Assessment of validation and antioxidant activities of novel 12 Korean strawberry cultivars

Songmi LEE Joong-Hyun CHO Ki Deok PARK Yong-Dae KIM Soon-Ho YIM About the authors

Abstract

In this study, antioxidant activities of extracts of 12 Korean strawberry cultivars were evaluated and validation of an analytical method for ellagic acid, a marker compound, was carried out. Cultivar 'Josaenghongshim' had the highest total polyphenol and flavonoid contents. In addition, cultivar 'Josaenghongshim' had the highest value and cultivar 'Seolhyang' showing the highest activity. Ellagic acid contents of 'Suhong', 'Okme', and 'Dahong' cultivars were the highest ellagic acid content. Correlation coefficient of the calibration curve was 0.9999, showing very high linearity. The HPLC-DAD method for the quantitation of ellagic acid content showed high linearity at various concentration ranges, with a limit of detection of 2.35 μg/mL. The limit of quantification was 7.13 μg/mL. Relative standard deviations from intra- and inter-day precision were less than 5.13%. Recovery rates of ellagic acid at 10, 50, and 100 μg/mL, respectively, were 99.0-100.1% with RSD values less than 5.30%. These results provide viable information for the validation of antioxidant capacities of 12 cultivars of Korean strawberries.

Keywords:
antioxidant; validation; marker compounds; Korean strawberries; ellagic acid

1 Introduction

Reactive oxygen species (ROS) such as superoxide anion radical (O2), hydroxy radical (·OH), and hydrogen peroxidase (H2O2) produced by various metabolic processes. There are known to cause human diseases and aging (Hwang et al., 2014Hwang, S. J., Yoon, W. B., Lee, O., Cha, S. J., & Kim, J. D. (2014). Radical-scavenging-linked antioxidant activities of extracts from block chokeberry and blueberry cultivated in Korea. Food Chemistry, 146, 71-77. http://dx.doi.org/10.1016/j.foodchem.2013.09.035. PMid:24176315.
http://dx.doi.org/10.1016/j.foodchem.201...
). Excessively produced ROS can lead to severe physiological disabilities by damaging tissues and cells, and inhibiting protein breakdown and DNA synthesis (Halliwell et al., 1992Halliwell, B., Gutteridge, J. M. C., & Cross, C. E. (1992). Free radicals, antioxidants and human disease: where are we now? The Journal of Laboratory and Clinical Medicine, 119(6), 598-620. PMid:1593209.). Living organisms have antioxidant systems such as superoxide dismutase (SOD), catalase, glutathione reductase, glutathione, and thioredoxin to protect cells against damages caused by ROS (Sies, 1993Sies, H. (1993). Strategies of antioxidant defense. European Journal of Biochemistry, 215(2), 213-219. http://dx.doi.org/10.1111/j.1432-1033.1993.tb18025.x. PMid:7688300.
http://dx.doi.org/10.1111/j.1432-1033.19...
). However, needs of safe and effective antioxidants are increased for stressed-out modern people (Lee & Lee, 2016Lee, S. H., & Lee, S. O. (2016). Polyphenol contents and antioxidant activities of lentil extracts from different cultivars. Journal of the Korean Society of Food Science and Nutrition, 45(7), 973-979. http://dx.doi.org/10.3746/jkfn.2016.45.7.973.
http://dx.doi.org/10.3746/jkfn.2016.45.7...
). Recently, due to improvement of living standard and change in consumer perception, functional food materials developed from natural product and related research are increased (Lee et al., 2015Lee, Y., Lee, J. H., Kim, S. D., Chang, M. S., Jo, I. S., Kim, S. J., Hwang, K. T., Jo, H. B., & Kim, J. H. (2015). Chemical composition, functional constituents, and antioxidant activities of berry fruits produced in Korea. Journal of the Korean Society of Food Science and Nutrition, 44(9), 1295-1303. http://dx.doi.org/10.3746/jkfn.2015.44.9.1295.
http://dx.doi.org/10.3746/jkfn.2015.44.9...
).

