Mónica
González
*a and
Venerando
González
b
aPost-harvest and Food Technology Laboratory, Department of Tropical Fruit Crops, Instituto Canario de Investigaciones Agrarias, Apdo. 60, 38200, La Laguna, Spain. E-mail: mgonzal@icia.es; Fax: +34 922 476303; Tel: +34 922 476310
bDepartment of Analytical Chemistry, Nutrition and Food Science, University of La Laguna, Campus de Anchieta, Astrofísico Francisco Sánchez s/n, 38205, La Laguna, Spain
First published on 2nd November 2010
This work is a review of the analytical strategies dealing with antioxidant phytochemicals in tropical and subtropical fruit by-products. The determination of bioactive compounds encompasses a number of different aspects and the analytical strategy employed depends on the biowaste, analyte and nature of the problem. In general, an analytical strategy involves recovering antioxidant phytochemicals from the sample matrix followed by separation, identification and measurement. For most phytochemicals, the recovery step typically involves extraction using a range of solvents. However, sample handling is often an ignored feature of the analysis. This review highlights the importance of sample preparation in the analysis of phytochemicals from tropical and subtropical fruit biowastes and the problems that can arise during this step. The various procedures are summarized and some typical “case studies” are presented.
Agro-industry waste is often utilized as feed or fertilizer. However, the cost of drying, storing and shipping biowastes limits the economic value of such products. One economically viable use for these wastes that is gaining interest is as food additives or supplements with high nutraceutical value.3
Multiple epidemiological studies have pointed out that consumption of fruits and vegetables imparts health benefits, such as reduced risk of coronary heart disease and stroke, as well as certain types of cancer. Apart from dietary fiber, these health benefits are mainly attributed to organic phytochemicals, such as polyphenolic compounds, carotenoids, tocopherols and sterols, among others, contained in fruits and vegetables. These antioxidant phytochemicals are also contained in the peels, seeds and stones of fruits and vegetables; in the majority of cases in greater quantities than in the edible part. These antioxidants are capable of slowing or preventing oxidation reactions that can produce free radicals by removing radical intermediates and inhibiting other oxidation reactions, but in order to do so they themselves must oxidize. Since oxidative stress might be an important part of many human diseases, the use of antioxidants is intensively studied, particularly in the prevention of stroke and neurodegenerative diseases. Other possible mechanisms of action by which polyphenols and other antioxidants may help to prevent disease include cell anti-proliferation, induction of cell cycle arrest and apoptosis, regulation of the host immune system, changes in cellular signalling, receptor sensitivity, enzyme activity, and gene regulation, etc., and they may have an anti-inflammatory effect.4,5
Furthermore, growing concern about food safety on the part of consumers, authorities and food industry producers has created a need to identify alternative natural and probably safer sources of food antioxidants. Replacing synthetic antioxidants with natural ones, such as phytochemicals, may have health benefits and increase functionality due to solubility in both oil and water, which could be useful for emulsions in food systems.
Unlike temperate fruits, tropical and subtropical fruits can be broadly defined as meeting all of the following criteria: crops that have their origin and commercial growing areas in the tropics or subtropics, plants that are evergreen and perennial, crops with a limited degree of frost resistance, and plants whose growth is practically nonexistent below 10 °C, with some exceptions according to species and individual age.6
Hundreds of tropical and subtropical fruits exist, but only around fifty are well known throughout most of the world. Production and trade figures allow the division of tropicals and subtropicals into three main categories with some overlapping: (i) major fruits, cultivated in most tropical (and subtropical) countries that are well known in both local and export-import markets (such as banana and plantain, coconut, mango, and pineapple); (ii) minor fruits, not so extensively cultivated, and of more limited consumption, both geographically and quantitatively; however, many are of considerable economic importance in their respective regional markets; examples of minor fruits are abiu, atemoya, avocado, breadfruit, carambola, cashew nut, cherimoya, durian, guava, jaboticaba, jackfruit, langsat, litchi, longan, macadamia, mangosteen, papaya, passion fruit, pulusan, rambutan, sapodilla, soursop, and white sapote and (iii) wild fruits belonging to diverse botanical families.6 Because wild fruits are not cultivated commercially in any country they are not used for processing and, therefore, not included in this review.
Fruits from the temperate zone are usually characterized by a large edible portion and moderate amounts of bio-waste material; in contrast, considerably higher ratios of by-products arise from tropical and subtropical fruit processing. Some data on worldwide production of the different tropical and subtropical fruits included in this review are presented in Table 1, alongside the different biowastes obtained from these fruits and the percentage of the total weight of the fruit that they represent.7–9
Developing rapid, rugged, robust and accurate analytical procedures is critical for the success of most of the steps necessary for designing, developing, and marketing value-added functional foods.10 Accurately identifying bioactive compounds is essential to finding relationships between different food components and health benefits. It is also critical to precisely quantify bioactive compounds to determine dietary intake levels and safety guidelines. Furthermore, assay procedures should be harmonized at the international level to analyze these value-added functional foods and facilitate their commerce in the global market.10
Analyzing phytochemicals from biowastes encompasses a number of distinct aspects and the analytical strategy will depend on the sample, analyte and nature of the problem. Although it is impossible for a single, global strategy to be valid for every by-product, given the diversity of analytes and plant materials, it is possible for a number of generalizations to be made. On the whole, the analytical procedure for determining phytochemicals from biowastes involves four common steps: sampling, sample storage, sample preparation (extraction from the plant material, hydrolysis and/or purification) and analytical determination (quantification and identification).
Most of the development made over the past few years in the analysis of phytochemicals from tropical and subtropical fruit biowastes has focused on the final analysis step. Remarkable innovations in instrumentation, spectroscopy, and chromatography have resulted in the rapid advancement of methods for high-throughput separation and detection of complex multi-component mixtures containing trace quantities of the analytes of interest. However, there has been limited research in the first three steps (sampling, sample storage, and sample preparation), which are the foundation for developing a quality, accurate, robust and rugged analytical procedure. In general, most uncertainty associated with analytic determination is usually related to these stages.10
This paper reviews analytical strategies dealing with antioxidant phytochemicals in tropical and subtropical fruit by-products: recovery of the phytochemicals from the sample matrix followed by separation, identification and quantification. It is focused on work published between 1990 and 2010, although most of the development made in the analysis of phytochemicals from tropical and subtropical fruit biowastes has been done over the past few years. The review emphasizes the importance of sample preparation in the determination of phytochemicals in these fruit by-products, as a critical step in the analysis of samples in which matrix components are biologically active and the analytes represent a diverse spectrum of numerous compounds, many of unknown identity.
