Software Original Scale total phenols Design points below predicted value 0.749713 0.232543 0.46 Total phenols (mg GAE/g DW) X1 = A: solvent volume X2 = B: power Actual Factor C: time = 105.00 (a) 0.405 0.35 0.295 0.24 735.29 5.65 617.65 6.82 500.00 8.00 A: solvent volume (ml) B: power (W) 382.35 9.18 10.35 264.71 Design-Expert® Software Original Scale total phenols Design points below predicted value 0.749713 0.232543 Actual Factor C: time = 105.00 (b) 0.46 Total phenols (mg GAE/g DW) X1 = A: solvent volume X2 = B: power 0.405 0.35 0.295 0.24 735.29 5.65 617.65 6.82 500.00 8.00 A: solvent volume (ml) 382.35 9.18 B: power (W) 10.35 264.71 Design-Expert® Software total phenols Design points below predicted value 1.1443 0.493787 0.96 Actual Factor C: time = 105.00 0.855 total phenols (mg GAE/g DW) X1 = A: solvent volume X2 = B: power (c) 0.75 0.645 735.29 0.54 617.65 500.00 5.64706 6.82353 B: power 382.35 8 9.17647 10.3529 264.71 A: solvent volume (ml) Figure 3.2. Response surface of extracts with 70% acetone - Effect of solvent volume and power on TPC on (a) Golden Delicious apple pomace (b) Red Delicious apple pomace and (c) Winesap apple pomace 61 Figure 3.3. Effect of solvent volume on TPC of Winesap apple pomace extracted with 70% acetone 62 Figure 3.4. Effect of solvent volume on DPPH inhibition percentage of Jonathan apple pomace extracted with 70% acetone 63 Figure 3.5. Interaction plot of Red Delicious apple pomace extracted with 60% ethanol Effect of microwave power and time on TPC 64 Design-Expert® Software inhibition percentage Design points above predicted value Design points below predicted value 93.0412 62.0275 Actual Factor C: time = 105.00 DPPH inhibition percentage (%) X1 = A: solvent volume X2 = B: power 91 86.75 82.5 78.25 74 735.29 617.65 5.64706 6.82353 500.00 8 382.35 9.17647 A: solvent volume (ml) 10.3529 B: power (W) 264.71 Figure 3.6. Response surface of Jonathan apple pomace extracted with 60% ethanol Effect of solvent volume and power on DPPH inhibition percentage 65 Design-Expert® Software Desirability Design points above predicted value Design points below predicted value 1 0.700 0 0.525 Actual Factor C: time = 149.12 0.350 Desirability X1 = A: solvent volume X2 = B: power 0.175 0.000 735.29 10.35 617.65 9.18 500.00 B: power (W) 8.00 382.35 6.82 264.71 A: solvent volume (ml) 5.65 Figure 3.7. Response surface of the effect of solvent volume and power on both TPC and DPPH inhibition percentage (desirability) for Red Delicious apple pomace extracted with 60% ethanol 66 Design-Expert® Software Desirability Design points below predicted value 1 0 0.940 X1 = A: solvent volume X2 = B: power 0.705 Desirability Actual Factor C: time = 149.12 0.470 0.235 0.000 735.29 617.65 500.00 5.65 6.82 8.00 382.35 9.18 10.35 B: power (W) 264.71 A: solvent volume (ml) Figure 3.8. Response surface of the effect of solvent volume and power on both TPC and DPPH inhibition percentage (desirability) for Red Delicious apple pomace extracted with 70% acetone 67 Figure 3.9. HPLC chromatogram at 280 nm of Red Delicious apple pomace extracted with 60% ethanol under optimum conditions 68 Table 3.1. Coded values and corresponding actual values of the optimization parameters Code -1 0 1 Solvent volume (A) (ml/g DW) 5.6 8.0 10.3 Power (B) (W) 264.7 500.0 735.3 Time (C) (s) 61 105 149 69 Table 3.2. Experimental design for extraction of phenolic compounds by MAE in terms of coded values Experiment no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Solvent volume (A) (ml/g DW) -1 1 -1 1 -1 1 -1 1 0 0 0 0 -1.7 +1.7 0 0 0 0 Power (B) (W) +1 +1 +1 +1 -1 -1 -1 -1 0 0 0 0 0 0 0 0 -1.7 +1.7 Time (C) (s) -1 -1 +1 +1 -1 -1 +1 +1 0 0 0 0 0 0 -1.7 +1.7 0 0 70 Table 3.3. Response surface models fitted based on TPC for different apple cultivars and solvent systems Solvent system 70% Acetone 60% EtOH 60% MeOH a Apple cultivar Model for TPC (in coded units)a F value p value R2 Red Delicious total phenols = 0.52 - 0.13A + 0.048B + 0.054C + 0.048A2 17.14 < 0.0001 0.90 Golden Delicious 1/(total phenols) = 2.84 + 0.62A - 0.28B - 0.35C - 0.31AB - 0.33AC 12.25 0.0003 0.87 Jonathan total phenols = 0.73 - 0.19A + 0.042B + 0.052C + 0.054A2 27.75 < 0.0001 0.92 Winesap total phenols = 0.69 - 0.16A + 0.052C + 0.053A2 17.72 < 0.0001 0.85 Red Delicious total phenols = 0.63 + 0.17B + 0.17C 3.58 0.0323 0.66 Golden Delicious 1/(total phenols) = 2.69 + 0.43A - 0.50B - 0.53C 7.55 0.0021 0.80 Jonathan (total phenols)-1.5 = 1.70 + 0.50A - 0.38B - 0.29C - 0.35AB 10.74 0.0004 0.82 Winesap 1/(total phenols) = 1.98 + 0.33A - 0.31B - 0.37C 8.57 