Strawberry (Fragaria ananassa Duch.), a perennial plant belonging to the rose family, has high contents of sugars, organic acids, and bioactive compounds such as ascorbic acid, polyphenols, quercetin, ferulic acid, ellagic acid, and flavonols (Kim et al., 2015Kim, S. K., Kim, D. S., Kim, D. Y., & Chun, C. H. (2015). Variation of bioactive compounds content of 14 oriental strawberry cultivars. Food Chemistry, 184, 196-202. http://dx.doi.org/10.1016/j.foodchem.2015.03.060. PMid:25872444.
http://dx.doi.org/10.1016/j.foodchem.201...
). Consuming strawberries can reduce the risk of chronic diseases such as cancers (Seeram et al., 2006Seeram, N. P., Adams, L. S., Zhang, Y., Lee, R., Sand, D., Scheuller, H. S., & Heber, D. (2006). Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro. Journal of Agricultural and Food Chemistry, 54(25), 9329-9339. http://dx.doi.org/10.1021/jf061750g. PMid:17147415.
http://dx.doi.org/10.1021/jf061750g...
), cardiovascular diseases (Azzini et al., 2010Azzini, E., Vitaglione, P., Intorre, F., Napolitano, A., Durazzo, A., Foddai, M. S., Fumagalli, A., Catasta, G., Rossi, L., Venneria, E., Raguzzini, A., Palomba, L., Fogliano, V., & Maiani, G.. (2010). Bioavailability of strawberry antioxidants in human subjects. The British Journal of Nutrition, 104(8), 1165-1173. http://dx.doi.org/10.1017/S000711451000187X. PMid:20487578.
http://dx.doi.org/10.1017/S0007114510001...
), and memory loss (Giampieri et al., 2012Giampieri, F., Tulipani, S., Alvarez-Suarez, J. M., Quiles, J. L., Mezzetti, B., & Battino, M. (2012). The strawberry: composition, nutritional quality, and impact on human health. Nutrition (Burbank, Los Angeles County, Calif.), 28(1), 9-19. http://dx.doi.org/10.1016/j.nut.2011.08.009. PMid:22153122.
http://dx.doi.org/10.1016/j.nut.2011.08....
). It can also decrease mutation and cholesterol level (Meyers et al., 2003Meyers, K. J., Watkins, C. B., Pritts, M. P., & Liu, R. H. (2003). Antioxidant and antiproliferative activities of strawberries. Journal of Agricultural and Food Chemistry, 51(23), 6887-6892. http://dx.doi.org/10.1021/jf034506n. PMid:14582991.
http://dx.doi.org/10.1021/jf034506n...
). Among all polyphenols in strawberries, ellagic acid has a strong antioxidant efficacy by scavenging free radicals (Türk et al., 2010Türk, G., Sönmez, M., Ceribaşi, A. O., Yüce, A., & Ateşşahin, A. (2010). Attenuation of cyclosporine A-induced testicular and spermatozoal damages associated with oxidative stress by ellagic acid. International Immunopharmacology, 10(2), 177-182. http://dx.doi.org/10.1016/j.intimp.2009.10.013. PMid:19883798.
http://dx.doi.org/10.1016/j.intimp.2009....
). It also possesses antiviral and antibacterial properties. It can also protect cells against apoptosis (Xu et al., 2003Xu, Y. M., Deng, J. Z., Ma, J., Chen, S. N., Marshall, R., Jones, S. H., Johnson, R. K., & Hecht, S. M. (2003). DNA damaging activity of ellagic acid derivatives. Bioorganic & Medicinal Chemistry, 11(7), 1593-1596. http://dx.doi.org/10.1016/S0968-0896(02)00452-2. PMid:12628683.
http://dx.doi.org/10.1016/S0968-0896(02)...
). Ellagic acid may produce positive effects in the control of estrogen receptor, collagen expression, and inhibition of photoresistance damage in fibroblasts (Kim et al., 2016Kim, K. S., Han, S. H., An, I. S., & Ahn, K. J. (2016). Protective effects of ellagic acid against UVA-induced oxidative stress in human dermal papilla. Asian Journal of Beauty and Cosmetology, 14(2), 191-200. http://dx.doi.org/10.20402/ajbc.2016.0048.
http://dx.doi.org/10.20402/ajbc.2016.004...
; Türk et al., 2010Türk, G., Sönmez, M., Ceribaşi, A. O., Yüce, A., & Ateşşahin, A. (2010). Attenuation of cyclosporine A-induced testicular and spermatozoal damages associated with oxidative stress by ellagic acid. International Immunopharmacology, 10(2), 177-182. http://dx.doi.org/10.1016/j.intimp.2009.10.013. PMid:19883798.
http://dx.doi.org/10.1016/j.intimp.2009....
; Papoutsi et al., 2005Papoutsi, Z., Kassi, E., Tsiapara, A., Fokialakis, N., Chrousos, G. P., & Moutsatsou, P. (2005). Evaluation of estrogenic/antiestrogenic activity of ellagic acid via the estrogen receptor subtypes ERα and ERβ. Journal of Agricultural and Food Chemistry, 53(20), 7715-7720. http://dx.doi.org/10.1021/jf0510539. PMid:16190622.
http://dx.doi.org/10.1021/jf0510539...
). In addition, ellagic acid can prevent tissue damage by repress peroxide lipid formation and control carcinogenesis by promoting the activity of detoxification enzyme (Maas et al., 1991Maas, J. L., Galletta, G. J., & Stoner, G. D. (1991). Ellagic acid, and anticarcinogen in fruits, especially in strawberries: a review. HortScience, 26(1), 10-14. http://dx.doi.org/10.21273/HORTSCI.26.1.10.
http://dx.doi.org/10.21273/HORTSCI.26.1....
). It also has protective effect against skin cancer (Mukhtar et al., 1986Mukhtar, H., Das, M., & Bickers, D. R. (1986). Inhibition of 3-methylcholanthrene-induced skin tumorigenicity in BALB/c mice by chronic oral feeding of trace amounts of ellagic acid in drinking water. Cancer Research, 46(5), 2262-2265. PMid:3486036.), and liver cancer (Shimogaki et al., 2000Shimogaki, H., Tanaka, Y., Tamai, H., & Masuda, M. (2000). In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibition. International Journal of Cosmetic Science, 22(4), 291-303. http://dx.doi.org/10.1046/j.1467-2494.2000.00023.x. PMid:18503416.
http://dx.doi.org/10.1046/j.1467-2494.20...
).