In many cases tropical fruit biowastes are obtained directly from agro-industries.12–21 However, most residues that will be analyzed are obtained from different kinds of fruit in the laboratory. These tropical and subtropical fruits can be obtained from fields belonging to the research institutes themselves or from commercial orchards,22–76 or local markets.77–100 However, when purchasing from local markets the researcher has no prior knowledge about the origins, variety and/or postharvest conditions (time elapsed after harvest and storage temperature of the fruit) of the samples. These factors must be characterized as they have an enormous impact on the quantity and even the type of bioactive compounds contained in plant material. The composition of bioactive compounds in tropical and subtropical fruit peel and seeds can vary depending on climate (temperatures, rainfalls and light hours), soil type and fertilization. Different varieties of tropical fruits, grown in different regions, in different years, can result in measurable differences in composition. The variety12,15,18,19,22–33,35–43,51–53,55,57–64,66,67,69–72,78–84,93,94,101–103 used in the analysis of tropical and subtropical fruit biowastes or the harvest index22,23,27 has been reported in some works, but rarely. The banana harvest index has been established as the caliber, measured in the middle finger of the outer whorl of the second hand from the distal end of the bunch.22,23 In mango, fruit maturity at harvest can be based on the days after full bloom, fruit shape and maturity of seed stone during destructive opening.27 Another aspect to keep in mind, especially when analyzing residues from climacteric fruit, is the ripening stage of the fruit at the moment the biowastes are obtained. Slight variability in the ripening stage could contribute to wide modifications in the bioactive compounds of climacteric fruit peel and seeds. For example, during the ripening process, there is a significant increase in the tocopherol content in the ripened banana peel.104 During ripening, the color of mango peel gradually changes from green to greenish-yellow, red, violet or yellow; these changes are related to an increase in carotenoids during ripening, ranging from 74 to 436 μg g−1 peel dry weight (DW).25 The carotenoid content was found to be greater in ripe mango peels (1400–4000 μg g−1 DW) compared to unripe peels (365–550 μg g−1 DW).26 The anthocyanin content (360–565 mg/100 g DW) was higher in ripe mango peel than in unripe peels (203–326 mg/100 g DW).25 Unripe mango peel had higher polyphenol content than ripe peels. Therefore, the total polyphenol content of unripe mango peels ranged from 90 to 110 mg g−1 peel DW, whereas it ranged from 55 to 100 mg g−1 DW in ripe peels, depending on the variety.25,26 Vitamin C and E contents ranged from 188 to 392 and 205 to 509 μg g−1 dry peel, respectively; and they were greater in ripe peels.26 The skin of “Hass” avocados changes color from green to purple and to black as the fruit ripens. This is the result of an initial decrease in chlorophyll content, followed by a dramatic increase in the level of the anthocyanin cyanidin 3-O-glucoside.58 The young mangosteen rind contains a higher content of phenolic compounds and tannins, promoting better free radical scavenging activity than the rind from the mature fruit. In contrast, the mature fruit rind contains higher amounts of total flavonoids and α-mangostin than the rind from the young mangosteen.105 The red colour of litchi fruit peel is mainly attributed to anthocyanins. The monomeric anthocyanin concentration of litchi pericarp (46 mg/100 g fresh weight (FW)) at ripeness makes them a potentially good source of anthocyanins.106 The individual anthocyanins identified were cyanidin-3-glucoside, cyanidin-3-rutinoside and malvidin-3-acetylglucoside. Only malvidin-3-acetylglucoside was detected in the unripe fruit. However, in the ripe fruit, cyanidin-3-rutinoside became the main anthocyanin (>75%),106–108 while cyanidin-3-glucoside represented less than 17%, and malvidin-3-acetylglucoside less than 9%.106 Therefore, it is essential that the ripening stage of the fruit be characterized when the fruit is analyzed. Climacteric fruits are usually harvested at physiological maturity,22,23,25,26,57,58,92,109 and then left to ripen at room temperature25,26,53,109 or in controlled conditions of temperature and relative humidity22,23,57,58,92 until full-ripening is reached, the stage at which the fruit biowaste is analyzed. Sometimes, biowastes are analyzed in fruits at physiological maturity24,25,84 or at different ripening stages,27,73,103,105 but normally antioxidant phytochemicals in biowastes are analyzed in ripe fruit.12,14,19,22–26,32,33,35–41,43,46–49,52,53,57,58,69,82,83,87,90,92,96,97,108–110 The ripening stage of banana is usually characterized in the middle finger of the outer whorl of each banana hand by using peel and pulp color, firmness, total soluble solid content and titratable acidity.22,23 The ripening stage of mangos and avocados has been characterized by measuring color and determining firmness.24,57,58 The characterization of cashew apples has been done on the basis of visual color, dry matter, total soluble solids, pH and titratable acidity.32,33 In tamarillo, the total soluble solids and moisture content were analyzed prior to obtaining the biowastes that were analyzed.49