0.0018 0.65 Red Delicious (total phenols)-1 = 2.18 + 0.33A - 0.39B - 0.39C - 0.24C2 10.03 0.0006 0.85 Golden Delicious 1/(total phenols) = 3.96 + 0.65A - 0.66B - 0.78C - 0.63AC - 0.52B2 - 0.48C2 7.45 0.0026 0.84 Jonathan 1/(total phenols) = 1.75 + 0.22A - 0.21B - 0.20C 6.48 0.0040 0.78 Winesap 1/(total phenols) = 2.04 - 0.40B - 0.37C 7.06 0.0069 0.49 only includes significant model terms (p < 0.05) 71 Table 3.4. Response surface models fitted based on DPPH activity for different apple cultivars and solvent systems Solvent system 70% Acetone 60% EtOH 60% MeOH a Apple cultivar Model for DPPH (in coded units)a F value p value R2 Red Delicious inhibition percentage = 69.32 - 13.46A + 2.57 B + 4.09C + 2.62B2 + 2.31C2 46.99 < 0.0001 0.95 Golden Delicious inhibition percentage = 48.61 - 12.12A + 3.52B + 5.13C + 4.23BC + 3.87A2 19.56 < 0.0001 0.93 Jonathan inhibition percentage = 79.85 - 6.76A + 1.98C 34.42 < 0.0001 0.82 Winesap inhibition percentage = 79.31 - 6.69A 31.10 < 0.0001 0.66 Red delicious inhibition percentage = 62.64 - 7.69A + 4.89B + 6.31C 11.03 0.0004 0.82 Golden delicious inhibition percentage = 32.74 + 0.98B + 4.18C + 8.86BC 3.51 0.0437 0.43 Jonathan inhibition percentage = 82.23 - 8.15A 45.52 < 0.0001 0.74 Winesap inhibition percentage = 46.80 - 5.29A + 6.09C 5.95 0.0078 0.56 Red Delicious inhibition percentage = 50.12 + 9.64B + 8.44C + 5.61B2 + 6.68C2 7.33 0.0024 0.80 Golden Delicious 1/(inhibition percentage) = 0.031 + 4.031 x 10-003A - 4.584 x 10-003B - 6.062 x 10-003C 5.156 x 10-003AC 3.38 0.0421 0.51 Jonathan not significant Winesap inhibition percentage = 62.12 + 7.38C 5.53 0.0159 0.42 only includes significant model terms (p < 0.05) 72 Table 3.5. Optimum conditions for the extraction of phenolic antioxidant compounds by MAE based on both TPC and DPPH activity Apple cultivar Solvent system Solvent volume (A) (ml/g DW) Power (B) (W) Time (C) (s) TPC (mg GAE/g DW) DPPH Inhibition Percentage (%) Red Delicious 70% Acetone 60% EtOH 60% MeOH 5.65 10.35 10.35 735.29 735.29 735.29 149.11 149.12 149.12 8.88 15.81 10.24 94.36 74.08 87.36 Golden Delicious 70% Acetone 60% EtOH 60% MeOH 5.65 10.35 10.35 735.28 735.29 735.29 149.12 149.12 149.12 2.90 8.64 9.92 71.29 46.75 46.75 Jonathan 70% Acetone 60% EtOH 60% MeOH 5.65 10.35 8.00a 735.29 735.29 500a 149.12 149.12 105a 5.79 7.65 - 88.60 90.38 - Winesap 70% Acetone 60% EtOH 60% MeOH 5.65 10.35 8.00a 735.29 735.29 735.29 149.12 149.12 149.12 5.67 5.83 6.28 88.60 62.20 75.47 a term not significant in model 73 Table 3.6. Predicted and experimental values of TPC and DPPH activity for Red Delicious apple pomace extracted under optimal conditions for 60% ethanol Solvent system Optimum conditions 60% ethanol Solvent volume (ml/g DW) 10.4 Power (W) 735 Time (s) 149 Solvent volume (ml/g DW) 70% acetone a Predicted yield Experimental yielda TPC (mg GAE/g DW) 15.8 16.8 ±0.97 DPPH activity (%) 74.1 77.1 ±3.44 5.65 TPC (mg GAE/g DW) 8.88 9.30 ±0.65 Power (W) 735 DPPH activity (%) 94.4 93.7 ±4.20 Time (s) 149 Mean ± standard deviation (n=3) Table.3.7. HPLC quantification of major apple polyphenols from optimized MAE extracts for each apple cultivar and solvent system Apple cultivar Concentration (mg/kg dry pomace)* Solvent system Catechin Epicatechin Procyanidin B2 Phloridzin c nd nd nd nd nd nd d nd nd nd nd nd nd 34.78 f 41.20 g 33.02 a nd nd nd 136.24 nd nd b nd nd nd 21.04 nd nd Red Delicious 60% MeOH 70% Acetone 60% EtOH 297.56 nd nd Golden Delicious 60% MeOH 70% Acetone 60% EtOH 192.51 nd nd Jonathan 60% MeOH 70% Acetone 60% EtOH 351.60 nd nd Winesap 60% MeOH 70% Acetone 60% EtOH 313.36 nd nd a b Gallic acid Caffeic acid 52.78 b 82.59 h 25.25 d nd nd b 189.09 32.71 f 10.00 d 27.63 c 38.40 nd nd a 80.83 f 46.33 i 59.20 g nd nd a 378.81 35.40 f 9.32 e 24.63 b 37.16 nd nd a 125.03 d 68.76 c 119.65 c nd nd c 23.08 33.89 f 9.77 b 34.67 b 38.00 nd e 22.56 a 169.52 c 115.15 a 211.34 e nd nd d 14.39 44.92 f 9.89 b 35.19 a 35.12 nd nd b 76.88 g 44.69 d 81.66 69.68 a 102.90 b 79.80 46.88 c 71.28 d 50.51 Chlorogenic acid Quercetrin d Quercetin nd nd nd c nd nd nd b nd nd 124.30 e nd nd nd * Data expressed as means; n=2 nd = not detected Means with different letters represent significant difference within a column 74 75 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 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ENCAPSULATION OF POLYPHENOL EXTRACTS FROM APPLE POMACE IN CYCLODEXTRINS † Vaishnavi Chandrasekar1, Tameshia S. Ballard1*, Fernanda San-Martín1 and Peter Hirst2 1 Department of Food Science, Purdue University, West Lafayette, IN 47907 2 