Although many studies have been conducted on ellagic acid using HPLC, only a few studies have been conducted on strawberry varieties (Yong et al., 2019Yong, Y. S., Lee, S., Byun, N. Y., Sun, S., Kim, M. J., Jang, S. W., Jang, W. S., Lee, S. Y., & Yim, S. H. (2019). Antioxidant activities and validation of analytical method of marker compounds in strawberry fruits from various cultivars. Korean Journal of Food Science Technology, 51, 517-523.). The objective of this study was to investigate antioxidant effects of 12 cultivars of strawberries grown and harvested by the National Institute of Horticultural and Herbal Science and chungcheongnamdo agricultural research & extension. In addition, we performed a validation of content analysis method for ellagic acid to obtain basic data that would aid the development of natural antioxidants and marker components of ellagic acid in various strawberry varieties.

2 Materials and methods

2.1 Chemical and reagents

Folin-Ciocalteu reagent, tannic acid, quercetin, ellagic acid, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and (+/-)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). For ellagic acid analysis, methanol and acetonitrile were obtained from J. T. Baker (Phillipsburg, NJ, USA). All other chemicals used were special grade reagents.

2.2 Plant materials and extraction

Twelve cultivars of Korean strawberries grown and harvested by the National Institute of Horticultural and Herbal Science and chungcheongnamdo agricultural research & extension in 2017 were used as samples in this study (Table 1). These 12 cultivars of strawberry samples used in this study were harvested from red-colored hard-boiled fruits with similar growth period. Strawberry samples were extracted with 10 vol (v/w) of water at 80 °C for 3 h, and extraction was repeated 3 times. Extracts were concentrated with vacuum evaporator (COSMOS660-50L, Kyungseo Machines Co, Incheon, Korea), and frozen in a deep freezer at -70 °C for 24 h, freeze-dried and stored at 4°C for use in the experiment. Each dried extraction, it was then dissolved in a pretreatment solvent (ethanol: distilled water: HCI = 3:1:1) and hydrolyzed for 3 h at 90°C. The extracted solution was then cooled to at room temperature and filtered with a syringe filter (0.45 µm, Hyundaimicro Co., Ltd, Korea) to obtain as a test solution.

Table 1
Parentage and Origin of 12 strawberry cultivars.

