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Phytochemical analysis of organic and conventionally cultivated Meyer lemons (Citrus meyeri Tan.) during refrigerated storage
Journal of Food Composition and Analysis 42 (2015) 63–70
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Original Research Article
Phytochemical analysis of organic and conventionally cultivated Meyer lemons (Citrus meyeri Tan.) during refrigerated storage Ram M. Uckoo, G.K. Jayaprakasha, Bhimanagouda S. Patil * Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, 1500 Research Parkway, Ste A120, College Station, TX 77845, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 May 2014 Received in revised form 1 January 2015 Accepted 1 January 2015 Available online 21 March 2015
The levels of phytochemicals in organically and conventionally cultivated Meyer lemons (Citrus meyeri Tan.) are unknown. In this study, Meyer lemons grown in south Texas under similar climatic conditions, using organic and conventional cultivation practices, were evaluated for their levels of phytochemicals. Mature fruits were harvested in two seasons, stored at market-simulated post-harvest conditions for four weeks, and periodically evaluated for levels of phytochemicals, including flavonoids, amines, organic acids and minerals. Results indicate that organically grown lemons contain significantly (P 0.05) higher levels of hesperidin, didymin and ascorbic acid than those cultivated in conventional system. Phenolic content was higher in organic lemons, whereas levels of citric acid and amines were higher in conventionally cultivated lemons. These results suggest that organically grown Meyer lemons are a good source of enhanced levels of flavonoids and ascorbic acid. Furthermore, storage of fruits at 10 8C up to four weeks helps maintain the levels of phytochemicals. To the best of our knowledge, this is the first report of phytochemicals evaluation of organic and conventionally grown Meyer lemons. ß 2015 Elsevier Inc. All rights reserved.
Chemical compounds studied in this article: Narirutin (PubChem CID: 442431) Hesperidin (PubChem CID: 10621) Didymin (PubChem CID: 16760075) Ascorbic acid (PubChem CID: 54670067) Citric acid (PubChem CID: 311) Octopamine (PubChem CID: 102484) Synephrine (PubChem CID: 7172) Keywords: Meyer lemon Food analysis Food composition Limonoids Flavonoids Amines Ascorbic acid Citric acid Nutrients Total phenolics
1. Introduction Lemons are among the most commonly consumed citrus fruits in the world. The United States ranks fifth in world in lemon production (Perez and Pollack, 2003) with an estimated acreage of approximately 63,000 acres (Spreen, 2001). Among citrus fruits, lemons have high citric acid content, rendering them unpalatable (Penniston et al., 2008). Therefore, these fruits are primarily consumed fresh along with other food materials, used as garnishes, or juiced to make lemonades. Meyer lemon is considered as the hybrid of a true lemon and an unknown citrus species
* Corresponding author. Tel.: +1 979 458 8090; fax: +1 979 862 4522. E-mail address: [email protected] (B.S. Patil). http://dx.doi.org/10.1016/j.jfca.2015.01.009 0889-1575/ß 2015 Elsevier Inc. All rights reserved.
(Citrus limon Citrus sinensis) (Uzun et al., 2014) and, unlike the more common lemon varieties, such as Lisbon or Eureka, Meyer lemon fruits have a sweeter, less acidic flavor. Meyer lemon is thornless, well adapted to warm climates and has the highest juice yield per box in comparison to several other common varieties of lemons (Lim, 2012; Moshonas et al., 1972). They also have high levels of both organic acids and health-promoting phytochemicals such as amines, flavonoids and limonoids (Del Rı´o et al., 2004; Lario et al., 2004; Uckoo et al., 2011). These phytochemicals are associated with several health benefits, such as antioxidant, anti-proliferative and anti-inflammatory activities and also with the prevention of coronary heart disease (Benavente-Garcı´a and Castillo, 2008; Kawaii et al., 1999; Patil et al., 2009). Furthermore, in human clinical trials, was implicated as a potential treatment for controlling bleeding from acute internal hemorrhoids (Misra and Parshad, 2000).
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Lemons also contain amines such as octopamine, synephrine and tyramine (Stewart and Wheaton, 1964). Metabolism of these amines forms epinephrine or norepinephrine; based on this, several formulations and extracts containing amines as the main ingredient have been promoted as weight-reducing dietary supplements (Stohs et al., 2011). Consumer interest in the health benefits of phytochemicals has increased to significant proportion worldwide. Accumulating evidence on the health benefits of phytochemicals and increasing interest of consumers in health-promoting foods requires strategies to enhance the levels of phytochemicals. The content of phytochemicals depends both quantitatively and qualitatively on plant genotype (Bhattacharyya et al., 2014; Grusak et al., 1999) and on environmental factors including water and mineral nutrition (Mena et al., 2013; Uckoo et al., 2009); pre- and post-harvest factors can cause wide variation in the levels of phytochemicals. However, little information is available on the effect of pre-harvest factors on variation of phytochemicals in lemons. Cultivation practice is one of the major pre-harvest factors that could influence phytochemical contents (Goldman et al., 1999; Wang, 2006). In addition, evolving consumer preferences have driven a dramatic increase in organic cultivation (Willer and Lukas, 2009). Sales of organic citrus have increased at an annual rate of 20% since 1990 (Liu, 2003). Organic cultivation integrates basic agronomic practices such as crop rotation, green manure, compost, biological pest control and mechanical cultivation, to sustain productivity and control pests. Due to the lack of use of synthetic fertilizers, pesticides and growth regulators in organic cultivation, various biotic and abiotic stresses seems to enhance synthesis of polyphenolics to provide plant defenses (Luttikholt, 2007; Matern and Kneusel, 1988; Smith et al., 2014). Currently, information related to the effect of organic cultivation on health-promoting phytochemicals in Mayer lemons is very limited. Also, the literature shows inconsistent findings on nutrient differences between organic vs. conventionally produced fruits and vegetables (Chassy et al., 2006). It is hypothesized that organically produced lemons could contain higher levels of phytochemicals than conventionally produced lemons. Determination of the levels of phytochemicals could help us to better understand the influence of cultivation systems and provide valuable information to consumers, enabling them to make better choices. 2. Materials and methods 2.1. Plant material and experimental design During 2008–2010, a field experiment located in the Lower Rio Grande Valley of south Texas was conducted to evaluate the influence of organic and conventional cultivation practices on the levels of phytochemicals in Meyer lemon fruits. Two orchards were selected: the conventional citrus orchard was located at Texas A&M University-Kingsville, Citrus Center in Weslaco, and approximately 24 miles away, the organic orchard (certified by the Department of Agriculture (USDA)) located at Mission, TX. Both orchards used flood irrigation with a common irrigation source, the Rio Grande River. Trees were spaced 4.6 m 7.3 m with a planting density of approximately 300 trees ha 1. Five fruit trees were grouped as a replicate and three replications were used for each cultivation practices. Agronomic operations, nutrient management and weather data were monitored and recorded during the experiment period (Supplementary Tables S1 and S2). For phytochemical analysis, mature Meyer lemon fruits of uniform size and shape were harvested in November 2008 (early harvest) and February 2010 (late harvest) from both the experimental orchards for the harvest seasons of 2008 and 2009, respectively. The color of the fruit was considered as the criterion for maturity.
