Comparative Analysis of Antioxidant Activity and Functional

Article pubs.acs.org/JAFC

Comparative Analysis of Antioxidant Activity and Functional Components of the Ethanol Extract of Lotus (Nelumbo nucifera) from Various Growing Regions Xu Zhao,† Jian Shen,‡ Kyung Ja Chang,† and Sung Hoon Kim*,‡ †

Department of Food and Nutrition, Inha University, Incheon 402-751, Korea Department of Chemistry, Konkuk University, Seoul 143-701, Korea

‡

S Supporting Information *

ABSTRACT: The variations in antioxidant activity and concentration of functional components in the ethanol extracts of lotus seeds and rhizomes based on the growing region and dryness were investigated. Free radical scavenging activity, total phenolic and flavonoid content, and concentration of several specific flavonoids and alkaloids in the ethanol extracts of lotus were measured. Antioxidant activity and its correlative total phenolic content varied characteristically depending on the growing region and dryness. High-perfomance liquid chromatography analysis showed that the ethanol extracts of lotus seeds from Vietnam (Ho Chi Minh City), raw rhizomes from Korea (Siheung), and dried rhizomes from Japan (Nigata) had the greatest specific flavonoid content. The ethanol extracts of seeds from China (Hubei), raw rhizomes from Japan (Nigata), and dried rhizomes from Korea (Siheung) had the greatest specific alkaloid content. Astragaline, rutin, isoquercetin, nuciferine, dauricine, isoliensinine, and neferine were identified in lotus rhizomes for the first time in this study. KEYWORDS: lotus (Nelumbo nucifera), seeds, rhizomes, ethanol extract, antioxidant, flavonoids, alkaloids

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INTRODUCTION Lotus (Nelumbo nucifera Gaertn), a member of the Nymphaeaceae family, is an aquatic plant widely cultivated in Asia. Most parts of the lotus are utilized as the medicinal herbs in traditional medicine. Lotus seeds, rhizomes, leaves, and flowers are also food ingredients, especially in Korea, China, Indian, Japan, Vietnam, and Thailand. The important functional components of the lotus are flavonoids and alkaloids, and their physiological properties have been extensively reported. Flavonoids, a group of polyphenols in plants, are usually subdivided into anthoxanthins, flavanones, flavanonols, flavans, anthocyanidins, and isoflavonoids. The total flavonoid intake of an average American is ≈20 mg/day and for Japanese it is ≈63 mg/day in food originating from plants.1 Flavonoids have been found to possess antiallergic,2 anti-inflammatory,3 antiviral,4,5 antiproliferative,6 anticarcinogenic,7 and antioxidant8 effects. Alkaloids, a group of compounds containing basic nitrogen atoms, are toxic to humans when consumed beyond a certain level. Previous studies have indicated that alkaloids possess antifibrotic,9 sedative,10 anti-HIV,11 antioxidant, anti-inflammatory,12 and antiobesity13 properties. Compared to the white lotus, which is distinguished by the color of the lotus flower, the red lotus is more commonly used as a food ingredient. The concentration of functional components differ in the red and white lotus.14 The methanol extracts of different parts of the lotus also contain different concentrations of functional components.15 Additionally, lotus leaves from different growing regions contain different concentrations of functional components and exert different levels of antioxidant effects.16 Therefore, the concentration of functional components and antioxidant effect of the lotus varies with the lotus type and parts, as well as the geographic location © 2014 American Chemical Society

where the lotus is grown. In addition, the concentration of functional components is also affected by the drying process.17 Recently, a lot of studies have been dedicated to the analysis of functional components of lotus leaves and embryos and their health-related properties. Lotus leaves and embryos are more commonly consumed as tea and medicines than as ingredients in meals. Lotus rhizomes, followed by lotus seeds, hold the largest market share as edible vegetables. Some previous studies indicated that lotus seeds and rhizomes possess antipyretic, antidiarrheal,18 antidiabetic,19 hepatoprotective,20,21 anti-inflammatory,12 and antioxidant22,23 effects. Flavonoids (flavonols and flavanols) and several alkaloids (neferine, liensinine, isoliensinine, and nuciferine) in lotus seeds have been extensively studied.24−27 However, only a few flavonoids such as catechin, quercetin, and gallocatechin and alkaloids from lotus rhizomes have been studied.28−30 Therefore, determination of several specific flavonoid and alkaloid contents in lotus rhizomes and seeds may be valuable for the development of functional foods derived from the lotus. In our preliminary studies, the ethanol extracts of lotus seeds and rhizomes showed an antiobesity effect.31,32 The lipid components in the ethanol extracts of lotus seeds and rhizomes, which were analyzed by our group,33 showed no antiobesity effect. Therefore, it was necessary to analyze the obesity-related functional components in the ethanol extracts of lotus seeds and rhizomes. In addition, the analysis of antioxidant activity and concentration of functional compounds, which vary with Received: Revised: Accepted: Published: 6227