2.3 Determination of total polyphenol content

Total polyphenol contents were measured applying the modified method of Folin-Ciocalteu method (Singleton et al., 1999Singleton, V. L., Orthofer, R., & Lamuela-raventos, R. M. (1999). Analysis of total phenols and other oxidation substrate and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology, 299, 152-178. http://dx.doi.org/10.1016/S0076-6879(99)99017-1.
http://dx.doi.org/10.1016/S0076-6879(99)...
). Each sample solution (500 µL) was mixed Folin-Ciocalteu reagent (500 µL) for 3 min, added with 10% Na2CO3 (500 µL), incubated at room temperature for 1 h, and then subjected to measurement of absorbance at 760 nm using a UV/VIS spectrophotometer (Neogen, Optizen 2120 UV, Sejong, Korea). Total polyphenol content (mg TAE/100 g fresh weight (FW)) was obtained using a standard curve prepared with tannic acid as the standard substance.

2.4 Determination of total flavonoid contents

Total flavonoid contents were determined using the Moreno method (Moreno et al., 2000Moreno, M. I. N., Isla, M. I., Sampietro, A. R., & Vattuone, M. A. (2000). Comparison of the free radical-scavenging activity of propolis from several regions of Argentina. Journal of Ethnopharmacology, 71(1-2), 109-114. http://dx.doi.org/10.1016/S0378-8741(99)00189-0. PMid:10904153.
http://dx.doi.org/10.1016/S0378-8741(99)...
). Briefly, each sample (10 µL) was diluted with 80% ethanol (990 µL). Then 100 µL of this sample solution was mixed with 80% ethanol solution (4.3 mL) containing of 10% aluminum nitrate and 1 M potassium acetate for 40 min. Absorbance was then measured at 415 nm using a spectrophotometer (Mecasys Co., Daejeon, Korea). Total flavonoid content (mg QE/100 g FW) was obtained using a standard curve prepared with quercetin as the standard substance.

2.5 DPPH radical scavenging activity

DPPH radical scavenging activity is one indicator of electron-donating ability. It was measured applying the modified method of Brand-Williams et al. (1995)Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft + Technologie, 28(1), 25-30. http://dx.doi.org/10.1016/S0023-6438(95)80008-5.
http://dx.doi.org/10.1016/S0023-6438(95)...
. The sample solution (10 μL/well) and 0.2 mM DPPH (190 μL/well) were added into a 96-well plate. After incubation at 25°C for 30 min, absorbance was measured at 515 nm using an ELISA reader (Thermo Fisher SCIENTIFIC, Multiskan Sky, KOREA).

2.6 ABTS radical scavenging activity

ABTS radical scavenging activity is one indicator of electron-donating ability. It was measured with modified method of Thaipong et al. (2006)Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., & Hawkins Byrne, D. (2006). Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. Journal of Food Composition and Analysis, 19(6-7), 669-675. http://dx.doi.org/10.1016/j.jfca.2006.01.003.
http://dx.doi.org/10.1016/j.jfca.2006.01...
. Briefly, 7 mM ABTS and 2.45 mM potassium persulfate were mixed at a ratio of 1: 1 (v /v) to react in a dark room at 25 °C for 24 h to generate radicals. Radical stock solution was diluted with distilled water so that the absorbance value at 734 nm was 0.70 ± 0.02. After incubation in a dark room at 25 °C for 5 min, absorbance was measured at 734 nm using a UV/VIS spectrophotometer (Neogen, Optizen 2120 UV, Sejong, Korea).

2.7 Prepare of standard solution

Ellagic acid analytical standard with a purity of 95% was dissolved in 1 × 10-2 N NaOH and store at 4°C. A standard solution was made by dilution so that the concentration of this solution was in the range of 10-100 μg/mL.

2.8 Method validation

Validation of analysis methods for marker components was performed by determining linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and precision according to guidelines of analysis methods used for medicines (Korean Food and Drug Administration, 2015Korean Food and Drug Administration – KFDA. (2015). Analytical method guideline about validation of drugs and etc. (pp. 1-18). Seoul, Korea: KFDA.).