Sample storage is an important step as there is often some delay between obtaining the biowaste and its analysis. For example, when biowastes are obtained from agro-industries, the storage conditions (temperature, time), from the time of collection to the time of analysis, are not usually specified. Improperly stored biowastes can rapidly lose their antioxidant properties, making them useless for extraction purposes. For example, pericarp browning, a common and important defect of harvested litchi fruit, is caused by anthocyanin degradation. The (−)-epicatechin is oxidized and oxidative products of (−)-epicatechin (o-quinones) catalyze other litchi flavanols and anthocyanin degradation, resulting in the pericarp browning of postharvest litchi fruit.107,108 Moreover, fresh biowaste has high moisture content (usually not reported) and therefore is at risk of microorganism growth unless stored in appropriate conditions. It is essential to inactivate all enzymatic and chemical reactions during storage so that the sample's identity does not change during this period.11 It is essential to dry tropical and subtropical fruit by-products to inactivate the enzymes responsible for degrading many active compounds and to decrease the rate of microbial growth. Drying time and temperature affect the activity and stability of bioactive compounds due to chemical and enzymatic degradation, losses by volatilization and/or thermal decomposition. Under natural drying conditions, biowastes are exposed to either direct sunlight for 3–7 days,14,15,18,102,111 or air-dried at room temperature for 1–2 weeks.47,78,112 Good air circulation is mandatory to reduce humidity as the biowastes dry. With artificial drying, the biowastes are oven-dried (with static or forced-air or under vacuum) at temperatures between 40–80 °C for 4–72 h12,13,15–17,30,35–36,38–41,45,52,53,71,72,85,88,90,97,105,110,113 or freeze-dried.22–24,27,28,32,33,37,42,51,55,68,80–83,86,87,89,96,100,101,114 Tamarind seeds have been dry heated along with acid washed sea sand on an open hot plate at 135 °C for 25 min.50
Some authors have described an increase (1.4 times) in the phenolic content of mango, longan and tamarind seeds when they are heated at temperatures between 105 and 160 °C prior to extraction.86,87 Coconut shell powder has been subjected to different toasting temperatures, from 100 to 200 °C, and toasting times, from 20 to 60 min, prior to the extraction of phenolic compounds.115 Thermal treatment, at 100 °C for 60 min, enhanced phenolic extraction from the coconut shell.111,115 The total phenol content in persimmon peel extracts (70% ethanol or water) increased between 4.2–6.9 times when the peel was heated at 150 °C for 20 min.112 This could be attributed to the formation of phenolic substances under milder heating temperature, probably due to the conversion of phenolic molecule precursors in this type of compound. This behavior may also be due to the fact that a large percentage of phenolic compounds are bound to cellular structures and heating treatments release bound phytochemicals from the matrix to make them more accessible in extraction.116
Reducing the particle size favors solvent extraction of phytochemicals,22,23 therefore, before extraction mechanical grinding of the biowastes is carried out to increase their surface area. Dried biowastes are usually ground with a ball, knife or hammer mills,12,13,16,18,22,23,27,28,30,55,80,82,83,85,88,90,111,115 blenders,35,36,38–41,53,78,110,112 grinders,20,21,42,52,86,87,96,117 mortars32,33,51 or processors.17,114 The particle size of the ground material is generally between 0.25 and 1 mm.13,14,17,18,22,23,32,50,52,53,55,78,87,90,91,105,111,113
Undoubtedly, the influence of storage temperature on the analyte of interest needs to be studied in detail in order to store samples under appropriate conditions. Dehydrated and ground samples are normally stored at room temperature,17,50,90,111 at 4 °C13,52 or below freezing at −20 °C12,22,23,27,37,47,51,67,81,86,87,96 or −80 °C.32,33,68 Generally, when anthocyanins are determined in plant residues, the samples do not dehydrate and are stored frozen at −20–−30 °C until extraction.62,106,107,118–120 Moreover, samples are usually stored protected from light,13,17,47,51,90,97 under nitrogen32,33 or in vacuum-packaged polyethylene pouches.51,52,81,86,87,90,96,97
Fig. 1 Techniques (extraction, hydrolysis and/or purification) used to prepare tropical and subtropical fruit biowastes for phytochemical analysis. |
Phytochemical compounds in plants can be distributed in various forms. For example, phenolic compounds exist as free aglycons or as conjugates with sugars or esters, or as polymers with multiple monomeric units. Moreover, phenolic compounds are not uniformly distributed and may be associated with cell walls, carbohydrates, or proteins.11,121 In addition, the task of phytochemical recovery is complicated as plant material is a natural matrix with high enzyme activity and the stability of bioactive compounds varies significantly, some are relatively stable while others are highly reactive;11,121 this complicates their extraction and quantitative recovery becomes particularly problematic. Some precautions are usually taken during extraction in order to prevent oxidation of bioactives and other deteriorative processes. Drying the plant by-product before extraction, immediately immersing the biowaste in methanol and using an acid extraction medium protects the material from oxidation.11 Moreover, the extractions can be carried out under reduced light22,23,57,58,82,92,95 or under an inert atmosphere.57,82,83,101 Some antioxidants can be used for this purpose as well.37,43,51,57,69,70,78,80,82,92
Extraction is the first and most important step in recovering and purifying active ingredients from plant biowastes. The objective when extracting phytochemical antioxidants from plant sources is to liberate these compounds from the structures where they are found. Solvent choice, contact time and temperature, number of extraction cycles, solvent to plant material ratio and extraction technique significantly influence the extraction efficiency. The role of each factor in the extraction process is not always obvious; the chemical characteristics of the solvent and the diverse structure and composition of natural products ensures that each biowaste-solvent system behaves differently and in an unpredictable manner.