Only completely yellow fruits were selected for harvest and considered mature. After harvesting, the fruits were washed with clean water, air dried and packed in cardboard boxes. The fruit boxes were stored at room temperature after harvest, for four days, and then moved to refrigerated storage, for both organic and conventionally grown lemons. After four days, the boxes were stored at the optimum temperature of 10 8C in an automated thermostat regulated refrigerator. Fruits were inspected every two days for any evidence of decay of fruits. For analysis of phytochemicals, 24 fruits from each replicate were randomly sampled at 4, 11, 18 and 25 days after storage. 2.2. Chemicals and reagents ()-Octopamine hydrochloride (98%), synephrine (95%), citric acid (99.5%), limonin (90%), meta-phosphoric acid (65%), Folin– Ciocalteu reagent (2N), glacial acetic acid (>99.5%) and high performance liquid chromatography (HPLC) grade phosphoric acid (85%), were purchased from Sigma–Aldrich (St. Louis, MO, USA). LAscorbic acid (>99.9) was purchased from Mallinckrodt (Paris, KY, USA). Authentic reference standards of narirutin (98%), didymin (98%) and hesperidin (90.8%), were purchased from ChromaDex Inc. (Irvine, CA, USA). Nanopure water (NANOpure, Barnstead/Thermolyne Corp., Dubuque, IA, USA) was used for sample preparation and HPLC analysis. Acetonitrile (HPLC grade, 99.9%) and N,N-dimethylformamide (HPLC grade, 99.7%) were purchased from Fisher Scientific (Pittsburgh, PA, USA). 2.3. Quantification of amines and organic acids The amines and organic acids were analyzed using the developed method reported earlier from our lab (Uckoo et al., 2011). Eight fruits were grouped as a subsample with three subsamples in each replication of individual treatment. Fruits from each subsample were peeled and blended using a household blender (Vita-prep, Cleveland, OH, USA). The blended juice was homogenized for 30 s using a Polytron homogenizer (Brinkmann Instruments Inc., Westbury, NY, USA). Three percent meta phosphoric acid (MPA) was used for extraction of amines and organic acids. In brief, 10 g of the homogenized juice sample was diluted with 30 mL of 3% MPA in a centrifuge tube and vigorously mixed. Three milliliters of sample mixture was filtered under vacuum using a 0.45 mm membrane filter (Millipore Corp., Bedford, MA, USA). The unfiltered juice residue was re-extracted with 3 mL of MPA in successive volumes of 1 mL each. All the extracts were pooled and 10 mL was injected into the HPLC for analysis. The HPLC system consisted of a Waters 1525 HPLC series (Milford, MA, USA) connected to a PDA detector. An Xbridge C18 column (3.54 mm, 4.6 mm 150 mm i.d.) from Waters (Milford, MA, USA) was used for all the separations. Elution was carried out at ambient temperature using the mobile phase composed of 3 mM phosphoric acid under isocratic condition. The flow rate was set at 1.0 mL/min, and detection was set at dual wavelengths of l223 nm and l254 nm with a total analysis time of 10 min. Three injections were performed for each sample. Peaks were identified on the basis of comparing and matching the ultraviolet spectra as well as the retention time (RT) of the individual standards. The results were further validated by spiking the sample extracts with pure standards (Uckoo et al., 2011). 2.4. Analysis of flavonoids Ten grams of blended juice sample were mixed with 10 mL of dimethyl formamide in a 50 mL centrifuge tube and homogenized for 30 s using a polytron homogenizer (Brinkmann Instruments Inc., Westbury, NY, USA). The homogenized juice was placed on a
R.M. Uckoo et al. / Journal of Food Composition and Analysis 42 (2015) 63–70
shaker for 3 h and later filtered to collect the extract. The procedure was repeated two more times and all the extracts were pooled together. The extract was filtered using a 0.45 mm membrane filter and 10 mL clear filtrate was injected into the HPLC for analysis. The HPLC system consisted of a Waters 1525 HPLC series (Milford, MA) connected to a PDA detector. Flavonoids were separated on an Xbridge C-18 column (3.54 mm, 4.6 mm 150 mm i.d.) from Waters (Milford, MA) and detected at l280 nm. The solvent system of acetonitrile/water plus 4% acetic acid, in a gradient starting at 15% and ending at 50% acetonitrile, was used. Flavonoids were identified by comparing their ultraviolet spectra and retention times with those of standards. Quantification of flavonoids was done by using known concentrations of external standards from the commercial source and all samples were run in triplicate (Uckoo et al., 2012). To further validate the identification of flavonoids, each homogenized lemon juice sample (10 mL) was extracted with acetonitrile in three successive steps consisting of 10 mL each. All the solvent extracts were pooled, filtered through a 0.45 mm membrane filter and analyzed by ultra-high performance liquid chromatography–time of flight-mass spectrometry (UHPLC– QTOF-MS) (maxis Impact, Bruker Daltonics, Billerica, MA, USA). Flavonoids were separated on a Kinetex C18 column (1.7 mm, 100 2.1 mm; Phenomenex, Torrance, CA, USA) using an Agilent 1290 UHPLC instrument (Agilent, Waldbronn, Germany). The separation was carried out at 50 8C with a flow rate of 0.2 mL/min using gradient elution of acetonitrile in 0.1% formic acid (solvent A) and acetonitrile (solvent B). Mass spectral analyses were performed using an electron spray ionization-quantitative-time of flight (ESI-Q-TOF) mass spectrometer equipped with an electrospray ionization source in negative ion mode. Capillary voltage was maintained at 3 kV, source temperature was set at 200 8C and nitrogen was used as the desolvation gas (12 L/min).
mature leaves were randomly harvested from trees in three rows for both treatments. The harvested leaf samples were washed with 1% hydrochloric acid solution, air dried and analyzed for macroand micronutrients. For juice mineral analysis, fruits harvested in both harvest seasons from the three replicates in each treatment were processed at 4, 11, 18 and 25 days after storage and juice was collected. The juice samples were homogenized and freeze-dried in a freeze-dryer (LabConco, Kansas City, MO). The lyophilized juice samples were blended to a fine powder, sieved and submitted for analysis. The mineral analysis was conducted at Texas A&M University’s Soil, Water and Forage Testing Laboratory at College Station, Texas. Briefly, the nitrite nitrogen was extracted from juice using 1 N potassium chloride (KCl) solution on a reciprocal shaker for 30 min followed by nitrite to nitrate reduction through a cadmium column in a colorimetric apparatus (FIA Lab Instruments Inc., Bellevue, WA, USA). Other juice minerals were quantified by inductive coupled plasma-atomic emission spectroscopy (ICP-AES) (Spectro Genesis, Deutschland, Germany). After digesting the juice samples in concentrated nitric acid, they were allowed to stay overnight at room temperature. The digested samples were heated to 125 8C for 4 h. After cooling and sample dilution, the intensity of the ion response was measured in ICP-AES. For the soil mineral analysis, the extractions were conducted using Mehlich III reagent and analyzed using ICP. 2.7. Statistical analysis Analysis of statistical differences between treatment groups was conducted using a general linear model procedure of SAS (SAS Institute, Cary, NC, USA). Mean comparisons were made using Duncan multiple range test with significant differences of means at the 95% confidence level (P 0.05).
2.5. Measurement of total phenolics
3. Results and discussion
The concentration of total phenolics in the extracts was determined using the Folin–Ciocalteu colorimetric method described by Negi and Jayaprakasha, with some modifications and the results are expressed as catechin equivalents (Negi et al., 2003). Briefly, 10 g of lyophilized fruit juice of each replicate of individual treatment was extracted exhaustively with 500 mL of methanol in Soxhlet type apparatus for 8 h. The extract was concentrated by roto-evaporation (Buchi Rotavapor; Bu¨chi Labortechnik, Flawil, Switzerland) and lyophilized in a freeze dryer (Labconco Freezone 4.5; Labconco Corp., Kansas City, MO, USA). The freeze-dried methanol extract was dissolved in a solvent mixture of methanol and water (80:20, v/v) to obtain a concentration of 5 mg/mL. Calibration curves were prepared for the working solutions of catechin (10, 20, 30, 40, 50, 75 and 100 mg) of standard by diluted in solvent mixture of methanol and water (80:20). Briefly, the dissolved sample extracts (100 mL) and standard concentrations of catechin were taken in test tubes and the volume was adjusted to 10 mL by addition of distilled water. One milliliter of 1-fold diluted Folin–Ciocalteu reagent and 1 mL of 7.5% sodium carbonate solution were added to all the tubes. The resultant samples were incubated for 30 min at room temperature, and the absorbance measured at l760 nm using a spectrophotometer. The estimation of total phenolics in all the extracts was carried out in triplicate and the mean results presented as a relative measure of catechin.
3.1. Contents of amines and organic acids
2.6. Soil, leaf and juice mineral analysis Soil core samples from the upper 30 cm depth were collected from three different rows of organic and conventional systems in the harvest seasons 2008 and 2009. For leaf nutrient analysis,
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Amines such as octopamine, synephrine and tyramine are compounds containing nitrogen functionality. These compounds are considered end products of nitrogen metabolism (Wheaton and Stewart, 1969), and thus these compounds are also studied as indicators for evaluation of the authenticity of organic and conventional citrus (Rapisarda et al., 2005), as application of fertilizer in conventional cultivation will produce higher levels of amines. Nitrogen management is an important factor of ‘‘Best Management Practices’’ in citrus crop production (Alva et al., 2006). Prior studies have demonstrated a positive correlation between nitrogen fertilization and yield (Uckoo et al., 2005). Among the detected amines, octopamine was the predominant amine present in both organic and conventionally grown lemons. In both the harvest years, conventionally grown lemons had a significantly higher content of octopamine. No significant difference was observed in the contents of synephrine and tyramine between organic and conventionally grown lemons (Table 1). The fruits harvested in 2008 had a higher content of amines compared to the fruits harvested in 2009. The significant variation in the levels of these amines among organic and conventionally produced lemons could be due to variation in the availability of soil nitrogen to the plants. The readily available N through fertilizer application in the conventional practice may have resulted in higher accumulation of these compounds. In the storage analysis, no significant variation was observed in the amines for both the treatments. These results suggest that storage of lemons at 10 8C could be a good strategy for maintaining the levels of amines in lemons.
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Table 1 Octopamine, synephrine and tyramine content in Meyer lemons cultivated under organic and conventional cultivation practices harvested in 2008 and 2009 analyzed at different intervals of storage.a Cultivation practice
Days after harvest
mg/100 g Harvest year 2008 (early)
Conventional Organic Conventional Organic Conventional Organic Conventional Organic
4 4 11 11 18 18 25 25
Harvest year 2009 (late)
Octopomine
Synephrine
Tyramine
Octopomine
Synephrine
Tyramine
22.5 0.80a 17.9 2.76b 19.2 1.53b 21.5 1.69a 19.9 1.09a 17.0 2.02b 19.3 1.60a 17.2 0.91b
10.9 1.62a 10.4 1.09a 12.4 0.52a 12.3 1.27a 12.0 0.79a 11.8 1.90a 10.8 0.52a 11.3 0.75a
16.3 1.58a 16.8 0.98a 15.3 0.37b 18.7 0.64a 15.2 2.81a 14.9 0.98a 15.6 1.37a 14.5 1.04b
6.1 1.84a 5.5 2.97b 6.8 2.28a 5.4 3.66b 5.9 4.51a 5.5 3.66a 5.4 3.44a 4.0 2.71b
3.8 2.25a 3.8 1.57a 3.8 3.20a 4.5 3.80a 3.3 1.48b 4.4 3.88a 3.8 1.39a 3.9 0.84a
4.4 1.68a 4.0 1.75b 4.7 2.39a 4.5 2.58a 4.7 2.80a 4.5 2.58a 5.6 4.41a 4.3 2.23b
a Each value is average of three replications standard deviation values. Mean separations within each harvest year and for similar storage interval between organic and conventional by Duncan’s multiple range test at P 0.05.