  1. Linearity, limit of detection, and limit of quantitation Ellagic acid standard solution (five concentrations) was used to determine coefficient (R2) using retention time and regression equation (y = ax + b, y: peak area, x: concentration (μg/mL)) shown on HPLC. Limit of detection and limit of quantitation for each component were determined based on the standard deviation and the slope of the calibration curve using chromatogram of the standard solution;

  2. Accuracy and precision Accuracy and precision were evaluated to determine inter-day and intra-day variations of the index component at the same concentration. Accuracy refers to the proximity of average test results obtained by the analysis method. Relative standard deviation was calculated by measuring each sample three times for each concentration (10 µg/mL, 50 µg/mL, and 100 µg/mL). Precision indicates the proximity between measured values when a sample is taken several times and measured under specified conditions. It was measured with relative standard deviation which was obtained by measuring the standard solution at each concentration (0, 50, and 100 µg/mL) three times;

  3. Stability To check chemical stability of ellagic acid according to the storage period, each sample was kept at room temperature for 24 h, and then ellagic acid content was measured.

2.9 Ellagic acid content analysis

Ellagic acid contents in strawberry extracts were analyzed using high performance liquid chromatography (HPLC, Agilent Technologies, Santa Clara, CA, USA) with an autosampler and a DAD detector. Analysis conditions are shown in Table 2. Analysis was done using YMC ODS column (4.6×250 mm, Kyoto, Japan) and tertiary distilled water containing 0.1% formic acid (solvent A) and 100% acetonitrile (solvent B) as mobile phase. Measurements were made at 254 nm wavelength with a flow rate of 1 mL/min and 10 μL of sample for injection.

Table 2
Analytical conditions of HPLC for ellagic acid analysis.

2.10 Statistical analysis

Experimental data of this study are expressed as mean ± standard deviation after three repeated experiments. Difference between groups was determined by one-way variance analysis and Duncan's multiple range test using SPSS (statistical package for the social sciences, ver. 25). Statistical significance was considered at p < 0.05.

3 Results and discussion

3.1 Determination of phenolic compounds

Phenolic compounds contained in fruits provide general quality (such as color and taste) and health functions (such as antioxidant) of fruits. They are important ingredients that can protect plants from infections by bacteria and viruses. They can also extend the storage period (Tosun et al., 2009Tosun, M., Ercisli, S., Karlidag, H., & Sengul, M. (2009). Characterization of red raspberry (Rubus idaeus L.) genotypes for their physicochemical properties. Journal of Food Science, 74(7), C575-C579. http://dx.doi.org/10.1111/j.1750-3841.2009.01297.x. PMid:19895463.
http://dx.doi.org/10.1111/j.1750-3841.20...
). In this study, total polyphenol contents present in strawberry extracts were measured using tannic acid as a reference substance for quercetin (Table 3). Total polyphenol contents ranged from 21.79 to 118.85 mg TAE/100 g FW, with 'Josaenghongsim' cultivar showing the highest total polyphenol contents among all cultivars tested, followed by 'Okme' and 'Chodong', whereas 'Manhyang', 'Sinseolmae', and 'Johong' cultivars had the lowest total polyphenol contents. Significant (p<0.05) differences in total polyphenol content were found among strawberry cultivars. Scalzo et al. (2005)Scalzo, J., Politi, A., Pellegrini, N., Mezzetti, B., & Battino, M. (2005). Plant genotype affects total antioxidant capacity and phenolic contents in fruit. Nutrition (Burbank, Los Angeles County, Calif.), 21(2), 207-213. http://dx.doi.org/10.1016/j.nut.2004.03.025. PMid:15723750.
http://dx.doi.org/10.1016/j.nut.2004.03....
have reported that total polyphenol contents in six Italian strawberry cultivars ('Don', 'Idea', 'Camarosa', 'Onda', 'Patty', and 'Svev') are in the range of 1,093~2,128 mg GAE/L. Pineli et al. (2011)Pineli, L. L. O., Moretti, C. L., dos Santos, M. S., Campos, A. B., Brasileiro, A. V., Córdova, A. C., & Chiarello, M. D. (2011). Antioxidants and other chemical and physical characteristics of two strawberry cultivars at different ripeness stages. Journal of Food Composition and Analysis, 24(1), 11-16. http://dx.doi.org/10.1016/j.jfca.2010.05.004.
http://dx.doi.org/10.1016/j.jfca.2010.05...
have also reported that total polyphenol contents in 'Osogrand' and 'Camino Real' are 2,234.62 mg GA/kg FW and 1,743.47 mg GA/kg FW, respectively. These results were higher than ours. However, Lim et al. (2016)Lim, S., Hwang, H., & Shin, Y. (2016). Physicochemical properties and antioxidant activities of ‘Selhyang’ and ‘Janghee’ strawberries from different ripening stages grown in Korea. J East Asian Soc Diet Life, 26(1), 80-87. http://dx.doi.org/10.17495/easdl.2016.2.26.1.80.
http://dx.doi.org/10.17495/easdl.2016.2....
have reported that total polyphenol contents in 'Sulhyang' and 'Janghee' are 229.18 and 39.11 mg GAE/100 g FW, respectively. Chaves et al. (2017)Chaves, V. C., Calvete, E., & Reginatto, F. H. (2017). Quality properties and antioxidant activity of seven strawberry (Fragaria×ananassa Duch) cultivars. Scientia Horticulturae, 225, 293-298. http://dx.doi.org/10.1016/j.scienta.2017.07.013.
http://dx.doi.org/10.1016/j.scienta.2017...
have shown that total polyphenol contents in seven strawberry cultivars are in the range of 1.6~2.54 mg TAE/g FW. These results were similar to ours.