April 10, 2014 June 9, 2014 June 16, 2014 June 16, 2014 dx.doi.org/10.1021/jf501644t | J. Agric. Food Chem. 2014, 62, 6227−6235

Journal of Agricultural and Food Chemistry

Article

Table 1. Growing Region, Part, and Storage Condition of Lotus Used in This Study no.

growing region

typea

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Korea (Muan) China (Guangzhou) China (Hubei) Vietnam (Ho Chi Minh City) Thailand (Lamphun) Korea (Siheung) Korea (Daegu) Korea (Haman) Korea (Muan) Japan (Nigata) Korea (Siheung) Korea (Daegu) Korea (Haman) China (Guangxi) Vietnam (Ho Chi Minh City) Korea (Muan) Japan (Nigata)

white red red red red red red red white white red red red red red white white

part

storage condition

seeds with embryo and seeds with embryo and seeds with embryo and seeds with embryo and seeds with embryo and rhizomes with knot rhizomes with knot rhizomes with knot rhizomes with knot rhizomes with knot rhizomes with knot rhizomes with knot rhizomes with knot rhizomes with knot rhizomes with knot rhizomes with knot rhizomes with knot

coat coat coat coat coat

driedb driedb driedb driedb driedb liquid nitrogen liquid nitrogen liquid nitrogen liquid nitrogen liquid nitrogen driedc driedc driedc driedb driedb driedc driedc

frozen frozen frozen frozen frozen

raw raw raw raw raw

Two types of lotus were distinguished by the color of the lotus flower. bDried lotus samples were purchased from different lotus farms and the Internet. cRaw lotus rhizomes were dried with a fan for 1 day in our laboratory.

a

the growing region and drying process, may play an important role in utilizing lotus extracts and developing functional foods. Therefore, in this study, we compared the antioxidant activity and concentration of several specific functional compounds in the ethanol extracts of dried lotus seeds and raw and dried lotus rhizomes from various growing regions.

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MATERIALS AND METHODS

Materials and Chemicals. Lotus samples were purchased from various lotus farms and via the Internet in September 2012 (Table 1). Dried lotus seeds were procured from Korea (Muan), China (Guangzhou and Hubei), Vietnam (Ho Chi Minh City), and Thailand (Lamphun) (Figure 1A). Raw lotus rhizomes were procured from Korea (Siheung, Daegu, Haman, and Muan), and Japan (Nigata) and frozen in liquid nitrogen (Figure 1B). Dried lotus rhizomes were obtained from China (Guangxi) and Vietnam (Ho Chi Minh City). Each raw rhizome from various growing regions was subdivided into two parts: one part for direct extraction and the other for drying. For the preparation of dried lotus rhizomes, air was fan-blown on the samples for 1 day at room temperature. The water content of the dried samples was normally less than 5%. The photographs of dried lotus rhizomes are shown in Figure 1C. All lotus samples were stored at −20 °C until they were used. Astragaline, rutin, isoquercetin, nuciferine, dauricine, isoliensinine, and neferine (≥98%) were obtained from Shenzhen Sungening BioTech Co. Ltd. (Shengzhen, China). Gallic acid (≥98%), (+)-catechin (≥98%), Folin-Ciocalteu’s phenol reagent, and 2,2-diphenyl-1picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). AB-8 macroporous adsorption resin was purchased from Anhui Sanxing Resin Technology Co. Ltd. (Anhui, China). Extraction of Lotus Seeds and Rhizomes. Each lotus sample (200 g) was blended with 70% ethanol solvent (1 L), extracted at 50 °C for 2 h and then filtered over a short pad of Celite. The filter cake was extracted with 70% ethanol three times (1 L × 3) at 50 °C for 2 h. The combined ethanol extract was evaporated to complete dryness under reduced pressure at 40 °C. The ethanol extract is referred to hereafter as LE. The crushed LE was continuously extracted with hexane (0.05 g of sample/(mL of hexane)) five times at room temperature for separating lipids and pigment components from the LE fractions. The combined hexane extract and residual fraction after hexane extraction were evaporated to dryness under reduced pressure at 40 °C. The hexane extract and residual fraction after hexane extraction from the LE fraction are respectively referred to hereafter as LEa and LEb. Each LE and LEb fraction was assayed to determine

Figure 1. Photographs of lotus seeds and raw and dried rhizomes used in this study. (A): Dried lotus seeds used in this experiment. (B): Raw lotus rhizomes used in this experiment. (C): Dried lotus rhizomes used in this experiment. Bar: 1 cm.