In general, no single solvent will provide optimum recovery of all phenols or even a limited range of phenols. Plant phenols are ionizable with typical pKa values ranging from 8 to 12.11,121 Thus, they exhibit considerable diversity in terms of acidity and polarity,and they also range from hydrophobic to hydrophilic. The polarity of the solvent and that of the different phenolic compounds affect extraction efficiency and the activity of the obtained extracts. In general, highly hydroxylated aglycone forms of phenolic compounds are soluble in alcohols. Less polar solvents such as ethyl acetate, acetone and chloroform are used for the less polar and highly methoxylated aglycone forms that are very common in fruit peel. The most polar phytochemicals can be extracted using water. Moreover, there are some important distinctions between fresh and dried samples. In the case of extractants using aqueous mixtures, the required proportion of water in the extractant is lower with fresh samples. Furthermore, with dried materials, low polarity solvents and ethyl acetate will simply leach the sample whereas alcoholic solvents presumably rupture cell membranes and enhance the extraction of endocellular materials.11,121
Solvent extraction of anthocyanins warrants special consideration. The fact that their chemistry is complicated by various pH-dependent equilibria11,121 has been exploited in traditional anthocyanin recovery strategies by extracting the flavylium cation form with methanol or ethanol containing hydrochloric (HCl) or formic acid (Table 2). The low pH value of the extraction solvent prevents the degradation of these pigments.122 However, pigment degradation occurs as the acids are concentrated during the evaporation of the alcohol-HCl or alcohol-formic acid mixtures. Small amounts of dilute acid may also cause partial or total hydrolysis of acylated anthocyanins that are present in plant tissues.122
In addition to the solvent, many other factors contribute to the efficiency of the extraction process. Extraction of phenolic compounds is typically conducted at room temperature, 20 to 30 °C (Table 2). However, increasing temperature improves extraction efficiency due to the enhanced diffusion rate and solubility of phytochemicals in solvents. Therefore, in some cases hot solvents (at temperatures between 37 and 90 °C) or heat reflux are used to extract bioactive compounds from tropical and subtropical fruit biowastes. However, it must be taken into account that the degradation rate of antioxidant compounds is time and temperature dependent. It has been widely reported that extraction temperature affects the stability of bioactive compounds. In addition, at high temperatures bioactive compounds can react with other components of the plant material, thus impeding extraction. In some cases cold extraction, at 4 °C, has been reported (Table 2). The mass transfer rate and the chemical solubility of the phytochemicals in the solvent can be significantly improved with agitation during the extraction procedure.14,18,27,30,32,33,37,38,75,78,83,90,95,101,114,123
The recovery of bioactive compounds from biowastes is also influenced by the extraction time and the number of extraction processes. Extraction times of a few minutes (between 1 and 30 min), hours (between 1 and 12 h) or days (between 1 and 3 days) have been used (Table 2). Usually a single extraction step is used, but sometimes from 2 to 8 extraction steps have been used (Table 2). Another aspect to take into account when extracting antioxidant compounds from biowastes is the solvent to plant material ratio. The most commonly reported ratios are between 1:1 and 10:1 (v:w).13–15,18,22,23,31,33,37,38,43,46,49,50,55,69,76–78,83,84,90,95,96,99,101,114,124–126 However, higher ratios between 20:1 and 100 :1 (v:w)12,27,30,32,55,59,61,64,65,67,75,80,82,89,127 or between 125:1 and 250:1 (v:w)49,52,86,87 have also been used.
In the majority of cases, the procedures employed to extract phytochemicals from tropical and subtropical fruit biowaste have not been specifically optimized for them, but have been optimized for the extraction of other types of plant material. Extraction methods differ between different biowastes because of their different matrices, with unique properties in terms of structure and composition (related to specie, variety, ripening stage). Therefore, considerable caution should be exercised when using methods that have been developed to analyze specific plant tissue types and phytochemical extraction should be optimized for each biowaste.
The effect of solvents on the extraction of phenolic compounds from banana peel has been evaluated (Table 2).22 Acetone (50%) was the most effective solvent for the extraction of phenolic compounds from this biowaste, extracting 1.5–3.5 times more than methanol, 50% methanol or 50% ethanol. Moreover, experimental design and response surface methodology was used in order to optimize the number of extraction steps, extraction time and temperature when extracting phenolics from banana peel (Table 2).23 The factor that had the greatest impact was the number of extraction steps: the extraction was improved by increasing the number of extractions. Optimum extraction of phenolic compounds was obtained with 3 extraction steps, with homogenization for 1 min at 25 °C and further centrifugation. The extraction of phenolics from mango kernel has been optimized by using 95% ethanol for 4.5 h or refluxing with ethanol for 3 h.15 The phenolic compounds extracted using reflux were 2.5 times higher than those extracted using shaking. The effects of solvent to sample ratio, temperature, extraction time, number of extractions and solvent type on the efficiency of phenolic extraction from date seeds has been also studied (Table 2).30,52 The optimum conditions were considered to be a two-stage extraction, each stage lasting 1 h at 45 °C with a solvent to sample ratio of 60:1 (v:w).30 Acetone (50%) was selected as a very efficient solvent for extraction.30 Extraction by dimethyl sulfoxide also gave a high total phenolic content in the extracts obtained from seeds of 14 date varieties.52 How the solvent (acetone, ethanol, methanol and water) affected the extraction of phenolic compounds from three different biowastes obtained from persimmon was evaluated.55 Acetone was the most effective solvent for the extraction of phenolics from persimmon calyx, seed and peel. Temperature and pH significantly influenced the extraction yield of anthocyanins from litchi fruit pericarp and antioxidant activity.67 Temperatures from 45–60 °C and pH from 3.0 to 4.0 exhibited a relatively high antioxidant activity. Five different solvents were used to evaluate total phenolic content, antioxidant capabilities and antityrosinase activity in litchi seeds (Table 2).38 There were no significant differences in the phenolic content among all the extracts except for those obtained using water. The effect of different solvents on phenolic extraction from longan pericarp has also been studied (Table 2).40 Extracting with methanol or ethanol obtained the highest total phenolic content, followed by water and ethyl acetate. Dried mangosteen pericarp was extracted with 95% ethanol by maceration, percolation, Soxhlet extraction, ultrasonic extraction and shaking.97 Soxhlet extraction promoted the maximum content of xanthones in the extracts and it was also recommended because of its low reagent consumption and its low extraction time. Moreover, the extraction solvent was also optimized.97 50% Ethanol was the appropriate solvent for extracting phenolic compounds and tannins from mangosteen pericarp, while 95% ethanol was recommended for α-mangostin extraction. The extraction of phenolics from carambola residues was carried out at different ratios of organic solvents and water, extraction temperatures and times (Table 2).89 The optimum solvent was 50% acetone. Moreover, the extraction efficiency increased with increased temperature and time. Considering the stability of antioxidants at high temperatures, 90 °C was chosen as a suitable extraction temperature and 45 min as the optimum extraction time. The effect of acetone concentration, extraction temperature and extraction time (Table 2) on phenolic content extracted from carambola residue was also studied by using response surface methodology.90 Acetone concentration was statistically the most significant factor and the optimal extraction conditions obtained were: 65% acetone concentration, 43 °C extraction temperature, and 234 min extraction time.