Table 3
Content of total polyphenols and flavonoids in 12 Korean strawberry cultivars.

In the present study, 'Josaenghongsim' had the highest total flavonoid contents at 599.56±9.82 mg QE/100 g FW, followed by 'Mihong' at 288.28±75.24 mg QE/100 g FW and 'Seolhyang' at 202.00±2.34 mg QE/100 g FW (Table 3). Significant (p < 0.05) differences in total flavonoid contents were found among strawberry cultivars. Lim et al. (2016)Lim, S., Hwang, H., & Shin, Y. (2016). Physicochemical properties and antioxidant activities of ‘Selhyang’ and ‘Janghee’ strawberries from different ripening stages grown in Korea. J East Asian Soc Diet Life, 26(1), 80-87. http://dx.doi.org/10.17495/easdl.2016.2.26.1.80.
http://dx.doi.org/10.17495/easdl.2016.2....
have reported that total flavonoid contents in 'Sulhyang' and 'Janghee' strawberrries are 52.32 and 39.11 mg CE/100 g, lower than ours. Bae et al. (2019)Bae, M. J., Kim, E. N., Choi, H. K., Byun, K. H., Chung, K. H., Yoon, J. A., & An, J. H. (2019). Quality characteristics and antioxidant activities of strawberries according to various extraction methods. Journal of the Korean Society for Food and Nutrition, 48(7), 728-738. http://dx.doi.org/10.3746/jkfn.2019.48.7.728.
http://dx.doi.org/10.3746/jkfn.2019.48.7...
have reported that total flavonoid content in 'Sulhyang' is 546.12 mg CHE/100 g sample, same as 'Josaenghongsim'. Kim et al. (2018)Kim, J. S., Moon, Y. S., & Kwak, E. J. (2018). Comparison of phenolic composition, content, and antioxidant activity in Raspberries and Blackberries. Weonye Gwahag Gisulji, 36, 115-127. have determined antioxidant activities of various berries and reported that total flavonoid contents in raspberry and wild raspberry are 731.08 and 362.41 mg QE/100 g FW, respectively, higher than our results. Lee et al. (2014)Lee, H. H., Moon, Y. S., Yun, H. K., Park, P. J., & Kwak, E. J. (2014). Contents of bioactive constituents and antioxidant activities of cultivated and wild Raspberries. Weonye Gwahag Gisulji, 32(1), 115-122. http://dx.doi.org/10.7235/hort.2014.13114.
http://dx.doi.org/10.7235/hort.2014.1311...
have shown that total flavonoid contents in wild raspberry were in the range of 10.91~14.90 mg QE/100 g FW, similar to our results. These findings indicate that total polyphenol and flavonoid contents are different according to the variety.