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dx.doi.org/10.1021/jf501644t | J. Agric. Food Chem. 2014, 62, 6227−6235


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DPPH radical scavenging activity and total phenolic and flavonoid content. Determination of Free Radical Scavenging Activity. Free radical scavenging activity was assessed using a modified DPPH radical decoloration assay.34 Various concentrations of samples (0−3.0 mg/ mL, 0.6 mL) were reacted with a DPPH methanol solution (100 μmol/L, 5 mL) in the dark for 30 min. The absorbance was measured at 517 nm using a UV−vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA), and methanol without DPPH was used as the blank. The antioxidant activity of each sample was expressed as an EC50 value (the efficient concentration of the sample that decreases the initial DPPH radical formation by 50%), which was estimated using linear regression analysis. In this decoloration assay, higher EC50 value refers to a lower antioxidant activity. Determination of Total Phenolic Content. The total phenolic content was analyzed using Folin-Ciocalteu assay.34 The sample (5 mg/mL, 0.1 mL) was mixed with distilled water (2 mL) and FolinCiocalteu’s phenol reagent (1 mL). After 5 min, a 20% aqueous sodium carbonate solution (5 mL) was added, and the mixture was incubated in the dark for 60 min. The absorbance was measured at 735 nm using a UV−vis spectrophotometer, and with distilled water was used as the blank. The total phenolic content was determined using gallic acid (0−0.8 mg/mL) as a calibration standard, and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram of sample. Determination of Total Flavonoid Content. Total flavonoid content was determined using a modified aluminum chloride coloration method.34 Each sample (10 mg/mL, 0.6 mL) was mixed with distilled water (3.75 mL) and 5% aqueous sodium nitrite solution (0.225 mL). After 6 min, a 10% aqueous aluminum chloride solution (0.45 mL) was added, and the mixture was incubated for 5 min, after which a sodium hydroxide solution (1 mol/mL, 1.5 mL) and distilled water were added for a total volume of 7.5 mL. The absorbance was measured at 510 nm using a UV−vis spectrophotometer, and distilled water was used as the blank. Total flavonoid content was determined using catechin (0−0.8 mg/mL) as a calibration standard. The results were expressed as milligrams of catechin equivalents (CE) per gram of sample. AB-8 Macroporous Adsorption Resin Column Chromatography. Each aliquot of the LEb fraction (1 g) was suspended in distilled water (10 mL) and centrifuged to the separate aqueous solution and precipitated fraction. The precipitated fraction was evaporated to complete dryness under reduced pressure. The aqueous solution was applied into an AB-8 macroporous adsorption resin column (190 mm × 30 mm i.d.) for 2 h and then eluted.35 Elution was performed as follows: first, polar compounds such as amino acids and carbohydrates were eluted with distilled water (500 mL) at a flow rate of 3 mL/min; second, nonpolar compounds such as flavonoids and alkaloids were eluted with 95% ethanol (500 mL) at a flow rate of 5 mL/min; third, the rest of the compounds were eluted with 5% ammonia solution (300 mL) at a flow rate of 5 mL/min. Water and ammonia fractions eluted from the column are referred to hereafter as LEb-1 and LEb-3, respectively. The precipitated fraction of the LEb separated by centrifugation and the ethanol fraction eluted from the column were combined and designated as LEb-2. The LEb-1, LEb-2, and LEb-3 fractions were evaporated to dryness under reduced pressure. The LEb-2 fraction was analyzed by high-performance liquid chromatography (HPLC) to determine the concentration of specific flavonoids and alkaloids. HPLC Analysis. Each LEb-2 fraction was dissolved in 50% aqueous methanol and filtered through a 0.45 μm filter. Analysis of flavonoids and alkaloids was performed by HPLC (Agilent Technologies 1200 series HPLC) using a Waters SYMMETRY C18 (T61421F) reversephase column (250 mm × 4.6 mm i.d., 5 μm particle size) with a column temperature of 30 °C. The flow rate was 1 mL/min, and the injection volume was 20 μL. For the flavonoid analysis, the mobile phase was composed of two solvents: 2% aqueous acetic acid solution (solvent A) and 0.5% aqueous acetic acid solution:acetonitrile (1:1, v/ v, solvent B).34 Gradient elution conditions were as follows: 0−10 min, 90% solvent A; 10−40 min, 90−60% solvent A; 40−55 min, 60−

45% solvent A; 55−60 min, 45−20% solvent A; 60−65 min, 20−0% solvent A; 65−70 min, 0−50% solvent A; 70−75 min, 50−70% solvent A; and 75−80 min, 70−90% solvent A. Elution was monitored by UV detection at 270 and 350 nm. For the alkaloid analysis, the mobile phase was composed of two solvents: acetonitrile (solvent A) and 0.1% aqueous triethylamine solution (solvent B).36 Gradient elution conditions were as follows: 0−15 min, 40−70% solvent A; 15−20 min, 70−100% solvent A; and 20−30 min, 100−40% solvent A. Elution was monitored by UV detection at 270 nm. The concentration of several specific flavonoids and alkaloids were calculated using the individual calibration curves. Statistical Analysis. Statistical analysis was performed using SPSS version 17.0. Results from three independent experiments were used to calculate the mean ± SD values for each sample. Significant differences among dried seeds and raw or dried rhizomes were determined using the Student’s t test and analysis of variance (ANOVA), respectively. Duncan’s multiple range tests were performed when data differences were identified at P < 0.05 by ANOVA analysis. Pearson’s correlation coefficient was used to analyze correlation between EC50 values and total phenolic and flavonoid content in the LE and LEb fractions.