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907 * Correspondence should be sent to: Tameshia S. Ballard, 745 Agriculture Mall Drive, Purdue University, West Lafayette, IN 47907 Phone: (765) 496-6545 Fax: (765) 494-7953 Email: email@example.com † Prepared for Submission to J. Agricultural and Food Chemistry 78 Abstract Extracts of apple pomace are rich in polyphenols that have high antioxidant activity. Encapsulation of Red Delicious apple pomace extracts obtained by microwaveassisted extraction was investigated in three types of cyclodextrins: α-cyclodextrin, βcyclodextrin and 2-hydroxypropyl-β-cyclodextrin. Quercitrin (84.5 mg/g dry weight of apple pomace), phloridzin (82.2 mg/g) and caffeic acid (18.22 mg/g) were the major phenolic compounds present in the acetone extracts as analyzed by HPLC. Inclusion complexes of the extract with cyclodextrins (CDs) were formed by dissolution in aqueous systems for 48 hours followed by freeze-drying to obtain dry complexes. Complex formation was verified by DSC and confirmed for all the three CD types. Inclusion complexes were found to be stable under oxidative conditions up to 250 °C. Phase solubility studies indicated an A L type diagram indicating the formation of a 1:1 complex of the extract with β-CD with a moderate binding constant (Kc = 1.02 mg GAE/g extract 1 ). α-CD and 2-hydroxypropyl-β-CD did not prove to be effective in forming soluble inclusion complexes from the current techniques employed. Encapsulation of the polyphenol rich apple pomace extract in β-CD results in the formation of stable complexes with enhanced solubility for use as functional food ingredients. Keywords: apple pomace extract, polyphenols, encapsulation, cyclodextrin, DSC, phase solubility 79 Introduction Apple pomace is the waste stream (seeds, skin, flesh) generated from apple juice or cider production. Currently it is used mainly as animal feed, although this application is limited due to its low protein and vitamin content (1). Apple pomace is rich in several polyphenols, such as quercetin glycosides, catechin, epicatechin, chlorogenic acid and phloridzin (2). These polyphenols have been shown to exhibit high antioxidant and radical scavenging activity in vitro (3-4), and may serve as potential replacers of synthetic antioxidants in lipid-based foods that are prone to oxidation. Therefore, the extraction of valuable natural antioxidant compounds from apple pomace may prove to be economically beneficial. In spite of these potential benefits, the use of these compounds as bioactive ingredients in foods is limited by their low water solubility and poor oxidative stability. Encapsulation of bioactive ingredients has been widely used in the food industry to protect these compounds from oxidation, thereby increasing their stability. Further, encapsulation in suitable wall materials may also serve to enhance the solubility, enabling their use in aqueous food systems (5). Encapsulation is commonly carried out by simply emulsifying the guest molecule with a suitable wall material in aqueous solution, after which the water may be removed by freeze-drying or spray-drying to obtain dry inclusion complexes (6). The use of cyclodextrins (CDs) as encapsulates in the food industry is steadily increasing (7). In the past, CDs have been primarily used in pharmaceutical applications for the encapsulation of hydrophobic drug molecules and stabilization of light or oxygen-sensitive compounds (8). However, do to their broad range of protective 80 effects and generally recognized as safe (GRAS) status in the US, CDs are now finding greater application in the food industry as well. CDs are cyclic oligosaccharides that consist of 6, 7 or 8 glucose units linked together by 1,4-α-glucosidic bonds, and are classified as α-CD, β-CD and λ-CD, respectively. These molecules are chemically and physically stable, and are obtained by the enzymatic modification of starch (9). They consist of a relatively hydrophobic cavity in the center, with water soluble hydroxyl groups located on the outer surface. This enables them to form inclusion complexes with hydrophobic guest molecules, while increasing their solubility and bioavailability. The size of the CD cavity determines the encapsulation efficiency of various phenolic compounds. Alpha-CD has the smallest cavity of the three native CDs followed by β- and γ-CD. Modified CDs have been developed with improved water solubility, such as 2-hydroxypropyl-β-CD (2-HP-β-CD). Limited studies have looked at the use of CDs for encapsulation of whole plant extracts containing