Ascorbic acid content was significantly higher (P 0.05) in organically grown lemons in comparison to conventionally grown in the 2008 harvest season (Table 2). The ascorbic acid content of organic fruits ranged from 272 mg/100 g to 321 mg/100 g whereas the conventionally grown fruit ranged from 194 mg/100 g and 243 mg/100 g. However in the late harvest of 2009, organically grown lemons had relatively lower contents of ascorbic acid ranging from 189 mg/100 g to 198 mg/100 g. Furthermore, conventionally grown lemons had a higher ascorbic acid in early harvest fruit during 2008 than the late harvest of 2009. Conventionally grown fruit, in 2009 had ascorbic acid contents ranging between 171 and 200 mg/100 g. The variation in ascorbic acid could be due to differences in maturity at harvest and climatic conditions during the growing season. Also, it is reported that mature citrus fruits harvested late in the season have lower concentrations of ascorbic acid in contrast to the fruits harvested early, which have higher concentrations (Nagy, 1980). Our results are consistent with previous results (Carbonaro et al., 2002; CarisVeyrat et al., 2004; Lombardi-Boccia et al., 2003). In the storage study, a gradual decline in ascorbic acid content was observed, with a loss of 13% and 12% in conventional and organically grown lemons, respectively, in the harvest year 2008. However, in 2009 the loss in ascorbic acid during storage was minimal in fruit from both organic and conventional production systems. The citric acid content was significantly (P 0.05) higher in conventional than organic fruit throughout the storage period in 2008 harvest (Table 2). The citric acid ranged from 70 g/100 g to 75 g/100 g in conventional lemons during the storage period of 25 days. A similar trend was also observed in the 2009 harvest year with conventional fruits having significantly higher citric acid contents at 4 days after harvest and 25 days after storage. The presence of high contents of ascorbic acid in organic
fruits suggests that organic cultivation practice could be used as a tool to obtain higher levels of ascorbic acid. 3.2. Flavonoids and their significance Lemons are a good source of flavonoids and characteristically contain high amounts of these health-promoting compounds. Flavonoids such as narirutin, hesperidin and didymin were detected in the lemon juice, with hesperidin as the major compound. Quantification of these flavonoids by HPLC indicated that organic fruits had significantly higher levels of hesperidin and didymin in both harvest years (Table 3). The level of hesperidin ranged from 82 mg/100 g to 155 mg/100 g in both harvest years for organic and conventionally grown lemons. In both harvest years the levels of hesperidin were significantly higher (P 0.05) in organic lemons than in conventionally grown lemons. Narirutin was relatively low in concentration, ranging from 0.97 mg/100 g to 1.63 mg/100 g in both harvest years for organic and conventionally grown lemons. The identities of the quantified flavonoids were confirmed by UHPLC–QTOF-MS analysis using ESI in negative ion mode. Fig. 1 illustrates the LC–MS spectra obtained for three flavonoids as [M H] ions, which identified these flavonoids as narirutin (M. Wt. 579), hesperidin (M. Wt. 609) and didymin (M. Wt. 593). Flavonoids are polyphenolic compounds which are implicated in the plant defensive mechanism against disease and pests (Treutter, 2006) and these results suggest that lemons are a good dietary source of flavonoids such as hesperidin. Cumulative evidence suggests that hesperidin, narirutin and didymin have antioxidant properties in vivo (Miyake et al., 1998), inhibit bone loss (Chiba et al., 2003) and possess antimicrobial properties (Yi et al., 2008). Due to these proven benefits, regular consumption of fruits and vegetables with high levels of flavonoids is
Table 2 Ascorbic acid and citric acid content of Meyer lemons cultivated under organic and conventional management practices harvested in 2008 and 2009 analyzed at different intervals of storage.a Cultivation practice
Days after harvest
Harvest year 2008 (early)
Harvest year 2009 (late)
Ascorbic acid (mg/100 g)
Citric acid (g/100 g)
Ascorbic acid (mg/100 g)
Citric acid (g/100 g)
Conventional Organic Conventional Organic Conventional Organic Conventional Organic
4 4 11 11 18 18 25 25
243 5.47b 310 34.4a 217 20.6b 321 14.0a 194 13.5b 307 30.7a 212 16.3b 272 34.1a
75.1 3.25a 53.6 4.76b 71.7 3.12a 50.9 0.88b 69.7 1.90a 54.5 2.62b 72.1 2.91a 52.3 2.55b
197 10.2a 198 18.8a 200 11.8b 209 13.7a 171 35.8b 209 13.7a 195 20.9a 192 18.4a
54.1 2.87a 50.8 2.62b 53.8 5.37a 51.6 2.15a 47.6 8.71b 51.6 2.15a 57.1 4.49a 54.1 5.50a
a Each value is an average of three replications standard deviation values. Mean separations within each harvest year and for similar storage interval between organic and conventional by Duncan’s multiple range test at P 0.05.
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Table 3 Levels of flavonoids (narirutin, hesperidin and didymin) present in Meyer lemons cultivated under organic and conventional cultivation practices harvested in 2008 and 2009 analyzed after different intervals of storage.a Cultivation practice
Days after harvest
mg/100 g Harvest year 2008 (early)
Conventional Organic Conventional Organic Conventional Organic Conventional Organic
4 4 11 11 18 18 25 25
Harvest year 2009 (late)
Narirutin
Hesperidin
Didymin
Narirutin
Hesperidin
Didymin
1.11 0.11a 1.14 0.07a 1.32 0.22a 1.44 0.12a 1.07 0.08a 1.26 0.16a 0.97 0.19a 1.01 0.22a
102 4.36b 134 11.9a 94 17.5b 155 8.66a 90 16.0b 130 19.4a 82 27.9b 115 21.7a
18.4 6.48b 27.8 2.87a 16.7 7.10b 32.3 3.98a 18.5 7.60b 31.3 9.93a 14.0 14.4b 27.8 9.71a
1.43 0.06a 1.63 0.06a 1.26 0.24a 1.33 0.11a 1.43 0.23a 1.31 0.30a 1.37 0.17a 1.57 0.09a
127 7.25b 150 8.27a 120 14.5b 145 14.7a 138 26.4b 152 26.7a 151 8.77a 148 9.77a
35.0 2.46b 45.1 2.48a 35.5 6.26b 43.6 4.06a 40.1 7.64b 46.8 7.34a 47.8 2.03b 52.7 3.72a
a Each value is average of three replications standard deviation values. Mean separations within each harvest year and for similar storage interval between organic and conventional by Duncan’s multiple range test at P 0.05.
field could have resulted in enhanced synthesis of polyphenolics. In contrast, fertilizer application in the conventional orchard could have resulted in less nutrient stress, which corresponds to low accumulation of these compounds. Similar trend in the levels of flavonoids were noticed in other studies comparing organic and conventional production systems. For example, in a three year comparison of the level of flavonoids in tomatoes, organically grown tomatoes had significantly higher levels of flavonoids than conventionally grown tomatoes (Chassy et al., 2006). In another study, organic and conventionally grown grapefruits were
recommended (Haldar et al., 2014). The per capita consumption of lemons is approximately 1.6 kg (USDA, 2012), which is lower other fruits. However, due to the high concentration of flavonoids compared to other fruits, lemons seem to have positive impact toward health benefits. It is possible that the higher level of certain phytochemicals including ascorbic acid, narirutin, hesperidin, didymin in lemons grown under organic cultivation, as compared with conventionally grown, occur due to the effect of nutrient stress conditions. The low nutrient levels (Table 4) observed in the soil analysis in the organic
(A) 0.15 AU
Hesperidin
0.10
Narirutin Didymin
0.05 0.00 0.00
5.00
10.00 15.00 Minutes
Intens. 461.1153 3
5000
Narirutin (M-H)Exact mass:579.17083
112.9871 1
25.00
-MS 609.1892 1
1.5
579.1803 4
4000 3000
Intens. x105
-MS
(B)
20.00
Hesperidin (M-H)Exact mass:609.18139
1.0
191.0223 2
2000
0.5 1000 0
100
200
300
400
500
600
700
800
900
Intens. x104
0.0
m/z
200
400
600
800
m/z
-MS 593 .18823
Didymin (M-H)Exact mass:593.18648
2.0
1.5
1.0 171.102 61 0.5
0.0
200
400
600
800
m/z
Fig. 1. (A) HPLC chromatogram of Meyer lemon, (B) negative ion mass spectra of flavonoids identified in Meyer lemon juice samples analyzed by liquid chromatography– quadrupole-time of flight (LC–QTOF) using electrospray ionization.