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RESULTS AND DISCUSSION Descriptions of lotus samples, including growing region, lotus parts used, and storage conditions, are presented in Table 1. The growing regions in Korea, China, Japan, Vietnam, and Thailand represent the main lotus-producing and lotusconsumption areas in Asia. Since many factors such as oxidants and the presence of microorganisms can lower the quality of raw lotus, the shelf life of raw lotus is very short. A semidrying process is adopted for long-term storage and ease of transportation, although the drying process can increase the oxidative browning of some antioxidants, such as flavonoids, and reduce antioxidant capacity.17 Dried lotus seeds and raw lotus rhizomes are commonly found in the market. In this study, raw lotus rhizomes from Korea (Siheung, Daegu, Haman, and Muan) and Japan (Nigata) were dried in order to compare the antioxidant effect and functional compound content of raw and dried rhizomes. The water content in raw lotus rhizomes was not statistically different (77.12%−81.60%, w/w). In general, organic solvents such as ethyl acetate, n-butanol, and methanol are widely used to extract functional components from natural products. However, such solvents are toxic and harmful for humans. A previous report indicated that 70% alcoholic solvents (either ethanol or methanol) exhibit a higher extraction efficiency for flavonoids from lotus leaves than 100% alcoholic solvents.37 Some of the hydrophilic functional compounds of lotus, such as certain alkaloids and polar phenolic content, are readily soluble in water and not organic solvents. Therefore, in this study, 70% aqueous ethanol was used as a nontoxic, safe, and highly efficient solvent to extract functional compounds from lotus seeds and rhizomes. The variations in the extract yield are presented in Table 2. The large quantity of water in raw lotus samples is likely to result in a lower yield under similar extraction conditions. Accordingly, the yield of the LE fractions showed obvious variation between raw and dried lotus rhizomes (5.30%− 34.85%, w/w), due to the variable water content and not the growing region. Lotus rhizomes are the richest in carbohydrates of all lotus parts studied, followed by the seeds.38−40 The high water-absorbing capacity of carbohydrates may result in a higher yield than the actual yield of the LE fractions, especially from starch-rich lotus rhizomes. Residual ethanol, absorbed water, and other volatile compounds in the LE fraction were 6229



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Table 2. Yields of the LE, LEa, and LEb Fractions of Lotus Seeds and Rhizomesa no. dried seeds 1 2 3 4 5 raw rhizomes 6 7 8 9 10 dried rhizomes 11 12 13 14 15 16 17

LE fraction

LEa fraction

Table 3. Yields of the Three Fractions of Lotus Seeds and Rhizomes Isolated by AB-8 Macroporous Adsorption Resin Column Chromatographya

LEb fraction

no. 20.53 19.35 18.20 20.84 19.42

4.47 7.55 6.93 10.99 8.37

95.84 92.45 92.96 90.25 92.61

5.30 5.51 6.49 5.85 6.84

2.87 2.23 1.23 2.15 1.79

94.09 93.75 94.32 97.83 95.31

20.52 22.31 26.52 23.65 34.85 27.99 23.38

3.04 3.19 1.01 2.40 1.49 2.28 2.42

94.87 94.60 97.29 95.80 95.19 94.98 92.37

dried seeds 1 2 3 4 5 raw rhizomes 6 7 8 9 10 dried rhizomes 11 12 13 14 15 16 17

a

Yields (%) of the LE, LEa, and LEb fractions were calculated as LE/ lotus, LEa/LE, and LEb/LE, respectively. LE, the ethanol extract; LEa, hexane fraction of the ethanol extract; LEb, residual fraction after separating the hexane fraction from the ethanol extract.

LEb-1 fraction

LEb-2 fraction

LEb-3 fraction

95.59 93.76 97.33 94.73 94.35

6.41 4.87 6.75 5.11 6.94

3.25 2.49 2.83 2.69 2.16

92.13 90.43 91.51 92.37 92.31

8.39 8.20 6.11 8.07 6.93

3.21 3.48 3.04 3.91 2.30

89.22 91.36 91.76 96.62 95.89 87.77 89.23

10.24 7.86 6.90 5.53 5.92 9.42 6.94

3.25 2.01 2.93 2.69 1.88 3.93 3.16

a

Yields (%) of the LEb-1, LEb-2, and LEb-3 fractions were calculated by LEb-1/LEb, LEb-2/LEb, and LEb-3/LEb, respectively. LEb-1, water fraction eluted from the AB-8 column; LEb-2, combined precipitated fraction of the LEb separated by centrifugation and the ethanol fraction eluted from the AB-8 column; LEb-3, ammonia fraction eluted from the AB-8 column.