a number of compounds, each likely having varying encapsulation efficiencies with the different types of cyclodextrins. The encapsulation of complex mixtures such as olive leaf extract (10) and propolis extract (6) have been achieved in β-CD. Complex formation of inclusion compounds may be confirmed using several techniques such as FTIR, NMR, SEM, X-ray diffraction and differential scanning calorimetry (DSC) (11-12). DSC is a thermal analysis tool that is commonly employed for confirming complex formation in the solid state, by comparing the thermogram of the prepared inclusion complex with the guest molecule (6, 10-12). Disappearance of thermal events associated with the guest molecule, in the thermogram of the inclusion complex is 81 indicative of complex formation (11). When carried out under an oxygen atmosphere, DSC may also be used to study the oxidative stability of the inclusion complex (13). In this work, encapsulation of the polyphenolic extract obtained from Red Delicious apple pomace was studied using three types of cyclodextrins: α-cyclodextrin, β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin. The main phenolic constituents in the extract were analyzed by HPLC. Complex formation and oxidative stability were analyzed by DSC. Further, the solubility of the inclusion complexes in water was investigated using phase solubility studies. Methods and materials Reagents and Chemicals α-CD, β-CD and 2-HP-β-CD, catechin, epicatechin, procyanidin B2, caffeic acid, chlorogenic acid, phloridzin, hydrocaffeic acid, hydrocoumaric acid, quercetin and quercetrin were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). Analytical grade methanol, ethanol and acetone, and HPLC grade acetic acid and water were obtained from Lilly Stores (West Lafayette, IN). Preparation of apple pomace extract Red Delicious apple pomace was provided by Biersdorfer Orchard (Guilford, IN). The pomace was stored at -20 °C in nitrogen-flushed Ziploc freezer bags until use. Samples were lyophilized for 48 hours in a freeze dryer (Virtis Co., Gardiner, NY) and ground to less than 1 mm particle size. Microwave-assisted extraction was carried out in 82 a Mars Xpress microwave extraction system (CEM Corp., Matthews, NC) using the optimized extraction conditions reported by Chandrasekar et al. (14). Briefly, apple pomace was extracted with 5.6 ml of 70% acetone for 149 seconds at 735 W microwave power. The extracts obtained were transferred to 15 ml centrifuge tubes and centrifuged at 1050 rpm for 20 minutes in a Dynac centrifuge (Clay Adams, Division of Becton Dickinson and Co., Parsippany, NJ). The supernatant was evaporated to remove the organic solvent in a rotary vacuum evaporator. The aqueous extract was frozen and lyophilized for 48 hours in a freeze-dryer (Virtis Co., Gardiner, NY) to obtain dry polyphenol extract. HPLC analysis The polyphenol extract was resuspended in an equal volume of methanol and analyzed by reverse-phase HPLC based on the method of Suarez et al.(15), using an Agilent 1200 Series HPLC (Santa Clara, CA) with a diode array detector. A Waters C-18 column (3.9 x 100 mm, I.D. 3.5 µm) equilibrated at 25 °C was employed, and the injection volumes was 10 µl. The mobile phase consisted of solvent A: 2% aqueous acetic acid and solvent B: 100% methanol. The gradient elution first consisted of a linear step from 0 to 45% of solvent B in 55 minutes, an isocratic step of 15 minutes, and finally another linear step from 45 to 55% of solvent B in 10 minutes. The mobile phase flow rate was set at 0.8 ml/min. Absorbance spectra was collected at 355 nm for detection of flavonol glycosides, 313 nm for hydroxycinnamic acids and 280 nm for the other phenolic compounds (procyanidins, dihydrochalcones, benzoic and 3-phenylpropionic 83 acids). External injected standards were used to identify and quantify some of the major phenolic compounds present. Preparation of inclusion complex The inclusion complexes of the polyphenol extract were prepared with α-CD, βCD and 2-HP-β-CD according to the method of Mourtzinos et al. (10) with some modification. 25 mg of polyphenol extract was dispersed in 10 ml of 9 mM aqueous solutions of the cyclodextrins and mixed at 30 °C for 48 hours. The suspension was then filtered through a 0.45 µm PVDF filter. The filtrate was frozen in liquid nitrogen and lyophilized for 48 hours in a freeze dryer (Virtis Co., Gardiner, NY). Preparation of physical mixture Physical mixtures of the polyphenol extract with α-CD, β-CD and 2-HP-β-CD were prepared by mixing the two components in the same weight ratio as the inclusion complex, using a mortar and pestle for about 5 min until a homogeneous blend was obtained. Study of complex formation by DSC Thermal analysis of the inclusion complexes was carried out using a Q2000 DSC (TA Instruments, New Castle, DE) under a nitrogen atmosphere according to the method of Mourtzinos et al.