3.2 Antioxidant activity

Antioxidant activity was measured based on DPPH and ABTS free radical scavenging activities widely used to find antioxidants from various natural materials (Lee et al., 2014Lee, H. H., Moon, Y. S., Yun, H. K., Park, P. J., & Kwak, E. J. (2014). Contents of bioactive constituents and antioxidant activities of cultivated and wild Raspberries. Weonye Gwahag Gisulji, 32(1), 115-122. http://dx.doi.org/10.7235/hort.2014.13114.
http://dx.doi.org/10.7235/hort.2014.1311...
). DPPH free radical scavenging activity consists in the reduction of DPPH radical by substances present in the extraxt, leading to the formation of reduced DPPH-H, which changes in color from deep purple to loss of color (Chaves et al., 2017Chaves, V. C., Calvete, E., & Reginatto, F. H. (2017). Quality properties and antioxidant activity of seven strawberry (Fragaria×ananassa Duch) cultivars. Scientia Horticulturae, 225, 293-298. http://dx.doi.org/10.1016/j.scienta.2017.07.013.
http://dx.doi.org/10.1016/j.scienta.2017...
). DPPH free radial scavenging activities were measured using DPPH and expressed as trolox equivalent (μmol TEAC/100 g FW). DPPH free radial scavenging activities of 12 cultivars of strawberries used in this experiment were in the range of 1,381.7±12.7 to 1,095.3±37.1 μmol TEAC/100 g FW, with 'Josaenghongsim', 'Johong', 'Sinseolmae', 'Seolhyang', and 'Berrystar' having the highest values (Table 4). Significant (p < 0.05) differences in DPPH free radial scavenging activity were found among strawberry cultivars. Nowicka et al. (2019)Nowicka, A., Kucharska, A. Z., Sokół-Łętowska, A., & Fecka, I. (2019). Comparison of polyphenol content and antioxidant capacity of strawberry fruit from 90 cultivars of Fragaria×ananassa Duch. Food Chemistry, 270, 32-46. http://dx.doi.org/10.1016/j.foodchem.2018.07.015. PMid:30174053.
http://dx.doi.org/10.1016/j.foodchem.201...
have determined DPPH free radical scavenging activities of 90 strawberries grown in Poland and found that the average activity is 751.57 μmol TEAC/100g. Pineli et al. (2011)Pineli, L. L. O., Moretti, C. L., dos Santos, M. S., Campos, A. B., Brasileiro, A. V., Córdova, A. C., & Chiarello, M. D. (2011). Antioxidants and other chemical and physical characteristics of two strawberry cultivars at different ripeness stages. Journal of Food Composition and Analysis, 24(1), 11-16. http://dx.doi.org/10.1016/j.jfca.2010.05.004.
http://dx.doi.org/10.1016/j.jfca.2010.05...
have reported that DPPH free radical scavenging activity of 'Osogrand' and 'Camino Real' are 12.83 μmol TEAC/g and 10.10 μmol TEAC/g, respectively, somewhat lower than our results.

Table 4
Antioxidant activities of 12 Korean strawberry cultivars.

Another effective method to measure radical scavenging activity is the ABTS radical cation decolorization assay. This method can be used to obtain results in a short time. It can be applied to both hydrophobic and hydrophilic sample (Kim et al., 2019Kim, H. H., Heo, M. R., Lee, S., & Yim, S. H. (2019). Validation of analytical method and antioxidant properties of Eriobotrya japonica Lindl. leaf extract according to extraction solvent. Korean Journal of Food Science Technology, 51, 301-308.). ABTS free radial scavenging activities of 12 strawberry extracts were compared. Results are shown in Table 4. ABTS free radical scavenging activities of these extracts were in the range of 9,179.0±1.0 to 4,262±15.3 μmol TEAC/100 g FW, with 'Seolhyang' showing the highest activity, followed by Suhong, Manhyang, and Johong. There were significant (p < 0.05) differences in ABTS free radical scavenging activity among strawberry cultivars. Kim et al. (2016)Kim, K. S., Han, S. H., An, I. S., & Ahn, K. J. (2016). Protective effects of ellagic acid against UVA-induced oxidative stress in human dermal papilla. Asian Journal of Beauty and Cosmetology, 14(2), 191-200. http://dx.doi.org/10.20402/ajbc.2016.0048.
http://dx.doi.org/10.20402/ajbc.2016.004...
have reported that ABTS free radical scavenging activities of raspberry and blackberry are 72 μmol TE/g and 20 μmol TE/g, respectively, lower than ours. Lee et al. (2015)Lee, Y., Lee, J. H., Kim, S. D., Chang, M. S., Jo, I. S., Kim, S. J., Hwang, K. T., Jo, H. B., & Kim, J. H. (2015). Chemical composition, functional constituents, and antioxidant activities of berry fruits produced in Korea. Journal of the Korean Society of Food Science and Nutrition, 44(9), 1295-1303. http://dx.doi.org/10.3746/jkfn.2015.44.9.1295.
http://dx.doi.org/10.3746/jkfn.2015.44.9...
have reported that ABTS free radical scavenging activities of black raspberry, blueberry, and raspberry are 2.99 mmol TEAC/100 g FW, 1.47 mmol TEAC/100 g FW, and 0.77 mmol TEAC/100 g FW, respectively. These results suggest that antioxidants present in these strawberry extracts could eliminate highly reactive free radicals to prevent oxidation of important biomolecules such as unsaturated fatty acids, genes, and proteins (Fan et al., 2011Fan, Z. L., Wang, Z. Y., & Liu, J. R. (2011). Cold-field fruit extracts exert different antioxidant and antiproliferative activities in vitro. Food Chemistry, 129(2), 402-407. http://dx.doi.org/10.1016/j.foodchem.2011.04.091. PMid:30634244.
http://dx.doi.org/10.1016/j.foodchem.201...
).