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Table 4 Nutrient analysis of soil amendments and soil in upper 45 cm in the conventional and organic lemon orchards. NO3
Cultivation
P
K
Ca
Mg
Na
Zn
Fe
0.01 0.75 0.083 0.100
0.09 1.59 0.345 0.348
0.03 0.77 0.220 0.310
0.01 0.50 0.413 0.322
0.03 0.29 0.202 0.088
0.00058 0.128 0.00053 0.00082
0.00611 1.68 0.00452 0.00316
0.0340 0.0515
0.408 0.262
6.44 5.26
0.519 0.389
0.241 0.146
0.00080 0.00298
0.00855 0.00452
% Organic Compost brew Compost Soil: 2008 Soil: 2009 Conventional Soil: 2008 Soil: 2009
0.02 3.29 9.00 1.96 12.0 4.24
evaluated for the levels of phytochemicals such as flavonoids for three consecutive harvest years. Organically cultivated grapefruits had significantly higher levels of naringin than the conventionally cultivated in all three years (Lester et al., 2007). These limited studies suggest a positive trend toward higher levels of flavonoids in organic cultivated fruits and vegetables, but further research is still needed. 3.3. Total phenolics
Harvest year 2008
8.00
Organic
7.00
Conventional
3.4. Juice and soil mineral analysis Mineral and nutrient analysis of the juice of organic and conventional lemons showed significant differences in the contents of total N (Fig. 3), P, K, Ca, Mg and protein (Table 5). However, these differences were inconsistent for the two harvest years. Significant differences (P 0.05) in the content of total N (Fig. 3) were only seen in the 2008 harvest year. No significant
6.00
1.4 5.00
(a)
1.2
4.00
1.0
Total N %
CA. Eq. G/ 100 G Juice dry wt.
In both harvest years, organic lemons (5.54–7.00 g/100 g of juice dry weight) had higher total phenolic contents as compared to conventional lemons, but the values were not significantly different (P 0.05) (Fig. 2). In the storage analysis, no significant (P 0.05) variation was noticed among the organic and conventionally grown lemons in both harvest years. Phenolic compounds
contribute significantly to fruit quality and health beneficial properties. Furthermore, studies suggest that polyphenols present in lemons inhibit diet-induced obesity in animal studies (Fukuchi et al., 2008). They are also an integral part of plant defense mechanisms, protecting plants from insects, pests and reactive oxygen species. Estimation of the total phenolic content provides a measure of the reducing capacity of the various bioactive compounds. Our results suggest that lemon fruits cultivated by organic or conventional management practices are a good source of phenolic compounds. Moreover, storage at 10 8C aids in maintaining the levels of phenolics in both organic and conventionally produced lemon fruits.
3.00 2.00 4
11 18 Days after storage
25
0.8 0.6 0.4 0.2 4
Harvest year 2009
1.2 8.00
11
6.00
4.00
2.00
18
25
Organic
(b)
Convenonal
1.1 Total N %
CA. Eq. G/ 100 G Juice dry wt.
0.0 10.00
1.0 0.9 0.8 0.7
4
11
18
25
Days after storage Fig. 2. Total phenolic content expressed as catechin (CA) equivalent (g/100 g of juice dry wt.) of Meyer lemon grown under organic and conventional management practices in different harvest years and analyzed after storage at 4, 11, 18 and 25 days.
0.6 4
11
18
25
Fig. 3. Total N% of Meyer lemons juice cultivated under organic and conventional management practices harvested in (a) 2008 and (b) 2009 analyzed after different intervals of storage (days after harvest).
R.M. Uckoo et al. / Journal of Food Composition and Analysis 42 (2015) 63–70
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Table 5 Mineral and nutrient analysis of Meyer lemons juice (dry weight basis) cultivated under organic and conventional management practices harvested and analyzed at different intervals of storage.a Cultivation
Days after harvest
P
K
Ca
Mg
Na
% 2008 harvest Conventional Organic Conventional Organic Conventional Organic Conventional Organic 2010 harvest Conventional Organic Conventional Organic Conventional Organic Conventional Organic
Zn
Fe
Cu
Mn
Protein
11.6a 13.2a 12.4a 12.4a 13.4a 12.8a 12.5a 13.1a
4.22a 3.87a 3.71a 3.45a 4.13a 3.91a 3.85a 4.27a
11.5a 11.9a 12.0a 11.8a 12.3a 12.7a 11.9a 11.9a
6.17a 4.42b 6.72a 2.54b 6.58a 4.82b 6.47a 5.36b
ppm
4 4 11 11 18 18 25 25
1.57a 1.80a 1.69a 1.73a 1.76a 1.97a 1.58b 1.96a
11.2b 13.1a 11.3b 12.4a 12.1b 13.6a 11.3b 13.2a
3.35b 3.89a 3.33b 4.40a 3.47b 4.61a 3.31b 4.29a
0.96a 1.00a 1.00a 0.93a 1.11a 1.13a 1.04a 1.09a
0.61a 0.54a 0.50a 0.47a 0.59a 0.45b 0.51a 0.55a
8.38a 8.91a 8.91a 7.93a 10.05a 8.74a 9.63a 8.68a
4 4 11 11 18 18 25 25
1.72b 2.05a 1.62b 2.01a 1.73b 2.07a 1.70b 2.04a
11.2b 13.6a 11.1b 13.5a 11.1b 13.2a 11.0b 13.0a
3.15a 3.63a 3.12b 3.92a 3.06b 4.20a 2.98b 4.17a
0.86b 0.98a 0.89a 0.96a 0.82b 0.96a 0.82b 0.96a
0.31a 0.19a 0.27a 0.22a 0.47a 0.24b 0.51a 0.28b
5.52a 5.08a 5.30a 5.29a 6.00a 4.99a 5.71a 5.00a
7.46a 9.13a 8.06a 9.00a 7.41a 8.43a 7.93a 9.75a
2.89a 2.95a 2.89a 3.02a 3.09a 2.98a 3.50a 2.99a
4.65a 3.88a 4.93a 3.72b 4.30a 3.76a 4.26a 3.77a
5.70a 5.88a 6.24a 5.89a 5.77a 5.39a 5.92a 5.46a
a Each value is average of three replications. Mean separations within each harvest year and for similar storage interval between organic and conventional by Duncan’s multiple range test at P 0.05.
differences were noticed for the content of Zn, Cu, Fe and Mn. Juice mineral content is influenced by the available soil nutrients. The high levels of N observed in the conventional lemons could be due to the readily available N applied as a fertilizer (46-0-0). This trend was noticed in both harvest years. In the storage analysis, no major differences were noticed, suggesting that lemon juice nutrients are not influenced by storage at 10 8C in both cultivation practices. The nutrient composition of the compost brew and the compost applied to the organic lemon orchard, as well as the soil nutrient analysis of the conventional and organic orchards, are given in Table 5. In both harvest years, the organic orchard had low NO3 contents (9.0–2.0%) in comparison to the conventional orchard (12.0% and 4.2%). The low NO3 content in organic orchard soil could be due to the absence of synthetic fertilization, while in the conventional orchard, synthetic fertilizers were applied in both harvest years. A similar trend of high levels of Ca, Mg, Fe and Zn was noticed in the conventional orchard soil in comparison to the soil from the organic orchard. Organic cultivation practice limits the availability of essential macronutrients due to reduced mineralization capacity by soil organisms (Zech et al., 1997). Moreover, the high temperature conditions prevalent in the south Texas result in high soil microbial activity, resulting in low organic matter content (Davidson and Janssens, 2006). Soil amendments such as fertilizer and compost are important factors for maintaining good plant health and also obtaining optimum yield (Fliessbach et al., 2007). Our previous results related to long-term evaluation of compost application on citrus suggest a positive correlation between compost application and root hair growth (Nelson et al., 2008). Therefore, compost application enables efficient water uptake and as well as nutrient uptake. Citrus crops require periodic nutrient management practices through fertilization to replenish the nutrients lost through the high yield of fruits harvested annually. Studies also suggest a positive correlation between fertilization and yield of citrus (Uckoo et al., 2005). Hence, in conventional production systems, inorganic fertilizers are commonly applied to obtain high fruit yields. In contrast, organic orchards substitute the application of organic amendments such as compost and compost brew. Compost brew is a nutrient culture obtained from agitating, aerating and extraction of compost using an Earth tea brewer (Sustainable Agricultural Technol., Cottage Grove, OR), or similar apparatus. The compost brew applied contains both macro- and micronutrients, and among these nutrients the brew contained
higher amounts of micronutrients (Fe: 128% and Zn: 1676%) as compared to macronutrients (NO3 : 0.02%, P: 0.01%, K: 0.09%). These results suggest that the soil nutrient contents differed in organic and conventional systems. 4. Conclusion The results from this study suggest that Meyer lemons are a good source of phytochemicals and that cultivation practice could be used as an effective tool for modulating phytochemical contents. Organic and conventionally cultivated lemons differ in their levels of ascorbic acid and flavonoids, although some inconsistencies do exist in different harvest seasons. No variation in the content of amines was noticed in lemons produced organically and conventionally. Storage at 10 8C helps to maintain the levels of these phytochemicals without any major adverse effects. Further long-term multi-location field studies are required to validate the variation in phytochemical contents in Meyer lemons. Acknowledgements The authors acknowledge the support of Mr. Dennis Holbrook, CEO, South Texas Organics (Mission, TX) for providing access to the organic Meyer lemon orchard. This project is based upon the work supported by the USDA-CSREES # 2009-34402-19831 ‘‘Designing Foods for Health’’ through the Vegetable & Fruit Improvement Center. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jfca.2015.01.009. References Alva, A.K., Paramasivam, S., Fares, A., Obreza, T.A., Schumann, A.W., 2006. Nitrogen best management practice for citrus trees: II. Nitrogen fate, transport, and components of N budget. Scientia Horticulturae 109 (3), 223–233. Benavente-Garcı´a, O., Castillo, J., 2008. Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity. Journal of Agricultural and Food Chemistry 56 (15), 6185–6205.