(10). Four types of samples were analyzed to compare their thermograms; pure apple polyphenol extract, pure cyclodextrin (α, β and 2-HP-β), physical mixtures and inclusion complexes. Calibration was carried out initially using indium metal and the scan rate used was 10 °C/min from 30-230 °C. Samples were 84 weighed with ±0.01 mg accuracy and placed in hermetically sealed aluminum pans. Experiments were conducted in duplicates for each sample. Study of oxidative stability by DSC Oxidative stability of the inclusion complexes was evaluated by thermal analysis using DSC under an oxygen atmosphere, as described by Mourtzinos et al. (10) . Inclusion complexes (with α, β and 2-HP-β-CDs) and pure polyphenol extract were weighed at ±0.01 mg accuracy in aluminum pans and sealed with lids containing a pinhole. Samples were heated from room temperature to 120 °C under an oxygen atmosphere at a heating rate of 90 °C/min. After 1 minute at 120 °C for temperature equilibration, the sample was heated up to 300 °C at a rate of 10 °C/min. DSC experiments were conducted in duplicates. Phase solubility study Phase solubility study was conducted based on the method of Higuchi and Connors (16). An excess amount of the polyphenol extract was added to increasing concentrations of α-CD, β-CD and 2-HP-β-CD aqueous solutions (3-15 mM). The inclusion complexes were allowed to form by continuous shaking for 48 hours at 30 °C. The solutions were filtered through 0.45 µm PVDF filter. The filtrate was frozen and lyophilized in a freeze dryer (Virtis Co., Gardiner, NY) to remove the water. The dried inclusion complexes were then extracted with 100% methanol by sonication for 5 minutes. The extract obtained was filtered and the total phenols in the filtrate was measured spectrophotometrically using the Folin-ciocalteu assay according to the method 85 of Spanos and Wrolstad (17). Gallic acid was used as the standard, and results were expressed in terms of gallic acid equivalents (GAE)/g pomace extract. The experiments were carried out in duplicate, and stability constants were calculated from the phasesolubility graph, using the Higuchi-Connors equation. Results and discussion HPLC analysis Extracts obtained by microwave-assisted extraction of Red Delicious apple pomace were analyzed by HPLC, and the phenolics present were identified based on the retention times of the standards. The major polyphenols present include quercitrin, phloridzin and caffeic acid, and their amounts are shown in Table 4.1. In addition, a significant peak (retention time 25.38 min) was observed in the spectra measured at 280 nm, but remained unidentified due to lack of standards and is suspected to be a quercetin glycoside. High amounts of quercetin glycosides were reported to be present in Gala apple pomace extracts analyzed by HPLC (4). Study of complex formation The DSC results obtained under inert nitrogen atmosphere to verify the formation of inclusion complexes between the apple polyphenol extract and the three types of CDs studied are shown in Fig 4.1. Thermograms were obtained for four types of samples: pure 86 polyphenol extract, pure cyclodextrin, the physical mixture and the inclusion complex. In Fig 4.1A, the pure polyphenol extract (a) shows an endothermic peak at about 160 °C, and several endothermic peaks after 180 °C. These correspond to melting points of caffeic acid (m.p. 195°C) and quercitrin (175°C), denoting degradation of the extract constituents. Pure α-CD (b) showed an endothermic peak at 190 °C, possibly due to the elimination of water. All these peaks are present in the physical mixture (c) as well. However, the endothermic peaks associated with the pure polyphenol extract as well as α-CD are absent in the thermogram of the inclusion complex (d), which is indicative of successful inclusion of the guest molecule. Once the inclusion complex is formed, the stability and melting characteristics of the resulting compound are altered, and as a result, they show disappearance of thermal events associated with the initial guest molecule and wall material. Figure 4.1B shows the thermograms obtained for the β-CD samples. Again, pure β-CD (b) shows endothermic peaks at 140 °C and 190 °C, possibly due to water elimination from the sample. The peaks associated with the pure polyphenol extract and pure β-CD are present in the physical mixture (c) but are absent in the thermogram of the inclusion complex (d). Thus, complex formation between β-CD and polyphenol extract may also be inferred from this result. β-CD has been shown to form complexes with other types of complex extracts as well, such as the flavonoid rich St John’s wort extract (Hypericum perforatum) (18), propolis ethanolic extract (6) and oleuropein rich olive leaf extract (10). 