3.4 Method validation

  1. Analysis conditions HPLC analyses of ellagic acid contents in strawberries were performed using an Agilent HPLC system (Agilent Technology, Santa Clara, CA, USA). The mobile phase consisted of solvent A (tertiary distilled water containing 0.1% formic acid) and solvent B (acetonitrile). The flow rate for the mobile phase was set at 1 mL/min. UV detection was performed at wavelength of 254 nm;

  2. Linearity, limit of detection (LOD), and limit of quantitation (LOQ) Standard calibration curves for ellagic acid standard by HPLC were obtained in the range of 10-100 µg/mL using five different concentrations (n = 3). Ellagic acid standard was used to calculate a linear regression equation with a correlation coefficient (R2) of 0.9999. LOD was found to be 2.35 µg/mL for ellagic acid standard and LOQ of the standard were found to be 7.13 µg/mL. Thus, detection and quantification of ellagic acid are possible even if its amount is small (Table 5). Results of this study can be used to verify quantitative and detection limits of marker component analysis for the standardization of strawberry extract;

    Table 5
    Calibration curve, linearity, limit of detection (LOD), limit of quantitation (LOQ) of ellagic acid.

  3. Accuracy, precision, and recovery The precision of the optimized method using standard compounds was verified at three concentrations (10, 50 and 100 µg/mL). Relative standard deviations (RSDs) were 0.20-5.13% and 0.82-5.31% by HPLC for inter-day and intra-day variations (n=3), respectively (Table 6). Regarding accuracy, the average recovery rate of ellagic acid was 99.0% to 100.1%. RSD value was in the range of 0.8% to 5.3% (Table 7). Thus, the analytic method was confirmed to have excellent precision and accuracy.
    Table 6
    Precision of ellagic acid
    Table 7
    Accuracy of ellagic acid.
  4. Stability and Contents Chemical stability of ellagic acid, a marker compound, was determined based on the change in content of ellagic acid at a concentration of 100 ug/mL after it was left at room temperature for 24 hours (Table 8). Results revealed that the average recovery rate of ellagic acid was 100.4%. Its content change rate was 1.50% after 24 hours of storage at room temperature. Thus, ellagic acid was confirmed to have chemical stability for 24 hours at room temperature. Also, the ellagic acid content in all varieties of strawberries was found to be in the range of 12.8±0.2 -16.6±0.3, respectivlely (Table 9).
    Table 8
    Analytical result of ellagic acid stability test
    Table 9
    Contents of ellagic acid in 12 Korean strawberry cultivars

5 Conclusions

In this study, strawberry fruit extracts from 12 Korean cultivars showed high antioxidant activities. Such high activities are thought to be due to their high contents of total polyphenol and flavonoid that can inhibit oxidation of free radicals. Results of this study could be used as basic data for establishing a standard for determining the development potential and performing content analysis of strawberry extracts based on quantitative analysis using ellagic acid as a marker compound for the validation and standardization of natural antioxidants.

  • Practical Application: Development of a new variety of strawberry with excellent antioxidant effect.
  • These two authors contributed equally to this work.

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Publication Dates

  • Publication in this collection
    04 Oct 2021
  • Date of issue
    2022

History

  • Received
    12 Aug 2021
  • Accepted
    21 Aug 2021
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