eliminated by hexane extraction followed by vacuum drying. Therefore, the sum of the yields of LEa and LEb fractions was less than the yield of the LE fraction. The yields of the three fractions isolated by AB-8 macroporous resin column are shown in Table 3. Similarly, the total yield of all fractions, including the LEb-1, LEb-2, and LEb-3 fractions, appeared to be higher than 100% because the LEb-1 fraction contained most of the carbohydrates presented in the LE fraction. DPPH radical scavenging activity varied significantly in the LE and LEb fractions of lotus seeds and rhizomes depending on the growing region and dryness (Table 4). The free radical scavenging activity was the highest in the LE fraction of lotus seeds from Vietnam and raw and dried rhizomes from Korea (Siheung). Phenolic compounds such as flavonoids are common plant antioxidants, and these are abundant in lotus seeds and rhizomes.23,41 In this study, the total phenolic and flavonoid content differed significantly in the LE and LEb fractions of lotus seeds and rhizomes from different growing regions (Table 5). The total phenolic content was the highest in the LE fraction of lotus seeds from Vietnam and raw and dried rhizomes from Korea (Siheung). In addition, the total flavonoid content was the highest in the LE fraction of lotus seeds from Korea (Muan), raw rhizomes from Korea (Daegu), and dried rhizomes from Korea (Siheung). A previous report demonstrated that the polyphenolic content determined in apple cultivars was affected by the growing region.42 Therefore, identical lotus parts may exhibit variations in antioxidant activity and total phenolic and flavonoid content depending on their growing region. The EC50 values and total phenolic content, not total flavonoid content, in the LE and LEb fractions of most raw rhizomes obviously increased after drying. Compared with the results for dried rhizomes, the extraction efficiency for nonpolar phenolic compounds in raw rhizomes was lower because of the

dilution of the ethanol solvent by the large water content in raw rhizomes. However, most of these nonpolar phenolic compounds (e.g., chlorogenic acid) exhibit low DPPH radical scavenging activity. The drying process led to an increase in oxidative browning, which in turn results in a decrease in antioxidant capacity.17 Therefore, this may explain why the extracts of dried rhizomes exhibited a lower antioxidant activity than did raw rhizomes. In addition, DPPH radical scavenging capacity and total phenolic content were lower in oven-dried papaya than freeze-dried papaya because of the decomposition of functional compounds at high temperatures.43 Dried brownish rhizomes purchased from China and Vietnam, which has been dried in a hot air oven, exhibited lower antioxidant activity and total phenolic content than other, less colored rhizomes dried under a fan in our laboratory. These results indicated that the drying process affects the antioxidant activity and total phenolic content of rhizome extract, not the total flavonoid content. As for the lotus part, the DPPH radical scavenging activity of seeds was lower than that of rhizomes. In addition, the total phenolic content in the LE fraction of seeds was obviously lower than that of the rhizomes. However, the total flavonoid content in lotus seeds and rhizomes was similar. These results did not correspond with a previous report, which indicated that the extract of lotus seeds showed similar DPPH radical scavenging activity with rhizomes and had higher total phenolic and flavonoid content than rhizomes.15 The ascorbic acid content in raw lotus seeds is under the detection limit,40 but its concentration in raw rhizomes is 0.44 mg/g39 and in dried rhizomes of Korea is from 15.5 to 22.5 mg/g.44 Other than phenolic compounds, ascorbic acid is a well-known potent 6230



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tively. The EC50 value significantly negatively correlated with the total phenolic content in lotus. A previous report also indicated that the total phenolic content in plant extracts significantly correlated with their free radical scavenging activity.47 Therefore, our finding indicated that the total phenolic content in lotus extracts contributes to their antioxidant potential. The HPLC analysis showed significant differences in the special flavonoids content for the LEb-2 fractions of dried lotus seeds and raw and dried rhizomes from various growing regions (Table 6). The highest concentrations of flavonoids identified in the LEb-2 fractions of lotus were as follows: catechin in seeds from Vietnam and raw and dried rhizomes from Japan; astragaline in seeds from China (Guangzhou), raw rhizomes from Korea (Muan), and dried rhizomes from Korea (Daegu); rutin in seeds from Vietnam, raw rhizomes from Japan, and dried rhizomes from China (Guangxi); and isoquercetin in seeds from Thailand, raw rhizomes from Korea (Siheung), and dried rhizomes from China (Guangxi). These results suggest that the variations in the concentration of special flavonoids in the LE fraction of lotus can be partly attributed to different growing conditions (e.g., weather and soil) and dryness. After drying, the concentration of catechin obviously increased and the concentration of other flavonoids obviously decreased in the LEb-2 fractions of rhizomes. A previous study showed that the concentration of special flavonoids such as catechin and isoquercetin in Ziziphus jujuba fruit was affected by the growing region, and the concentrations decreased after drying.17 Catechin in lotus embryos was determined by Geng et al.48 A previous study showed that fresh seed coats from China (Wuhan) contained astragaline (7.2 mg/kg), rutin (11.8 mg/ kg), and isoquercetin (55.8 mg/kg); while dried embryos contained rutin (4079.0 mg/kg) and isoquercetin (637.8 mg/ kg).24 However, in this study, the concentrations of astragaline, rutin, and isoquercetin (1.0−3.3 mg/kg, 81.7−166.5 mg/kg, and 17.8−36.1 mg/kg, respectively) were measured in dried lotus seeds, which included the seed coat, embryo, and endosperm. In dried lotus rhizomes from China (Hubei), the concentration of catechin was 25 mg/kg in rhizomes and 110 mg/kg in knots of rhizomes.49 In this study, the concentration of catechin in dried rhizomes with knots varied from 13.2 to 133.7 mg/kg depending on the growing region and dryness. Lotus seeds that include the seed coat, embryo, and endosperm are rich in starch, lipids, and protein, and lotus rhizomes with knots contain more carbohydrates than isolated knots. This may be one of the reasons for the differences in specific flavonoid content between current and previous studies; the differences in the lotus-growing regions and analysis methods could also have contributed to the observed differences. Although many flavonoids found in lotus leaves and seeds have been previously reported,24,34,49 the presence of astragaline, rutin, and isoquercetin in lotus rhizomes was first identified in this study. Because of the lower flavonoids contents in lotus rhizomes,15 the levels of special flavonoids in rhizomes obtained from some growing regions were under the detection limit. In future studies, the unknown peaks in HPLC chromatograms are expected to be identified. There were significant differences in alkaloid contents in the LEb-2 fraction of lotus seeds and raw and dried rhizomes from various growing regions (Table 7). After drying, the concentration of dauricine significantly increased and the concentration of neferine decreased in the LEb-2 fractions of rhizomes. The concentration of specific alkaloids in the LEb-2