Once extracts have been obtained they are usually concentrated at low temperature (35–45 °C) or lyophilized. The residues are then redissolved in an adequate solvent for subsequent purification and analysis.13–16,20,21,24,31,38,43,44,50,54,62,64,65,69,70,72,75,77–87,89,93,94,96,98,99,101,105,123,124,128
Sometimes mango kernels,81,82 pineapple by-products,16 longan seeds44 and tamarind seed coats50,100 and tamarind pericarp100 are defatted (Table 2), prior to extracting antioxidant compounds from them, with n-hexane16,81,82,100 or petroleum ether.37,43,44,50 Other times, the extract obtained from mango kernel83,84 or mangosteen pericarp114 is extracted with n-hexane,84 dichloromethane83 or diethyl ether114 to remove residual lipid material.
Carotenoids exhibit pronounced photo- and thermal sensitivity. These phytochemicals, in their natural environment, are incorporated in lipoproteins or membranes, and are relatively well protected. However, if they are isolated in extractants, they readily undergo trans-cis isomerization catalyzed by light, acids and bases, oxygen, heat, traces of metal ions, etc.129 Therefore, some measures must be taken during the sample pre-treatment: antioxidants must be used, laboratory operations must be carried out in dimmed, yellow or red light, evaporation should be carried out under a protective nitrogen or argon atmosphere at temperatures below 40 °C and the samples should be stored in darkness, under nitrogen or argon, at temperatures around −20 °C.129
Extraction of cashew nut shell liquid (CNSL) from split cashew shell with SFC-CO2 is an interesting approach because it could allow for much more of the CNSL to be used by the food industry.117,133,134 The composition of the supercritical carbon dioxide and solvent extracted CNSL from the raw cashew nut shells were comparable.134 Philip et al.133 evaluated the isolation of anacardic acid from natural CNSL by SFC-CO2 under a range of operating conditions of pressure, temperature and CO2 flow rate. The best working conditions were found to be 50 °C and 300 bar at a flow rate of 5 g min−1.133 Under these conditions it was possible to quantitatively isolate high purity anacardic acid from crude natural CNSL within 150 min. Two extraction methods were also used to extract anacardic acid compounds from the cashew shell:117 (i) the typical extraction method, in which CO2 flows through the extractor at constant temperature, and (ii) the pressure profile method, in which the extractor content is subjected to pressurization with CO2 and depressurization steps before beginning the extraction. The pressure profile method promoted CO2 dissolution into the shell material because of the changes produced in its structure, giving yields 10 times greater than those of the typical extraction method for the same amount of CO2. Under these conditions, the composition of the extracted oil was virtually the same as that obtained by expressed CNSL, without chemical degradation of the anacardic acid.117
The xanthones from mangosteen pericarp have been extracted without ethanol and with 2–3% ethanol at 30 MPa and 50 °C in SFC-CO2.88 SFE using ethanol as a modifier significantly increased the extraction yields of xanthones. The performance of SFE with pure CO2 when extracting phytochemicals from tamarind seed coat was very low.91 However, adding an ethanol co-solvent increased the extraction of (−)-epicatechin substantially (500 times) and the antioxidant activity of the extracts. Luegthanaphol et al. optimized the extraction temperature and pressure over the range 35–80 °C and 10–30 MPa, respectively, and highlighted that the amount of (−)-epicatechin extracted decreased when temperature and pressure increased.91 SFC-CO2 extraction was studied as a method to extract antioxidant compounds from tamarind seed coat.21 Different combinations of pressure and temperature were used with and without ethanol as modifier. As temperature and pressure were increased (80 °C and 30 MPa optimum conditions), more phenolic compounds were extracted.21
The extraction of carotenoids from persimmon peels using SFE at 60 °C and 30 MPa, was carried out.132 CO2 was used as the SCF and 5–20% of ethanol was added as a co-solvent. When increasing the ethanol amount from 0 to 10%, the carotenoid yield in the extraction was improved, although the selectivity of the extracted carotenoids was drastically depressed.
The use of ultrasound (at an intensity of 4,870 W m−2) with 50% ethanol has been evaluated to extract phenolic compounds from coconut shell.111,115 The effect of extraction temperature and time, solvent to biowaste ratio and water pH was evaluated using response surface methodology. Phenolic extraction was maximized when pH values of 6.5, high solvent to solid ratio (50:1, v:w) and low temperature (30 °C) were employed. Increasing extraction time did not increase the extraction of phenolics (optimum 15 min). At optimum operating conditions, sonication enhanced the phenolic extraction yield 3 fold. Flavanols have been extracted from fresh litchi pericarp using acidified methanol in an ultrasonic bath at room temperature for 0.5 h (Table 2).108 Longan pericarp or seed in 70% acetone (solvent to solid ratio 125:1, v:w) was also subjected to ultrasonic treatment (input power 700 W) for 20 min.42
HPE has been used to extract bioactive compounds from litchi and longan pericarp. Prasad et al. studied the effect of extraction conditions on the extraction yield, including type of solvent and concentration, solvent to plant material ratio, acidic medium, extraction pressure, time and temperature.35,36,39–41,110 These authors did not find significant differences in the phenolic content when the litchi pericarp was extracted using HPE, UE or conventional extraction.35,36 However, the HPE technique increased the flavonoid extraction yield from litchi pericarp up to 2.6 times in comparison with UE and up to 10 times compared with conventional extraction. In longan pericarp, the optimum HPE conditions for phenolic compounds39 and corilagin41 were determined to be 500 MPa for 2.5 min at 30 °C, 50% ethanol and a solvent:solid ratio of 50:1 (v:w). The HPE technique resulted in the highest extraction of total phenolic and corilagin content (1.2–1.4 times) and was faster than either UE or conventional extraction.39,41 Xanthones have also been extracted from mangosteen pericarp with ethanol using HPE at 100 °C for 15 min.137 The extraction yield using HPE was twice that obtained by UE; moreover, both solvent consumption and extraction time were reduced.