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Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Research Article
Phytochemical analysis of organic and conventionally cultivated Meyer lemons (Citrus meyeri Tan.) during refrigerated storage Ram M. Uckoo, G.K. Jayaprakasha, Bhimanagouda S. Patil * Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, 1500 Research Parkway, Ste A120, College Station, TX 77845, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 May 2014 Received in revised form 1 January 2015 Accepted 1 January 2015 Available online 21 March 2015
The levels of phytochemicals in organically and conventionally cultivated Meyer lemons (Citrus meyeri Tan.) are unknown. In this study, Meyer lemons grown in south Texas under similar climatic conditions, using organic and conventional cultivation practices, were evaluated for their levels of phytochemicals. Mature fruits were harvested in two seasons, stored at market-simulated post-harvest conditions for four weeks, and periodically evaluated for levels of phytochemicals, including flavonoids, amines, organic acids and minerals. Results indicate that organically grown lemons contain significantly (P 0.05) higher levels of hesperidin, didymin and ascorbic acid than those cultivated in conventional system. Phenolic content was higher in organic lemons, whereas levels of citric acid and amines were higher in conventionally cultivated lemons. These results suggest that organically grown Meyer lemons are a good source of enhanced levels of flavonoids and ascorbic acid. Furthermore, storage of fruits at 10 8C up to four weeks helps maintain the levels of phytochemicals. To the best of our knowledge, this is the first report of phytochemicals evaluation of organic and conventionally grown Meyer lemons. ß 2015 Elsevier Inc. All rights reserved.
Chemical compounds studied in this article: Narirutin (PubChem CID: 442431) Hesperidin (PubChem CID: 10621) Didymin (PubChem CID: 16760075) Ascorbic acid (PubChem CID: 54670067) Citric acid (PubChem CID: 311) Octopamine (PubChem CID: 102484) Synephrine (PubChem CID: 7172) Keywords: Meyer lemon Food analysis Food composition Limonoids Flavonoids Amines Ascorbic acid Citric acid Nutrients Total phenolics
1. Introduction Lemons are among the most commonly consumed citrus fruits in the world. The United States ranks fifth in world in lemon production (Perez and Pollack, 2003) with an estimated acreage of approximately 63,000 acres (Spreen, 2001). Among citrus fruits, lemons have high citric acid content, rendering them unpalatable (Penniston et al., 2008). Therefore, these fruits are primarily consumed fresh along with other food materials, used as garnishes, or juiced to make lemonades. Meyer lemon is considered as the hybrid of a true lemon and an unknown citrus species
* Corresponding author. Tel.: +1 979 458 8090; fax: +1 979 862 4522. E-mail address: [email protected] (B.S. Patil). http://dx.doi.org/10.1016/j.jfca.2015.01.009 0889-1575/ß 2015 Elsevier Inc. All rights reserved.
(Citrus limon Citrus sinensis) (Uzun et al., 2014) and, unlike the more common lemon varieties, such as Lisbon or Eureka, Meyer lemon fruits have a sweeter, less acidic flavor. Meyer lemon is thornless, well adapted to warm climates and has the highest juice yield per box in comparison to several other common varieties of lemons (Lim, 2012; Moshonas et al., 1972). They also have high levels of both organic acids and health-promoting phytochemicals such as amines, flavonoids and limonoids (Del Rı´o et al., 2004; Lario et al., 2004; Uckoo et al., 2011). These phytochemicals are associated with several health benefits, such as antioxidant, anti-proliferative and anti-inflammatory activities and also with the prevention of coronary heart disease (Benavente-Garcı´a and Castillo, 2008; Kawaii et al., 1999; Patil et al., 2009). Furthermore, in human clinical trials, was implicated as a potential treatment for controlling bleeding from acute internal hemorrhoids (Misra and Parshad, 2000).
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Lemons also contain amines such as octopamine, synephrine and tyramine (Stewart and Wheaton, 1964). Metabolism of these amines forms epinephrine or norepinephrine; based on this, several formulations and extracts containing amines as the main ingredient have been promoted as weight-reducing dietary supplements (Stohs et al., 2011). Consumer interest in the health benefits of phytochemicals has increased to significant proportion worldwide. Accumulating evidence on the health benefits of phytochemicals and increasing interest of consumers in health-promoting foods requires strategies to enhance the levels of phytochemicals. The content of phytochemicals depends both quantitatively and qualitatively on plant genotype (Bhattacharyya et al., 2014; Grusak et al., 1999) and on environmental factors including water and mineral nutrition (Mena et al., 2013; Uckoo et al., 2009); pre- and post-harvest factors can cause wide variation in the levels of phytochemicals. However, little information is available on the effect of pre-harvest factors on variation of phytochemicals in lemons. Cultivation practice is one of the major pre-harvest factors that could influence phytochemical contents (Goldman et al., 1999; Wang, 2006). In addition, evolving consumer preferences have driven a dramatic increase in organic cultivation (Willer and Lukas, 2009). Sales of organic citrus have increased at an annual rate of 20% since 1990 (Liu, 2003). Organic cultivation integrates basic agronomic practices such as crop rotation, green manure, compost, biological pest control and mechanical cultivation, to sustain productivity and control pests. Due to the lack of use of synthetic fertilizers, pesticides and growth regulators in organic cultivation, various biotic and abiotic stresses seems to enhance synthesis of polyphenolics to provide plant defenses (Luttikholt, 2007; Matern and Kneusel, 1988; Smith et al., 2014). Currently, information related to the effect of organic cultivation on health-promoting phytochemicals in Mayer lemons is very limited. Also, the literature shows inconsistent findings on nutrient differences between organic vs. conventionally produced fruits and vegetables (Chassy et al., 2006). It is hypothesized that organically produced lemons could contain higher levels of phytochemicals than conventionally produced lemons. Determination of the levels of phytochemicals could help us to better understand the influence of cultivation systems and provide valuable information to consumers, enabling them to make better choices. 2. Materials and methods 2.1. Plant material and experimental design During 2008–2010, a field experiment located in the Lower Rio Grande Valley of south Texas was conducted to evaluate the influence of organic and conventional cultivation practices on the levels of phytochemicals in Meyer lemon fruits. Two orchards were selected: the conventional citrus orchard was located at Texas A&M University-Kingsville, Citrus Center in Weslaco, and approximately 24 miles away, the organic orchard (certified by the Department of Agriculture (USDA)) located at Mission, TX. Both orchards used flood irrigation with a common irrigation source, the Rio Grande River. Trees were spaced 4.6 m 7.3 m with a planting density of approximately 300 trees ha 1. Five fruit trees were grouped as a replicate and three replications were used for each cultivation practices. Agronomic operations, nutrient management and weather data were monitored and recorded during the experiment period (Supplementary Tables S1 and S2). For phytochemical analysis, mature Meyer lemon fruits of uniform size and shape were harvested in November 2008 (early harvest) and February 2010 (late harvest) from both the experimental orchards for the harvest seasons of 2008 and 2009, respectively. The color of the fruit was considered as the criterion for maturity.