Table 4. DPPH Radical Scavenging Activities of the LE and LEb Fractions of Lotus Seeds and Rhizomesa EC50 value (μg/mL) no. dried seeds 1 2 3 4 5 raw rhizomes 6 7 8 9 10 dried rhizomes 11 12 13 14 15 16 17 ascorbic acid

LE fraction

LEb fraction

211.88 255.76 191.67 134.92 138.40

± ± ± ± ±

10.06a 18.88b 10.19c 4.11d 5.98d

145.56 237.21 162.62 103.26 137.99

± ± ± ± ±

5.27a 15.22b 6.02c 1.57d 5.08a

45.79 45.89 82.07 74.99 63.63

± ± ± ± ±

2.25a 0.28ad 3.22bc 3.72cd 3.36db

35.57 41.83 73.63 58.22 57.30

± ± ± ± ±

1.04ad 3.80b 3.99db 1.81cc 0.67cd

48.34 69.32 108.80 98.89 110.35 111.69 73.28 5.79

± ± ± ± ± ± ± ±

3.12a 3.28b 0.69c 0.63d 4.99c 2.56c 3.83b 0.11

45.94 45.72 87.97 92.88 86.91 80.32 71.23

± ± ± ± ± ± ±

1.03a 1.70a 4.49bc 3.40c 3.81b 3.44d 0.35e

Data shown are mean ± standard deviation (SD) from SPSS; each data point was derived from three independent repetitions. LE, the ethanol extract; LEb, residual fraction after separating hexane fraction from the ethanol extract. Values with different letters within the column were significantly different (P < 0.05) as observed by Duncan’s multiple range test for dried seeds and raw and dried rhizomes. Values with characters within raw and dried lotus rhizomes from the same growing region were significantly different as observed by the Student’s t test (see P values in footnotes b, c, and d). bP < 0.05. cP < 0.01. dP < 0.001. a

antioxidant, and its EC50 value of DPPH in this study was 5.79 μg/mL. Folin-Ciocalteu’s phenol reagent is sensitive to not only phenolic compounds but also nonphenolic compounds such as vitamins and certain amino acids.45 Studies in our laboratory showed that the Folin-Ciocalteu assay is very sensitive toward ascorbic acid while the aluminum chloride assay is not. In the Folin-Ciocalteu assay, 1 g of ascorbic acid shows 596.19 mg of GAE, but in the aluminum chloride assay, 1 g of ascorbic acid shows only 1.12 mg of CE. Therefore, the higher DPPH radical scavenging activity and total phenolic content may be attributed to a higher concentration of ascorbic acid in lotus rhizomes. In addition, the differences in the results between the current and previous study15 may be due to potential differences in dryness and extraction conditions, which can affect the degree of oxidation and extraction efficiency of phenolic compounds and ascorbic acid. Because the aluminum chloride coloration method is specific only for the analysis of anthoxanthins, including flavones and flavonols, estimation of total flavonoid content using this method likely underestimates the actual content.46 In this study, similar total flavonoid content in lotus seeds and rhizomes indicated similarity in the concentration of anthoxanthins, not of other flavonoids. Similar correlation coefficients were observed for DPPH radical scavenging activity and total phenolic and flavonoid content in the extracts of dried seeds and raw and dried rhizomes (Tables S1−S3, Supporting Information), respec6231



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Table 5. Total Phenolic and Flavonoid Content of the LE and LEb Fractions of Lotus Seeds and Rhizomesa total flavonoid content

total phenolic content LE fraction (mg of GAE/(g of LE))

no. dried seeds 1 2 3 4 5 raw rhizomes 6 7 8 9 10 dried rhizomes 11 12 13 14 15 16 17 ascorbic acid