The phenolic compounds from longan pericarp were extracted with 95% ethanol (solvent:plant material ratio 10:1, v:w) employing MAE, at 80 °C for 30 min (Table 2).113 The phenolic content of extracts obtained from longan pericarp was similar when using either MAE or Soxhlet extraction. However, the antioxidant activity of microwave-assisted extracts was superior to that of Soxhlet extracts, had faster extraction times (2 h for Soxhlet and 30 min for MAE) and required less solvent. All of which are clear advantages to using this technique.113
Alkaline hydrolysis conditions have been employed to isolate phenolic acids from the biowastes obtained from tropical and subtropical fruits in order to determine bound phenols.30,31,85,114 Hydrolysis of esterified ferulic acid from pineapple peel with sodium hydroxide (NaOH) has been described by Tilay et al.,85 who performed alkaline treatment with 2 M NaOH for 24 h at 22 °C. The residue of date seeds obtained after extraction was hydrolyzed with 2.5 M NaOH and stirred at room temperature for 4 h.30 The residue obtained after extracting the soluble phenolic compounds from cashew apple bagasse31 or mangosteen pericarp114 was hydrolyzed with 4 M NaOH for 4 h at room temperature. Next, the residue obtained after hydrolysis of mangosteen pericarp was heated with 2 M HCl for 30 min at 95 °C to liberate the phenolic acids from glycosidic bonds.114 Sometimes the use of an inert atmosphere,30,31,114 antioxidant,85 or the acidification of the hydrolyzates30,31,85,114 are essential precautions due to the poor stability of some phenolic acids in alkaline conditions. The hydrolysis method of choice is always a compromise between efficiently producing aglycones from plant material and degrading aglycones. Enzyme-assisted hydrolysis of plant material is a very interesting option due to its high specificity, the fact that it uses mild reactive conditions and because it achieves highly stable extracts with antioxidant activity and without organic solvent residues.141,142 However, its use has not been reported in the hydrolysis of tropical and subtropical biowastes.
Saponification is usually recommended to analyze the carotenoids, sterols or tocopherols of plant tissues129 as it simplifies chromatographic profiles by removing potentially interfering compounds such as chlorophyll degradation products and chlorophyll-esters. The disadvantage of this approach is that it involves extra handling steps including partitioning out of the saponified analytes, evaporation of the extracts to dryness and making up to solution before injection into the chromatograph, thereby increasing both the time and the expense of the analysis. To saponify lipid extract or oil prior to carotenoid, sterol or tocopherol determination, an aliquot of the sample is stirred with methanol25,26,104,109 or ethanol13,47,102,143 containing potassium hydroxide (KOH). Saponification is usually carried out at high temperature for 10–120 min (Table 2).13,47,51,102,104,143
Unlike most C–C single bonds that are often chemically resistant, the inter-flavanol C–C bonds in the B-type linkage of the proanthocyanidins can be readily broken under mild acidic conditions such as 1% HCl in ethanol118,119 or methanol,44,49,144 at room temperature for 2 h44,49 or at 4 °C for more than 6 h.118,119,144 This procedure has been carried out to hydrolyze anthocyanins from litchi pericarp,44,118,119 mangosteen pericarp144 and tamarillo pomace.49 The depolymerization of proanthocyanidins from litchi68 or mangosteen145 pericarp was carried out with thiol reagents.68,145 Tannins from litchi pericarp have been mixed with toluene-a-thiol 5% in 0.2 N HCl in methanol, stirred and heated for 2 min at 90 °C.68 Thiolytic degradation, followed by mass spectrometric and nuclear magnetic resonance analysis indicated the presence of catechin, epicatechin and proanthocyanidin A-type constitutive units.The mangosteen pericarp was mixed with methanol, 36% HCl and mercaptoacetic acid and heated at 65 °C for 12 h.145 This depolymerization process gave rise to 4β-(carboxymethyl)sulfanyl-(−)-epicatechin methyl ester, with a yield (calculated as epicatechin) 6 times higher than the proanthocyanidins extracted from mangosteen pericarp (without hydrolysis).
Amberlite XAD-series or Sephadex LH-20 column chromatography is commonly used for anthocyanin purification. The concentrated extract obtained from litchi pericarp was purified with an XAD-7 (50 × 2.0 cm) column.118,119 Liu et al. purified the concentrated extract obtained from litchi pericarp on an Amberlite XAD-16 (30 × 2.5 cm i.d.) column and eluted the fraction with 40% ethanol; after the ethanol was removed, the fraction was extracted with ethyl acetate.107 Subsequently, the anthocyanin fraction, in water phase, was applied to a Toyopearl HW-40S (20 × 1.6 cm i.d.) column and the pigments were eluted using a methanol:water:formic acid mixture (50:48:2, v:v:v) at a flow rate of 0.8 ml min−1. PVPP was also used to purify the anthocyanin extracts obtained from litchi pericarp.106 The absorbed anthocyanins were eluted with methanol:HCl. The anthocyanin extracts from tamarillo peelings were applied to an XAD-7 (800 mg, 40 mm i.d.) column. The pigments were eluted with a mixture of methanol:acetic acid (19:1, v:v).48 Then, the XAD-7 isolates of peelings were fractionated by high-speed counter-current chromatography.
Fractionation of polymers and oligomers from proanthocyanidin of persimmon peel was carried out by column separation.147 Previously, a mixture of freshly crushed persimmon peel and dried green tea leaves in water containing citric acid was boiled for 3 h. At this stage, substitution at the C-4 positions of polymeric proanthocyanidin with monomeric tea catechins occurred, and consequently, the polymeric molecules were converted into oligomers. The filtrate was directly applied to a Sephabeads SP 825 (45 × 10 cm i.d.) column. Elution with water washed out non-phenolic compounds. Further elution with water containing increasing amounts of ethanol (20–80% ethanol) yielded a mixture of oligomeric proanthocyanidin and tea catechins. The mixture was subsequently subjected to Sephadex LH-20 CC with ethanol. The monomeric tea catechins were eluted out with ethanol, and further elution with 50% aqueous acetone yielded oligomers.147 To prepare polymeric proanthocyanidin from persimmon peel, an aqueous acetone extract was subjected to MCI-gel CHP 20P CC with water containing methanol (0–80%) to yield polymers.