87 Results obtained for 2-HP-β-CD are shown in figure 4.1C, and also shows complex forming ability with the apple pomace extract. Pure 2-HP-β-CD shows an endothermic peak at 220 °C, which is also present in the thermogram of the physical mixture (c), with a slight shift to the left, indicating no complex formation by this process. However, the DSC scan of the inclusion complex did not show any endothermic peak in this temperature range, confirming the formation of a complex with the CD. Thus, all the three types of CDs seem to have the ability to form a complex with the polyphenolic extract obtained from apple pomace. Study of oxidative stability The thermograms of pure apple pomace extract and the inclusion complexes (with α, β and 2-HP-β-CDs) under oxidative conditions are shown in figure 4.2. The pure extract shows exothermic peaks at 185 °C and higher, which relates to the oxidation of polyphenols in the extract. Phenolic compounds are sensitive to oxidation, and typically degrade at temperatures over 100 °C, giving rise to various decomposition products (1920). Exothermic peaks were also present in the thermogram of the inclusion complexes beyond 250 °C. This indicates the ability of the inclusion complexes obtained with all 3 types of CD’s to protect the pure extract against oxidation up to temperatures of 250 °C. Phase solubility study Phase solubility studies were carried out to investigate the dissolution of the inclusion complexes formed using the three types of cyclodextrins in water, and the results are shown in Fig. 4.3. Since apple pomace extract is a complex mixture of several 88 phenolic compounds, the results were described in terms of total phenols measured using the Folin-ciocalteu assay, and expressed as mg GAE/g pure extract. This approach is commonly employed when forming inclusion complexes with whole plant extracts (6) as compared to encapsulation of a single compound. The solubility of the inclusion complexes formed using β-CD was found to increase linearly (R2 = 0.9802) with increase in cyclodextrin concentration. The slope of this line was estimated to be < 1 (0.0191) indicating an A L type diagram and 1:1 binding between the guest molecule and wall material (10). The stability constant for the extract-β-CD complex was calculated based on the Higuchi-Connors equation (16) and was determined as K c = 1.02 (mg GAE/g extract)-1. Several studies have confirmed the formation of soluble inclusion complexes using β-CD with similar A L type phase solubility diagrams (6, 10, 12). Inclusion complexes formed using α-CD and 2-HP-β-CD did not show any increase in solubility, and the total phenols measured was found to remain constant and low, indicative of a B I type phase solubility diagram. It is possible that these results are due to the strong binding of the guest molecules within the cavities of α-CD and 2-HP-βCD, and thus inability to extract out the complexed polyphenols from the cavity. However, another possibility is that these CDs have poor complex forming ability with apple polyphenols resulting in very small amounts of phenolics being encapsulated. One of the key factors for inclusion complex formation is steric effects, and the size of the hydrophobic cavity plays an important role in forming the inclusion complex with guest molecules. α-CD is smaller and can typically complex with low molecular weight guest molecules, or compounds with aliphatic side chains (7), and is known to be less effective 89 than β-CD with regard to the formation of inclusion complexes with phenolic compounds (21). DSC studies carried out under nitrogen atmosphere qualitatively indicate the formation of an inclusion complex between the apple extract and α-CD/2-HP-β-CD, but do not provide a quantitative estimation of encapsulation efficiencies. It is not possible to come to a definite conclusion regarding this, and further studies are required in order to identify the cause for this discrepancy. Conclusion Extracts obtained from Red Delicious apple pomace is rich in