LEb fraction (mg of GAE/(g of LEb))

LE fraction (mg of CE/(g of LE))

LEb fraction (mg of CE/(g of LEb))

21.63 13.99 18.65 31.22 24.70

± ± ± ± ±

0.57a 0.18b 0.75c 0.70d 0.73e

23.27 14.61 19.67 32.57 28.04

± ± ± ± ±

0.81a 0.63b 0.33c 1.01d 0.85e

13.88 8.21 8.51 13.34 10.91

± ± ± ± ±

0.35a 0.22b 0.37b 1.30a 0.90c

16.52 8.18 8.57 14.62 10.48

± ± ± ± ±

0.36a 0.14b 0.32b 0.48c 0.59d

70.01 61.64 41.65 31.63 49.69

± ± ± ± ±

5.94ab 0.75bb 0.88c 1.37dd 1.43e

74.72 67.24 41.94 38.68 50.19

± ± ± ± ±

2.46ab 0.54b 0.48cd 1.23dd 1.54ec

12.71 13.21 8.16 7.33 11.34

± ± ± ± ±

1.23a 0.69bb 0.15c 0.18d 0.66ac

13.39 15.96 8.81 7.27 12.21

± ± ± ± ±

0.62a 0.67bc 0.66c 0.02d 0.30e

82.21 64.63 43.19 29.20 31.21 52.17 53.38 596.19

± ± ± ± ± ± ± ±

4.28a 0.95b 1.99c 0.64d 0.26d 1.19e 2.37e 21.44

80.05 65.70 54.47 33.41 31.14 51.81 60.46

± ± ± ± ± ± ±

1.43a 3.43b 1.25c 1.17d 0.85d 1.53c 1.11e

13.05 11.41 7.83 7.94 8.74 6.82 9.33 1.12

± ± ± ± ± ± ± ±

0.62a 0.27b 0.53c 0.41c 0.38d 0.16e 0.22d 0.09

14.24 11.60 9.25 13.44 9.02 7.12 12.11

± ± ± ± ± ± ±

0.41a 0.82b 0.54c 0.60a 0.59c 0.23d 0.51b

Data shown are mean ± SD from SPSS; each data point was derived from three independent repetitions. LE, the ethanol extract; LEb, residual fraction after separating hexane fraction from the ethanol extract. Values with different letters within the column were significantly different (P < 0.05) as observed by Duncan’s multiple range test of dried seeds and raw and dried rhizomes. Values with characters within raw and dried lotus rhizomes from the same growing region were significantly different as observed by the Student’s t test (see P values given in footnotes b, c, and d). bP < 0.05. cP < 0.01. dP < 0.001. a

Table 6. Concentrations of Individual Flavonoids in the LEb-2 Fractions of Lotus Seeds and Rhizomes (mg/(g of LEb-2))a no. dried seeds 1 2 3 4 5 raw rhizomes 6 7 8 9 10 dried rhizomes 11 12 13 14 15 16 17

catechin

astragaline

3.48 3.89 0.79 9.16 2.70

± ± ± ± ±

0.78ab 0.55b 0.04c 0.45d 0.34a

0.08 0.38 0.11 0.26 0.15

± ± ± ± ±

3.02 3.23 1.43 1.65 3.25

± ± ± ± ±

0.01a 0.44ad 0.03bd 0.10bd 0.46ad

0.07 ± 0.01a

3.71 8.06 6.83 1.45 0.67 2.98 9.53

± ± ± ± ± ± ±

0.45a 0.30b 0.30c 0.08d 0.05e 0.14f 0.52g

0.05 0.09 0.03 0.08 0.04 0.07 0.06

rutin

0.01a 0.10b 0.01a 0.01c 0.02a

11.29 14.76 7.15 17.32 7.14

± ± ± ± ±

0.62a 1.25b 0.51c 2.74b 0.79c

isoquercetin 0.16a 0.80a 0.23b 0.66a 0.38a

17.43 21.74 9.60 29.40 12.88

± ± ± ± ±

1.39a 1.59b 0.74c 3.53d 0.91c

0.54 ± 0.14b

3.64 3.23 1.49 1.76 3.96

± ± ± ± ±

0.15ab 0.44ad 0.04cd 0.11cd 0.46bd

3.89 8.15 6.93 2.15 1.13 3.05 9.96

± ± ± ± ± ± ±

0.45a 0.29b 0.30c 0.16d 0.05e 0.14f 0.50g

2.58 2.71 1.56 2.66 2.89

± ± ± ± ±

0.06 ± 0.01ac 0.11 ± 0.02b

± ± ± ± ± ± ±

0.01ab 0.01c 0.01d 0.01c 0.01ad 0.03bc 0.01ab

total

0.22 ± 0.06

0.49 ± 0.02d

0.10 ± 0.01a

0.03 ± 0.01a

0.43 ± 0.09b 0.30 ± 0.01c

0.07 ± 0.01b 0.18 ± 0.02c 0.12 ± 0.01d

0.22 ± 0.03c

0.16 ± 0.01c

Data shown are mean ± SD from SPSS; each data point was derived from three independent repetitions. Values with different letters (a∼g) within the column were significantly different (P < 0.05) as observed by Duncan’s multiple range test for dried lotus seeds and raw and dried lotus rhizomes, respectively. Values with characters within raw and dried lotus rhizomes from the same growing region were significantly different as observed by the Student’s t test (see P values given in footnotes c and d). bP < 0.05. cbP < 0.01. dP < 0.001. a