Carotenoid extracts from banana peel were purified with an alumina column and eluted with acetone: n-hexane (2:1, v: v)77 or successively with petroleum ether, ether:petroleum ether (9:1, v: v), ether:petroleum ether (1:4, v :v), ether:petroleum ether (1:3, v:v) and methanol:ether (1:9, v:v).92 A silica gel column was used to purify sterols from mango seed kernel oil.143
The anthocyanins contained in the extracts obtained from tamarillo peel were also fractionated by HSCCC, with a solvent system consisting of n-butanol:tert-butyl methyl ether:acetonitrile:water (2:2:1:5, v:v:v:v) acidified with 0.1% trifluoroacetic acid.48 The organic phase was used as the stationary phase and elution mode was head-to-tail.
Total flavonoids are usually determined by using a colorimetric method (with measurement at 510 nm) based on the formation of acid-stable complexes between aluminium chloride and the C-4 keto group and either the C-3 or C-5 hydroxyl group of flavones and flavonols. In addition, aluminium chloride forms acid-labile complexes with the orthodihydroxyl groups in the A- or B-ring of flavonoids. This method has been used to determine flavonoids in banana peel,45,78 mango peel,24,45 date seeds,30 litchi pericarp,64,68–70 mangosteen pericarp,105 passion fruit peel45 and tamarillo pomace.49 Results are usually expressed as quercetin,49,105 catechin30,78 and rutin24,45 equivalents. However, only flavones and flavonols are found to form stable complexes with aluminium chloride; none of the colorimetric methods can detect all types of flavonoids.
The analysis of anthocyanins is complex due to their ability to undergo structural transformations and complexation reactions. The total anthocyanin content in crude extracts containing other phenolic materials has been determined by measuring solution absorbance at a single wavelength, usually 520–535 nm.59,67,106,120,144 This is possible because the typical absorption band, in the 490 to 550 nm region of the visible spectra, of the anthocyanins is far from the absorption bands of other phenolics, which have spectral maxima in the UV range. In many instances, however, this simple method is inappropriate because of interference from anthocyanin degradation products from browning reactions. In those cases, the approach has been to use differential and/or subtractive methods to quantify anthocyanins and their degradation products. The monomeric anthocyanin concentration is widely evaluated by the pH differential method, based on the structural transformation of anthocyanins that occurs with a change in pH (colored at pH 1.0 and colorless at pH 4.5).22,25,48,49,61,63,66,99,106,109 Results are usually expressed as cyanidin 3-glucoside22,25,59–61,63,66,99,109,144 or as delphinidin 3-glucoside48 equivalents.
The determination of condensed tannins (proanthocyanidins) is based on oxidative depolymerization of condensed tannins in butanol-HCl reagent.42 The presence of iron is considered to increase the reproducibility and sensitivity of the assay. Vanillin-HCl is also used to determine condensed tannins.49,99
Due to the presence of a long system of conjugated double bonds, carotenoids are intensely colored and thus strongly absorb in the visible region, between 400 and 500 nm, usually exhibiting three absorption bands or two bands plus a shoulder, with vibrational fine structure, the middle band having the highest intensity.129 These absorption properties provide a simple and cheap possibility for direct analytical determination of the total carotenoid content by measuring the absorption of the extracts at 450 nm; the results are usually expressed as trans-β-carotene equivalents.51,53
Some phenolic compounds possess native fluorescence. This characteristic has been used to determine major phenolics in persimmon peel.54 Therefore, fluorescence emission has been measured at excitation (λexc) and emission (λem) wavelengths suitable for each phenolic acid that has been determined: gallic acid λexc = 260 nm, λem = 357 nm, pH = 4.63; protocatechuic acid λexc = 290 nm, λem = 363 nm, pH = 10.7; vanillic acid λexc = 305 nm, λem = 378 nm, pH = 9.3; p-coumaric acid λexc = 330 nm, λem = 443 nm, pH = 10.7; and ferulic acid λexc = 340 nm, λem = 460 nm, pH = 11.2.
HPLC is currently the most popular and reliable technique for the analysis of phenolic compounds obtained from biowastes of tropical and subtropical fruits (Table 4). A typical system involves reversed phase liquid chromatography (RP-HPLC) comprising a C18 stationary phase, in a column ranging from 15 to 30 cm in length and usually with a 4.6 mm internal diameter (i.d.) and 5 μm packing. In some instances, isocratic elution has provided adequate resolution due to selectivity effects of one or more components of the mobile phase, although gradient elution has usually been mandatory because of the complexity of the phenolic profile of most samples (Table 4). Elution systems are usually binary, with an aqueous acidified polar solvent and a less polar organic solvent such as acetonitrile or methanol. Occasionally tetrahydrofuran and 2-propanol have also been used as the organic solvent. The greatest alteration observed in the mobile phases is the acid type used as modifier to minimize peak tailing. Most often acetic or formic acid are employed; however, phosphoric acid and trifluoroacetic acid have also been used. Flow rates are usually 1.0 ml min−1 and columns are kept at ambient or slightly above ambient temperature. Phenolic elution is typical of RP-HPLC, that is, polar compounds elute first, followed by those of decreasing polarity. Hence, an elution order can be developed as phenolic acids < cinnamic acids < flavonoids although overlap of the individual members of different classes is inevitable because of the diversity of compounds.121 In phenolic and cinnamic acids, polarity is increased most by hydroxyl groups at the 4-position, followed by those at the 3- and 2-positions. The elution pattern for flavonoids containing equivalent substitution patterns is flavanone glycoside followed by flavonol and flavone glycosides and then the free aglycones in the same order.121
HPLC has been shown to be the most convenient and reliable technique for the analysis of carotenoids. Both normal phase and reversed phase HPLC methods, operating with isocratic or gradient elution and a wide variety of mixtures of different organic solvents as mobile phases, have been used to separate and quantify carotenoids in fruit biowastes. Separation has been more effective using a RP-C30 stationary phase instead of RP-C18, because the resolution of geometrical isomers with this column is outstanding (Table 4). However, for plant material with a relatively simple carotenoid profile there seems to be little advantage gained by using RP-C30 columns rather than conventional RP-C18 columns. The high price of RP-C30 columns combined with a limited column lifetime for this type of analysis significantly increases overall costs.51 In reversed-phase systems, non-aqueous mobile phases are recommended because the pronounced hydrophobicity of carotenoids makes it difficult or impossible to separate them in mobile phases containing water. Various mixtures of solvents, mostly of methanol, acetonitrile and tetrahydrofuran, have been successfully used for this purpose.129
Carotenoids from banana peel were separated in an ODS-A column with different isocratic solvent systems: methanol:ethyl acetate (62:38, v:v), methanol:ethyl acetate (88:12, v:v) or methanol:water:ethyl acetate (80:10:10, v:v:v).92 The analysis of these analytes in banana peel was carried out using a RP-C18 column with acetonitrile and methanol:ethyl acetate (1:1, v:v), containing triethylamine and BHT, as mobile phase (Table 4).51 Carotenoids in persimmon peel have been analyzed using a RP-C30 column with a mobile phase consisting of methanol:methyl tert-butyl ether:water (81:15:4, v:v::v) (eluant A) and methanol:methyl tert-butyl ether (10:90, v:v) (eluant B), both containing ammonium acetate.102 Tocopherols in pitaya seeds have been analyzed by HPLC with fluorescence detection.47 The separation of α- and γ-tocopherols was performed in a LichroCart Purospher STAR Si column with n-hexane:2-propanol (99:1, v:v) as mobile phase (Table 4).