Only completely yellow fruits were selected for harvest and considered mature. After harvesting, the fruits were washed with clean water, air dried and packed in cardboard boxes. The fruit boxes were stored at room temperature after harvest, for four days, and then moved to refrigerated storage, for both organic and conventionally grown lemons. After four days, the boxes were stored at the optimum temperature of 10 8C in an automated thermostat regulated refrigerator. Fruits were inspected every two days for any evidence of decay of fruits. For analysis of phytochemicals, 24 fruits from each replicate were randomly sampled at 4, 11, 18 and 25 days after storage. 2.2. Chemicals and reagents ()-Octopamine hydrochloride (98%), synephrine (95%), citric acid (99.5%), limonin (90%), meta-phosphoric acid (65%), Folin– Ciocalteu reagent (2N), glacial acetic acid (>99.5%) and high performance liquid chromatography (HPLC) grade phosphoric acid (85%), were purchased from Sigma–Aldrich (St. Louis, MO, USA). LAscorbic acid (>99.9) was purchased from Mallinckrodt (Paris, KY, USA). Authentic reference standards of narirutin (98%), didymin (98%) and hesperidin (90.8%), were purchased from ChromaDex Inc. (Irvine, CA, USA). Nanopure water (NANOpure, Barnstead/Thermolyne Corp., Dubuque, IA, USA) was used for sample preparation and HPLC analysis. Acetonitrile (HPLC grade, 99.9%) and N,N-dimethylformamide (HPLC grade, 99.7%) were purchased from Fisher Scientific (Pittsburgh, PA, USA). 2.3. Quantification of amines and organic acids The amines and organic acids were analyzed using the developed method reported earlier from our lab (Uckoo et al., 2011). Eight fruits were grouped as a subsample with three subsamples in each replication of individual treatment. Fruits from each subsample were peeled and blended using a household blender (Vita-prep, Cleveland, OH, USA). The blended juice was homogenized for 30 s using a Polytron homogenizer (Brinkmann Instruments Inc., Westbury, NY, USA). Three percent meta phosphoric acid (MPA) was used for extraction of amines and organic acids. In brief, 10 g of the homogenized juice sample was diluted with 30 mL of 3% MPA in a centrifuge tube and vigorously mixed. Three milliliters of sample mixture was filtered under vacuum using a 0.45 mm membrane filter (Millipore Corp., Bedford, MA, USA). The unfiltered juice residue was re-extracted with 3 mL of MPA in successive volumes of 1 mL each. All the extracts were pooled and 10 mL was injected into the HPLC for analysis. The HPLC system consisted of a Waters 1525 HPLC series (Milford, MA, USA) connected to a PDA detector. An Xbridge C18 column (3.54 mm, 4.6 mm 150 mm i.d.) from Waters (Milford, MA, USA) was used for all the separations. Elution was carried out at ambient temperature using the mobile phase composed of 3 mM phosphoric acid under isocratic condition. The flow rate was set at 1.0 mL/min, and detection was set at dual wavelengths of l223 nm and l254 nm with a total analysis time of 10 min. Three injections were performed for each sample. Peaks were identified on the basis of comparing and matching the ultraviolet spectra as well as the retention time (RT) of the individual standards. The results were further validated by spiking the sample extracts with pure standards (Uckoo et al., 2011). 2.4. Analysis of flavonoids Ten grams of blended juice sample were mixed with 10 mL of dimethyl formamide in a 50 mL centrifuge tube and homogenized for 30 s using a polytron homogenizer (Brinkmann Instruments Inc., Westbury, NY, USA). The homogenized juice was placed on a
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shaker for 3 h and later filtered to collect the extract. The procedure was repeated two more times and all the extracts were pooled together. The extract was filtered using a 0.45 mm membrane filter and 10 mL clear filtrate was injected into the HPLC for analysis. The HPLC system consisted of a Waters 1525 HPLC series (Milford, MA) connected to a PDA detector. Flavonoids were separated on an Xbridge C-18 column (3.54 mm, 4.6 mm 150 mm i.d.) from Waters (Milford, MA) and detected at l280 nm. The solvent system of acetonitrile/water plus 4% acetic acid, in a gradient starting at 15% and ending at 50% acetonitrile, was used. Flavonoids were identified by comparing their ultraviolet spectra and retention times with those of standards. Quantification of flavonoids was done by using known concentrations of external standards from the commercial source and all samples were run in triplicate (Uckoo et al., 2012). To further validate the identification of flavonoids, each homogenized lemon juice sample (10 mL) was extracted with acetonitrile in three successive steps consisting of 10 mL each. All the solvent extracts were pooled, filtered through a 0.45 mm membrane filter and analyzed by ultra-high performance liquid chromatography–time of flight-mass spectrometry (UHPLC– QTOF-MS) (maxis Impact, Bruker Daltonics, Billerica, MA, USA). Flavonoids were separated on a Kinetex C18 column (1.7 mm, 100 2.1 mm; Phenomenex, Torrance, CA, USA) using an Agilent 1290 UHPLC instrument (Agilent, Waldbronn, Germany). The separation was carried out at 50 8C with a flow rate of 0.2 mL/min using gradient elution of acetonitrile in 0.1% formic acid (solvent A) and acetonitrile (solvent B). Mass spectral analyses were performed using an electron spray ionization-quantitative-time of flight (ESI-Q-TOF) mass spectrometer equipped with an electrospray ionization source in negative ion mode. Capillary voltage was maintained at 3 kV, source temperature was set at 200 8C and nitrogen was used as the desolvation gas (12 L/min).
mature leaves were randomly harvested from trees in three rows for both treatments. The harvested leaf samples were washed with 1% hydrochloric acid solution, air dried and analyzed for macroand micronutrients. For juice mineral analysis, fruits harvested in both harvest seasons from the three replicates in each treatment were processed at 4, 11, 18 and 25 days after storage and juice was collected. The juice samples were homogenized and freeze-dried in a freeze-dryer (LabConco, Kansas City, MO). The lyophilized juice samples were blended to a fine powder, sieved and submitted for analysis. The mineral analysis was conducted at Texas A&M University’s Soil, Water and Forage Testing Laboratory at College Station, Texas. Briefly, the nitrite nitrogen was extracted from juice using 1 N potassium chloride (KCl) solution on a reciprocal shaker for 30 min followed by nitrite to nitrate reduction through a cadmium column in a colorimetric apparatus (FIA Lab Instruments Inc., Bellevue, WA, USA). Other juice minerals were quantified by inductive coupled plasma-atomic emission spectroscopy (ICP-AES) (Spectro Genesis, Deutschland, Germany). After digesting the juice samples in concentrated nitric acid, they were allowed to stay overnight at room temperature. The digested samples were heated to 125 8C for 4 h. After cooling and sample dilution, the intensity of the ion response was measured in ICP-AES. For the soil mineral analysis, the extractions were conducted using Mehlich III reagent and analyzed using ICP. 2.7. Statistical analysis Analysis of statistical differences between treatment groups was conducted using a general linear model procedure of SAS (SAS Institute, Cary, NC, USA). Mean comparisons were made using Duncan multiple range test with significant differences of means at the 95% confidence level (P 0.05).
2.5. Measurement of total phenolics
3. Results and discussion
The concentration of total phenolics in the extracts was determined using the Folin–Ciocalteu colorimetric method described by Negi and Jayaprakasha, with some modifications and the results are expressed as catechin equivalents (Negi et al., 2003). Briefly, 10 g of lyophilized fruit juice of each replicate of individual treatment was extracted exhaustively with 500 mL of methanol in Soxhlet type apparatus for 8 h. The extract was concentrated by roto-evaporation (Buchi Rotavapor; Bu¨chi Labortechnik, Flawil, Switzerland) and lyophilized in a freeze dryer (Labconco Freezone 4.5; Labconco Corp., Kansas City, MO, USA). The freeze-dried methanol extract was dissolved in a solvent mixture of methanol and water (80:20, v/v) to obtain a concentration of 5 mg/mL. Calibration curves were prepared for the working solutions of catechin (10, 20, 30, 40, 50, 75 and 100 mg) of standard by diluted in solvent mixture of methanol and water (80:20). Briefly, the dissolved sample extracts (100 mL) and standard concentrations of catechin were taken in test tubes and the volume was adjusted to 10 mL by addition of distilled water. One milliliter of 1-fold diluted Folin–Ciocalteu reagent and 1 mL of 7.5% sodium carbonate solution were added to all the tubes. The resultant samples were incubated for 30 min at room temperature, and the absorbance measured at l760 nm using a spectrophotometer. The estimation of total phenolics in all the extracts was carried out in triplicate and the mean results presented as a relative measure of catechin.
3.1. Contents of amines and organic acids
2.6. Soil, leaf and juice mineral analysis Soil core samples from the upper 30 cm depth were collected from three different rows of organic and conventional systems in the harvest seasons 2008 and 2009. For leaf nutrient analysis,
65
Amines such as octopamine, synephrine and tyramine are compounds containing nitrogen functionality. These compounds are considered end products of nitrogen metabolism (Wheaton and Stewart, 1969), and thus these compounds are also studied as indicators for evaluation of the authenticity of organic and conventional citrus (Rapisarda et al., 2005), as application of fertilizer in conventional cultivation will produce higher levels of amines. Nitrogen management is an important factor of ‘‘Best Management Practices’’ in citrus crop production (Alva et al., 2006). Prior studies have demonstrated a positive correlation between nitrogen fertilization and yield (Uckoo et al., 2005). Among the detected amines, octopamine was the predominant amine present in both organic and conventionally grown lemons. In both the harvest years, conventionally grown lemons had a significantly higher content of octopamine. No significant difference was observed in the contents of synephrine and tyramine between organic and conventionally grown lemons (Table 1). The fruits harvested in 2008 had a higher content of amines compared to the fruits harvested in 2009. The significant variation in the levels of these amines among organic and conventionally produced lemons could be due to variation in the availability of soil nitrogen to the plants. The readily available N through fertilizer application in the conventional practice may have resulted in higher accumulation of these compounds. In the storage analysis, no significant variation was observed in the amines for both the treatments. These results suggest that storage of lemons at 10 8C could be a good strategy for maintaining the levels of amines in lemons.
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Table 1 Octopamine, synephrine and tyramine content in Meyer lemons cultivated under organic and conventional cultivation practices harvested in 2008 and 2009 analyzed at different intervals of storage.a Cultivation practice
Days after harvest
mg/100 g Harvest year 2008 (early)
Conventional Organic Conventional Organic Conventional Organic Conventional Organic
4 4 11 11 18 18 25 25
Harvest year 2009 (late)
Octopomine
Synephrine
Tyramine
Octopomine
Synephrine
Tyramine
22.5 0.80a 17.9 2.76b 19.2 1.53b 21.5 1.69a 19.9 1.09a 17.0 2.02b 19.3 1.60a 17.2 0.91b
10.9 1.62a 10.4 1.09a 12.4 0.52a 12.3 1.27a 12.0 0.79a 11.8 1.90a 10.8 0.52a 11.3 0.75a
16.3 1.58a 16.8 0.98a 15.3 0.37b 18.7 0.64a 15.2 2.81a 14.9 0.98a 15.6 1.37a 14.5 1.04b
6.1 1.84a 5.5 2.97b 6.8 2.28a 5.4 3.66b 5.9 4.51a 5.5 3.66a 5.4 3.44a 4.0 2.71b
3.8 2.25a 3.8 1.57a 3.8 3.20a 4.5 3.80a 3.3 1.48b 4.4 3.88a 3.8 1.39a 3.9 0.84a
4.4 1.68a 4.0 1.75b 4.7 2.39a 4.5 2.58a 4.7 2.80a 4.5 2.58a 5.6 4.41a 4.3 2.23b
a Each value is average of three replications standard deviation values. Mean separations within each harvest year and for similar storage interval between organic and conventional by Duncan’s multiple range test at P 0.05.