several polyphenols that have shown high antioxidant activity. Some of the compounds identified by HPLC include quercetrin, caffeic acid and phloridzin. The encapsulation of these compounds was prepared by dissolution in three types of cyclodextrins: α-CD, β-CD and 2-HP-β-CD followed by freeze-drying to obtain dry complexes. DSC studies verified the formation of inclusion complexes between the apple polyphenol extract with all three types of cyclodextrins. The formation of inclusion complexes was also found to be effective in protecting the polyphenol extract from oxidation up to temperatures of 250 °C. Phase solubility studies indicate a 1:1 binding of the polyphenol rich extract with β-CD, and increasing water solubility of the complex with increase in CD concentration. Given the antioxidant potential of these phenolic compounds, the solid inclusion complexes obtained with β-CD may prove valuable for use as a stable and water-soluble food ingredient. 90 Figure 4.1. Complex formation study of apple extract with (A) α-CD (B) β-CD and (C) 2hydroxypropyl-β-CD 4.1A. Thermograms of (a) pure apple pomace extract (b) pure α-CD (c) physical mixture (d) inclusion complex 91 4.1B. Thermograms of (a) pure apple pomace extract (b) pure β-CD (c) physical mixture (d) inclusion complex 92 4.1C. Thermograms of (a) pure apple pomace extract (b) pure 2-hydroxypropyl-β-CD (c) physical mixture (d) inclusion complex 93 Figure 4.2. DSC thermograms of pure apple pomace extract and inclusion complexes under oxidative conditions 94 Total phenols (mg GAE/g extract) 0.4 0.35 0.3 0.25 β-CD 0.2 α-CD 0.15 2-hydroxypropyl-β-CD 0.1 0.05 0 0 3 6 9 12 15 Cyclodextrin concentration (mM) Figure 4.3. Phase solubility study of apple pomace extract with α-, β, and γ-CD at 30 °C (n=2) 95 Table 4.1. HPLC quantification of apple polyphenols from Red Delicious apple pomace extract Polyphenol Amount (mg/kg dry apple pomace)a Quercitrin 46.33 ± 1.98 Phloridzin 82.59 ± 2.61 Caffeic acid 10.01 ± 0.23 Gallic acid 0.33 ± 0.08 Catechin 0.15 ± 0.21 a expressed as mean ± S.D. (n=2) 96 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Vendruscolo, F.; Albuquerque, P. M.; Streit, F.; Esposito, E.; Ninow, J. L., Apple Pomace: A Versatile Substrate for Biotechnological Applications. Critical Reviews in Biotechnology 2008, 28, (1), 1-12. Foo, L. Y.; Lu, Y., Isolation and identification of procyanidins in apple pomace. Food chemistry 1999, 64, (4), 511-514. Lee, K. W.; Kim, Y. J.; Kim, D. O.; Lee, H. J.; Lee, C. Y., Major phenolics in apple and their contribution to the total antioxidant capacity. Journal of Agricultural and Food Chemistry 2003, 51, (22), 6516-6520. Lu, Y.; Yeap Foo, L., Antioxidant and radical scavenging activities of polyphenols from apple pomace. 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Pralhad, T.; Rajendrakumar, K., Study of freeze-dried quercetin-cyclodextrin binary systems by DSC, FT-IR, X-ray diffraction and SEM analysis. Journal of pharmaceutical and biomedical analysis 2004, 34, (2), 333-339. Karathanos, V.; Mourtzinos, I.; Yannakopoulou, K.; Andrikopoulos, N., Study of the solubility, antioxidant activity and structure of inclusion complex of vanillin with [beta]-cyclodextrin. Food Chemistry 2007, 101, (2), 652-658. Rudnik, E.; Szczucinska, A.; Gwardiak, H.; Szulc, A.; Winiarska, A., Comparative studies of oxidative stability of linseed oil. Thermochimica Acta 2001, 370, (1-2), 135-140. Chandrasekar, V.; Ballard, T.; Gonzalez, F. S.-M.; Hirst, P. Optimizing the microwave-assisted extraction of phenolic antioxidants from apple pomace and microencapsulation in cyclodextrins. Purdue University, West Lafayette, 2010. Suarez, B.; Palacios, N.; Fraga, N.; Rodriguez, R., Liquid chromatographic method for quantifying polyphenols in ciders by direct injection. Journal of Chromatography A 2005, 1066, (1-2), 105-110. Higuchi, T.; Connors, K., Phase-solubility techniques. Adv. Anal. Chem. Instrum 1965, 4, (2), 117–212. 97 17. 18. 19. 20. 