Other separation techniques, such as capillary electrophoresis, have not been used to detect antioxidant phytochemicals in tropical and subtropical biowastes.
The complementary nature of fluorescence detection has been demonstrated and used in series with a UV detector to detect tannins. Michodjehoun-Mestres et al. used a fluorescence detector (275 nm for excitation and 322 nm for emission) connected in series to a PDA detector (280 nm) to detect tannins in cashew apple skin.33 However, this technique, and others such as electrochemistry, have not been commonly used to detect antioxidant phytochemicals in this type of biowaste.
Mass spectrometry (MS) is a powerful tool for elucidating phenolic structures. MS can be carried out either on-line in combination with chromatographic techniques or off-line. The on-line coupling of chromatography with MS has been the single-most important advance in the analysis of bioactive compounds in biowastes obtained from tropical and subtropical fruits (Table 4). Moreover, the number of applications involving direct inlet introduction of phytochemicals to the mass spectrometer has increased significantly in the last years.
In coupled mode, the mass spectrometer may function simply as a highly selective detector but it is in qualitative analysis that it excels providing unsurpassed opportunities for compound “identification”. GC-MS has been used, with electron impact ionization (EI), to identify phenolic compounds in cashew nut shell34 or phenolic and cinnamic acids in mangosteen pericarp114 previously derivatizing the phenolic compounds. Moreover, GC-MS has been used to identify sterols and tocopherols from banana peel104 or sterols from mango seed kernel oil.143 However, it is the hyphenation of LC with MS that has revolutionized the analysis of non-volatile species (Table 4). The development of soft ionization techniques, such as atmospheric pressure ionization (API), to analyze polar, non-volatile, thermolabile molecules has facilitated the analysis of high molecular mass molecules such as plant phenols by LC-MS. API-based interfacing systems are mainly based on electrospray ionization (ESI). API mass spectra typically comprise protonated molecular ions [M + H]+ in positive ion mode or deprotonated molecular ions [M − H]− in negative ion mode with few fragment ions and thus have a low structure information content. An acid (acetic or formic) is often added to mobile phases in positive ion ESI as a source of protons to assist ionization.
On rare occasions, MS can provide sufficient data for full structure analysis. Generally, the analyte fragmentation in mass spectra is used to determine molecular mass, elemental formula and to establish the distribution of substituents on the phenolic rings. In less favorable situations, the mass spectrum will assist in structural elucidation although other techniques such as nuclear magnetic resonance (NMR) are used for a definitive structural assignment.28,34,43,44,48,69,70,73,77,79,93,94,124–126,128 NMR spectra of phenols are frequently complex and identifying isolated compounds is complicated by the absence of suitable reference standards. In many instances, the combination of UV, MS and 1H NMR will provide adequate information for structural elucidation.20,74,100,148 In other cases, information on the 13C NMR20,34,43,44,69,70,73,74,77,100,124,125,128,149 signals is necessary along with 2D correlation experiments involving 1H–1H correlations such as COSY (Correlation Spectroscopy)20,48,100 or 1H–13C correlation experiments such as HMBC (Heteronuclear Multiple Quantum Coherence)20,73 and HSQC (Heteronuclear Multiple Bond Coherence)48 as applied in the structural identification of anthocyanins isolated from tamarillo peel,48 xanthones from mangosteen pericarp73 or phenolic compounds isolated from tamarind seeds and tamarind seed coats.20
From this global perspective, it is vital to develop analytical methods designed specifically to quantify phytochemicals. One of the current difficulties with phytochemical analysis is lack of confidence in methodology. There is no discussion on how the recovery of bioactive compounds from tropical and subtropical fruit biowastes may be influenced by many factors, e.g., specie, variety, ripening stage, preharvest and postharvest conditions and extraction conditions. Moreover, there are no standard procedures to isolate phytochemicals and, in the majority of cases, the procedures employed to extract phytochemicals from tropical and subtropical fruits biowaste have not been specifically optimized for them. Therefore, any quantitative data on phytochemical concentration must be evaluated in this light. In this sense, developing more effective and selective extraction techniques will significantly improve the process of obtaining bioactive compounds from tropical and subtropical fruit biowastes. Intense work needs to be carried out to increase the commercial availability of bioactive compounds as suitable reference compounds and of reference materials. In addition, it is necessary to carry out validation studies on the analytical methods developed specifically to analyze bioactive compounds from tropical and subtropical fruit biowastes.
This journal is © The Royal Society of Chemistry 2010 |