Ascorbic acid content was significantly higher (P 0.05) in organically grown lemons in comparison to conventionally grown in the 2008 harvest season (Table 2). The ascorbic acid content of organic fruits ranged from 272 mg/100 g to 321 mg/100 g whereas the conventionally grown fruit ranged from 194 mg/100 g and 243 mg/100 g. However in the late harvest of 2009, organically grown lemons had relatively lower contents of ascorbic acid ranging from 189 mg/100 g to 198 mg/100 g. Furthermore, conventionally grown lemons had a higher ascorbic acid in early harvest fruit during 2008 than the late harvest of 2009. Conventionally grown fruit, in 2009 had ascorbic acid contents ranging between 171 and 200 mg/100 g. The variation in ascorbic acid could be due to differences in maturity at harvest and climatic conditions during the growing season. Also, it is reported that mature citrus fruits harvested late in the season have lower concentrations of ascorbic acid in contrast to the fruits harvested early, which have higher concentrations (Nagy, 1980). Our results are consistent with previous results (Carbonaro et al., 2002; CarisVeyrat et al., 2004; Lombardi-Boccia et al., 2003). In the storage study, a gradual decline in ascorbic acid content was observed, with a loss of 13% and 12% in conventional and organically grown lemons, respectively, in the harvest year 2008. However, in 2009 the loss in ascorbic acid during storage was minimal in fruit from both organic and conventional production systems. The citric acid content was significantly (P 0.05) higher in conventional than organic fruit throughout the storage period in 2008 harvest (Table 2). The citric acid ranged from 70 g/100 g to 75 g/100 g in conventional lemons during the storage period of 25 days. A similar trend was also observed in the 2009 harvest year with conventional fruits having significantly higher citric acid contents at 4 days after harvest and 25 days after storage. The presence of high contents of ascorbic acid in organic
fruits suggests that organic cultivation practice could be used as a tool to obtain higher levels of ascorbic acid. 3.2. Flavonoids and their significance Lemons are a good source of flavonoids and characteristically contain high amounts of these health-promoting compounds. Flavonoids such as narirutin, hesperidin and didymin were detected in the lemon juice, with hesperidin as the major compound. Quantification of these flavonoids by HPLC indicated that organic fruits had significantly higher levels of hesperidin and didymin in both harvest years (Table 3). The level of hesperidin ranged from 82 mg/100 g to 155 mg/100 g in both harvest years for organic and conventionally grown lemons. In both harvest years the levels of hesperidin were significantly higher (P 0.05) in organic lemons than in conventionally grown lemons. Narirutin was relatively low in concentration, ranging from 0.97 mg/100 g to 1.63 mg/100 g in both harvest years for organic and conventionally grown lemons. The identities of the quantified flavonoids were confirmed by UHPLC–QTOF-MS analysis using ESI in negative ion mode. Fig. 1 illustrates the LC–MS spectra obtained for three flavonoids as [M H] ions, which identified these flavonoids as narirutin (M. Wt. 579), hesperidin (M. Wt. 609) and didymin (M. Wt. 593). Flavonoids are polyphenolic compounds which are implicated in the plant defensive mechanism against disease and pests (Treutter, 2006) and these results suggest that lemons are a good dietary source of flavonoids such as hesperidin. Cumulative evidence suggests that hesperidin, narirutin and didymin have antioxidant properties in vivo (Miyake et al., 1998), inhibit bone loss (Chiba et al., 2003) and possess antimicrobial properties (Yi et al., 2008). Due to these proven benefits, regular consumption of fruits and vegetables with high levels of flavonoids is
Table 2 Ascorbic acid and citric acid content of Meyer lemons cultivated under organic and conventional management practices harvested in 2008 and 2009 analyzed at different intervals of storage.a Cultivation practice
Days after harvest
Harvest year 2008 (early)
Harvest year 2009 (late)
Ascorbic acid (mg/100 g)
Citric acid (g/100 g)
Ascorbic acid (mg/100 g)
Citric acid (g/100 g)
Conventional Organic Conventional Organic Conventional Organic Conventional Organic
4 4 11 11 18 18 25 25
243 5.47b 310 34.4a 217 20.6b 321 14.0a 194 13.5b 307 30.7a 212 16.3b 272 34.1a
75.1 3.25a 53.6 4.76b 71.7 3.12a 50.9 0.88b 69.7 1.90a 54.5 2.62b 72.1 2.91a 52.3 2.55b
197 10.2a 198 18.8a 200 11.8b 209 13.7a 171 35.8b 209 13.7a 195 20.9a 192 18.4a
54.1 2.87a 50.8 2.62b 53.8 5.37a 51.6 2.15a 47.6 8.71b 51.6 2.15a 57.1 4.49a 54.1 5.50a
a Each value is an average of three replications standard deviation values. Mean separations within each harvest year and for similar storage interval between organic and conventional by Duncan’s multiple range test at P 0.05.
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Table 3 Levels of flavonoids (narirutin, hesperidin and didymin) present in Meyer lemons cultivated under organic and conventional cultivation practices harvested in 2008 and 2009 analyzed after different intervals of storage.a Cultivation practice
Days after harvest
mg/100 g Harvest year 2008 (early)
Conventional Organic Conventional Organic Conventional Organic Conventional Organic
4 4 11 11 18 18 25 25
Harvest year 2009 (late)
Narirutin
Hesperidin
Didymin
Narirutin
Hesperidin
Didymin
1.11 0.11a 1.14 0.07a 1.32 0.22a 1.44 0.12a 1.07 0.08a 1.26 0.16a 0.97 0.19a 1.01 0.22a
102 4.36b 134 11.9a 94 17.5b 155 8.66a 90 16.0b 130 19.4a 82 27.9b 115 21.7a
18.4 6.48b 27.8 2.87a 16.7 7.10b 32.3 3.98a 18.5 7.60b 31.3 9.93a 14.0 14.4b 27.8 9.71a
1.43 0.06a 1.63 0.06a 1.26 0.24a 1.33 0.11a 1.43 0.23a 1.31 0.30a 1.37 0.17a 1.57 0.09a
127 7.25b 150 8.27a 120 14.5b 145 14.7a 138 26.4b 152 26.7a 151 8.77a 148 9.77a
35.0 2.46b 45.1 2.48a 35.5 6.26b 43.6 4.06a 40.1 7.64b 46.8 7.34a 47.8 2.03b 52.7 3.72a
a Each value is average of three replications standard deviation values. Mean separations within each harvest year and for similar storage interval between organic and conventional by Duncan’s multiple range test at P 0.05.
field could have resulted in enhanced synthesis of polyphenolics. In contrast, fertilizer application in the conventional orchard could have resulted in less nutrient stress, which corresponds to low accumulation of these compounds. Similar trend in the levels of flavonoids were noticed in other studies comparing organic and conventional production systems. For example, in a three year comparison of the level of flavonoids in tomatoes, organically grown tomatoes had significantly higher levels of flavonoids than conventionally grown tomatoes (Chassy et al., 2006). In another study, organic and conventionally grown grapefruits were
recommended (Haldar et al., 2014). The per capita consumption of lemons is approximately 1.6 kg (USDA, 2012), which is lower other fruits. However, due to the high concentration of flavonoids compared to other fruits, lemons seem to have positive impact toward health benefits. It is possible that the higher level of certain phytochemicals including ascorbic acid, narirutin, hesperidin, didymin in lemons grown under organic cultivation, as compared with conventionally grown, occur due to the effect of nutrient stress conditions. The low nutrient levels (Table 4) observed in the soil analysis in the organic
(A) 0.15 AU
Hesperidin
0.10
Narirutin Didymin
0.05 0.00 0.00
5.00
10.00 15.00 Minutes
Intens. 461.1153 3
5000
Narirutin (M-H)Exact mass:579.17083
112.9871 1
25.00
-MS 609.1892 1
1.5
579.1803 4
4000 3000
Intens. x105
-MS
(B)
20.00
Hesperidin (M-H)Exact mass:609.18139
1.0
191.0223 2
2000
0.5 1000 0
100
200
300
400
500
600
700
800
900
Intens. x104
0.0
m/z
200
400
600
800
m/z
-MS 593 .18823
Didymin (M-H)Exact mass:593.18648
2.0
1.5
1.0 171.102 61 0.5
0.0
200
400
600
800
m/z
Fig. 1. (A) HPLC chromatogram of Meyer lemon, (B) negative ion mass spectra of flavonoids identified in Meyer lemon juice samples analyzed by liquid chromatography– quadrupole-time of flight (LC–QTOF) using electrospray ionization.