21. Spanos, G. A.; Wrolstad, R. E., Influence of Processing and Storage on the Phenolic Composition of Thompson Seedless Grape Juice. Journal of Agricultural and Food Chemistry 1990, 38, (7), 1565-1571. Kalogeropoulos, N.; Yannakopoulou, K.; Gioxari, A.; Chiou, A.; Makris, D., Polyphenol characterization and encapsulation in [beta]-cyclodextrin of a flavonoid-rich Hypericum perforatum (St John's wort) extract. LWT-Food Science and Technology 2010. Hamama, A. A.; Nawar, W. W., Thermal decomposition of some phenolic antioxidants. Journal of Agricultural and Food Chemistry 1991, 39, (6), 10631069. Larrauri, J. A.; Ruperez, P.; Saura-Calixto, F., Effect of Drying Temperature on the Stability of Polyphenols and Antioxidant Activity of Red Grape Pomace Peels. Journal of Agricultural and Food Chemistry 1997, 45, (4), 1390-1393. Calabro, M. L.; Tommasini, S.; Donato, P.; Raneri, D.; Stancanelli, R.; Ficarra, P.; Ficarra, R.; Costa, C.; Catania, S.; Rustichelli, C.; Gamberini, G., Effects of alpha and beta-cyclodextrin complexation on the physico-chemical properties and antioxidant activity of some 3-hydroxyflavones. Journal of pharmaceutical and biomedical analysis 2004, 35, (2), 365-377. 98 CHAPTER 5. CONCLUSIONS Millions of apples are processed globally into juice, cider and other products, generating large amounts of apple pomace as a waste stream. This waste stream is severely under-utilized, and is currently being used as animal feed or compost. Apple pomace is rich in polyphenols such as quercetin glycosides and hydroxycinnamic acids that have very good antioxidant and radical scavenging activity. The use of these natural phenolic extracts as replacers of synthetic antioxidants in foods may prove valuable from a consumer standpoint. Removal of these compounds from the apple matrix has conventionally been done by solid-liquid extraction, using mainly organic solvents in combination with a small amount of water. However, these methods require extensive amounts of solvent and extraction time, and consideration of alternate extraction techniques may prove more efficient. Additionally, these compounds are hydrophobic and prone to oxidation, limiting their use as stable food ingredients. Therefore, their encapsulation in cyclodextrins (CDs) was also studied to enhance stability and water solubility. The microwave-assisted extraction of phenolic antioxidants was optimized from apple pomace of four apple varieties (Red Delicious, Golden Delicious, Winesap and 99 Jonathan) using response surface methodology. Optimum extraction conditions were based on the total polyphenol content (TPC), as well as the antioxidant activity measured by the inhibition percentage (IP) of the DPPH free radical. The important parameters that were optimized included solvent type (70% acetone, 60% methanol and 60% ethanol), solvent volume to sample ratio (4-12 ml/g), extraction time (30 - 180 s) and microwave power (100-900 W). Good correlation (R2>0.80) was obtained between TPC and DPPH activity in all extracts. All solvent systems seemed to show identical extraction conditions, independent of pomace variety. Red Delicious apple pomace extracts contained highest TPC (15.8 mg GAE/g DW) and DPPH radical scavenging activity (94.4 % inhibition) obtained under the optimum conditions of 735 W and 149 s, with 10.4 mL of 60% ethanol and 5.65 mL of 70% acetone, respectively. Encapsulation of phenolic extracts obtained from Red Delicious apple pomace was carried out using α-CD, β-CD and 2-hydroxypropyl-β-CD by dissolution in aqueous solutions followed by freeze-drying to obtain dry inclusion complexes. Complex formation was confirmed by DSC for all three CD types. The inclusion complexes formed were also found to be stable and resistant to oxidation up to temperatures of 250 °C, compared to the pure apple pomace extract. Phase solubility studies were carried out to study the water solubility of the complexes. A 1:1 binding of the polyphenol rich extract with β-CD was observed, demonstrating increasing water solubility of the complex with increase in CD concentration. However, complexes formed with α-CD and 2-hydroxypropyl-β-CD did not show increased water solubility. It is concluded that β-CD 100 inclusion complexes were effective in enhancing both stability and solubility of the phenolic extracts, and serves as a potential food ingredient. Future work Recommended future work could involve extension of the MAE optimization study to other potential waste streams that are rich in phenolic compounds. In the encapsulation study, examining the mechanisms for dissolution of the inclusion complexes and identification of the compounds encapsulated may be worthwhile. This would help identify which phenolic compounds present in the extracts are involved in binding with the hydrophobic cavity of the CD and forming inclusion complexes. In addition, structural characterization of the guest-host complex using NMR can be carried out. It is also suggested that release kinetics of the encapsulated guest molecules be explored. Additionally, the application of these inclusion complexes as antioxidants could be investigated in model food systems.