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Table 4 Nutrient analysis of soil amendments and soil in upper 45 cm in the conventional and organic lemon orchards. NO3
Cultivation
P
K
Ca
Mg
Na
Zn
Fe
0.01 0.75 0.083 0.100
0.09 1.59 0.345 0.348
0.03 0.77 0.220 0.310
0.01 0.50 0.413 0.322
0.03 0.29 0.202 0.088
0.00058 0.128 0.00053 0.00082
0.00611 1.68 0.00452 0.00316
0.0340 0.0515
0.408 0.262
6.44 5.26
0.519 0.389
0.241 0.146
0.00080 0.00298
0.00855 0.00452
% Organic Compost brew Compost Soil: 2008 Soil: 2009 Conventional Soil: 2008 Soil: 2009
0.02 3.29 9.00 1.96 12.0 4.24
evaluated for the levels of phytochemicals such as flavonoids for three consecutive harvest years. Organically cultivated grapefruits had significantly higher levels of naringin than the conventionally cultivated in all three years (Lester et al., 2007). These limited studies suggest a positive trend toward higher levels of flavonoids in organic cultivated fruits and vegetables, but further research is still needed. 3.3. Total phenolics
Harvest year 2008
8.00
Organic
7.00
Conventional
3.4. Juice and soil mineral analysis Mineral and nutrient analysis of the juice of organic and conventional lemons showed significant differences in the contents of total N (Fig. 3), P, K, Ca, Mg and protein (Table 5). However, these differences were inconsistent for the two harvest years. Significant differences (P 0.05) in the content of total N (Fig. 3) were only seen in the 2008 harvest year. No significant
6.00
1.4 5.00
(a)
1.2
4.00
1.0
Total N %
CA. Eq. G/ 100 G Juice dry wt.
In both harvest years, organic lemons (5.54–7.00 g/100 g of juice dry weight) had higher total phenolic contents as compared to conventional lemons, but the values were not significantly different (P 0.05) (Fig. 2). In the storage analysis, no significant (P 0.05) variation was noticed among the organic and conventionally grown lemons in both harvest years. Phenolic compounds
contribute significantly to fruit quality and health beneficial properties. Furthermore, studies suggest that polyphenols present in lemons inhibit diet-induced obesity in animal studies (Fukuchi et al., 2008). They are also an integral part of plant defense mechanisms, protecting plants from insects, pests and reactive oxygen species. Estimation of the total phenolic content provides a measure of the reducing capacity of the various bioactive compounds. Our results suggest that lemon fruits cultivated by organic or conventional management practices are a good source of phenolic compounds. Moreover, storage at 10 8C aids in maintaining the levels of phenolics in both organic and conventionally produced lemon fruits.
3.00 2.00 4
11 18 Days after storage
25
0.8 0.6 0.4 0.2 4
Harvest year 2009
1.2 8.00
11
6.00
4.00
2.00
18
25
Organic
(b)
Convenonal
1.1 Total N %
CA. Eq. G/ 100 G Juice dry wt.
0.0 10.00
1.0 0.9 0.8 0.7
4
11
18
25
Days after storage Fig. 2. Total phenolic content expressed as catechin (CA) equivalent (g/100 g of juice dry wt.) of Meyer lemon grown under organic and conventional management practices in different harvest years and analyzed after storage at 4, 11, 18 and 25 days.
0.6 4
11
18
25
Fig. 3. Total N% of Meyer lemons juice cultivated under organic and conventional management practices harvested in (a) 2008 and (b) 2009 analyzed after different intervals of storage (days after harvest).
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Table 5 Mineral and nutrient analysis of Meyer lemons juice (dry weight basis) cultivated under organic and conventional management practices harvested and analyzed at different intervals of storage.a Cultivation
Days after harvest
P
K
Ca
Mg
Na
% 2008 harvest Conventional Organic Conventional Organic Conventional Organic Conventional Organic 2010 harvest Conventional Organic Conventional Organic Conventional Organic Conventional Organic
Zn
Fe
Cu
Mn
Protein
11.6a 13.2a 12.4a 12.4a 13.4a 12.8a 12.5a 13.1a
4.22a 3.87a 3.71a 3.45a 4.13a 3.91a 3.85a 4.27a
11.5a 11.9a 12.0a 11.8a 12.3a 12.7a 11.9a 11.9a
6.17a 4.42b 6.72a 2.54b 6.58a 4.82b 6.47a 5.36b
ppm
4 4 11 11 18 18 25 25
1.57a 1.80a 1.69a 1.73a 1.76a 1.97a 1.58b 1.96a
11.2b 13.1a 11.3b 12.4a 12.1b 13.6a 11.3b 13.2a
3.35b 3.89a 3.33b 4.40a 3.47b 4.61a 3.31b 4.29a
0.96a 1.00a 1.00a 0.93a 1.11a 1.13a 1.04a 1.09a
0.61a 0.54a 0.50a 0.47a 0.59a 0.45b 0.51a 0.55a
8.38a 8.91a 8.91a 7.93a 10.05a 8.74a 9.63a 8.68a
4 4 11 11 18 18 25 25
1.72b 2.05a 1.62b 2.01a 1.73b 2.07a 1.70b 2.04a
11.2b 13.6a 11.1b 13.5a 11.1b 13.2a 11.0b 13.0a
3.15a 3.63a 3.12b 3.92a 3.06b 4.20a 2.98b 4.17a
0.86b 0.98a 0.89a 0.96a 0.82b 0.96a 0.82b 0.96a
0.31a 0.19a 0.27a 0.22a 0.47a 0.24b 0.51a 0.28b
5.52a 5.08a 5.30a 5.29a 6.00a 4.99a 5.71a 5.00a
7.46a 9.13a 8.06a 9.00a 7.41a 8.43a 7.93a 9.75a
2.89a 2.95a 2.89a 3.02a 3.09a 2.98a 3.50a 2.99a
4.65a 3.88a 4.93a 3.72b 4.30a 3.76a 4.26a 3.77a
5.70a 5.88a 6.24a 5.89a 5.77a 5.39a 5.92a 5.46a
a Each value is average of three replications. Mean separations within each harvest year and for similar storage interval between organic and conventional by Duncan’s multiple range test at P 0.05.
differences were noticed for the content of Zn, Cu, Fe and Mn. Juice mineral content is influenced by the available soil nutrients. The high levels of N observed in the conventional lemons could be due to the readily available N applied as a fertilizer (46-0-0). This trend was noticed in both harvest years. In the storage analysis, no major differences were noticed, suggesting that lemon juice nutrients are not influenced by storage at 10 8C in both cultivation practices. The nutrient composition of the compost brew and the compost applied to the organic lemon orchard, as well as the soil nutrient analysis of the conventional and organic orchards, are given in Table 5. In both harvest years, the organic orchard had low NO3 contents (9.0–2.0%) in comparison to the conventional orchard (12.0% and 4.2%). The low NO3 content in organic orchard soil could be due to the absence of synthetic fertilization, while in the conventional orchard, synthetic fertilizers were applied in both harvest years. A similar trend of high levels of Ca, Mg, Fe and Zn was noticed in the conventional orchard soil in comparison to the soil from the organic orchard. Organic cultivation practice limits the availability of essential macronutrients due to reduced mineralization capacity by soil organisms (Zech et al., 1997). Moreover, the high temperature conditions prevalent in the south Texas result in high soil microbial activity, resulting in low organic matter content (Davidson and Janssens, 2006). Soil amendments such as fertilizer and compost are important factors for maintaining good plant health and also obtaining optimum yield (Fliessbach et al., 2007). Our previous results related to long-term evaluation of compost application on citrus suggest a positive correlation between compost application and root hair growth (Nelson et al., 2008). Therefore, compost application enables efficient water uptake and as well as nutrient uptake. Citrus crops require periodic nutrient management practices through fertilization to replenish the nutrients lost through the high yield of fruits harvested annually. Studies also suggest a positive correlation between fertilization and yield of citrus (Uckoo et al., 2005). Hence, in conventional production systems, inorganic fertilizers are commonly applied to obtain high fruit yields. In contrast, organic orchards substitute the application of organic amendments such as compost and compost brew. Compost brew is a nutrient culture obtained from agitating, aerating and extraction of compost using an Earth tea brewer (Sustainable Agricultural Technol., Cottage Grove, OR), or similar apparatus. The compost brew applied contains both macro- and micronutrients, and among these nutrients the brew contained
higher amounts of micronutrients (Fe: 128% and Zn: 1676%) as compared to macronutrients (NO3 : 0.02%, P: 0.01%, K: 0.09%). These results suggest that the soil nutrient contents differed in organic and conventional systems. 4. Conclusion The results from this study suggest that Meyer lemons are a good source of phytochemicals and that cultivation practice could be used as an effective tool for modulating phytochemical contents. Organic and conventionally cultivated lemons differ in their levels of ascorbic acid and flavonoids, although some inconsistencies do exist in different harvest seasons. No variation in the content of amines was noticed in lemons produced organically and conventionally. Storage at 10 8C helps to maintain the levels of these phytochemicals without any major adverse effects. Further long-term multi-location field studies are required to validate the variation in phytochemical contents in Meyer lemons. Acknowledgements The authors acknowledge the support of Mr. Dennis Holbrook, CEO, South Texas Organics (Mission, TX) for providing access to the organic Meyer lemon orchard. This project is based upon the work supported by the USDA-CSREES # 2009-34402-19831 ‘‘Designing Foods for Health’’ through the Vegetable & Fruit Improvement Center. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jfca.2015.01.009. References Alva, A.K., Paramasivam, S., Fares, A., Obreza, T.A., Schumann, A.W., 2006. Nitrogen best management practice for citrus trees: II. Nitrogen fate, transport, and components of N budget. Scientia Horticulturae 109 (3), 223–233. Benavente-Garcı´a, O., Castillo, J., 2008. Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity. Journal of Agricultural and Food Chemistry 56 (15), 6185–6205.
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