fraction of seeds was higher than that of rhizomes. This finding may be because of the higher concentration of alkaloids detected in lotus embryos.12,50 The highest concentrations of alkaloids identified in the LEb-2 fractions of lotus were as

follows: nuciferine in seeds from Korea (Muan), raw rhizomes from Japan, and dried rhizomes from China (Guangxi); dauricine in seeds from China (Guangzhou) and raw and dried rhizomes from Japan; isoliensinine in seeds from 6232



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Table 7. Concentrations of Individual Alkaloids in the LEb-2 Fractions of Lotus Seeds and Rhizomes (mg/(g of LEb-2)) no. dried seeds 1 2 3 4 5 raw rhizomes 6 7 8 9 10 dried rhizomes 11 12 13 14 15 16 17

nuciferine 4.97 2.06 0.99 1.58 1.48

± ± ± ± ±

0.13a 0.03b 0.02c 0.06d 0.16d

0.25 ± 0.09

0.26 ± 0.03 0.15 ± 0.01a 0.22 ± 0.03b 0.48 ± 0.01c 0.29 ± 0.04d 0.27 ± 0.01d

dauricine 3.55 3.91 1.93 2.18 3.53

± ± ± ± ±

0.21a 0.05a 0.13b 0.71b 0.99a

2.87 1.64 1.37 1.33 5.43

± ± ± ± ±

0.04ac 0.17bb 0.34bb 0.21b 0.08cb

2.60 2.13 2.26 1.77 2.37 1.44 6.00

± ± ± ± ± ± ±

0.06a 0.14bc 0.20ac 0.26bd 0.40ac 0.02d 0.21e

isoliensinine 1.49 19.37 19.02 19.48 9.61

± ± ± ± ±

0.15a 0.50b 1.39b 0.95b 1.62c

1.49 ± 0.03

neferine 0.56a 1.01b 0.27c 1.39c 0.29d

24.24 45.48 49.29 51.64 23.20

± ± ± ± ±

0.77a 1.52b 1.06c 3.06c 2.89a

1.59 ± 0.32a 2.21 ± 0.15ac 3.07 ± 0.27b

4.46 5.60 4.44 1.33 9.78

± ± ± ± ±

0.29a 0.35bc 0.26a 0.21cd 0.14d

4.91 6.57 4.80 7.43 14.48 3.36 9.50

± ± ± ± ± ± ±

0.45a 0.25b 0.68a 0.26b 2.12c 0.06a 0.41d

14.24 20.14 27.35 28.40 8.58

± ± ± ± ±

4.09 ± 0.06cc

1.53 ± 0.06a 1.40 ± 0.17a 4.69 ± 0.80b

total

2.16 2.69 2.54 3.78 7.13 1.93 3.23

± ± ± ± ± ± ±

0.39a 0.07ab 0.56ab 0.23c 0.89d 0.06a 0.25bc

Data shown are mean ± SD from SPSS; each data point was derived from three independent repetitions. Values with different letters (a∼e) within the column were significantly different (P < 0.05) as observed by Duncan’s multiple range test for dried lotus seeds and raw and dried lotus rhizomes, respectively. Values with characters within raw and dried lotus rhizomes from the same growing region were significantly different as observed by the Student’s t test (see P values in footnotes b, c, and d). bP < 0.05. cP < 0.01. dP < 0.001. a

Vietnam, raw rhizomes from Korea (Daegu), and dried rhizomes from Vietnam; and neferine in seeds from Vietnam, raw rhizomes from Japan, and dried rhizomes from Vietnam. These results suggested that the concentration of special alkaloids in the lotus extracts can vary depending on the growing region and dryness. The presence of dauricine in lotus embryos was first reported in 1961.51 In previous reports from China, the concentration of nuciferine in dried lotus embryos from Hubei was reported to be 10.2 mg/kg,52 while the concentrations of isoliensinine and neferine in dried lotus embryos from Shanghai were reported as 2466.7 and 226.4 mg/kg, respectively.26 These concentrations were different from those obtained in this study for nuciferine, isoliensinine, and neferine in dried lotus seeds (11.3−62.7, 18.8−217.2, and 93.7−312.4 mg/kg). These differences between current and previous studies may be attributed to the fact that we used whole lotus seeds containing endosperm rich in starch, lipids, and protein, and this may have affected the concentration of alkaloids. Moreover, the concentration of alkaloids in lotus seeds may vary depending on the growing region and analysis methods. In addition, the presence of dauricine, nuciferine, isoliensinine, and neferine in lotus rhizomes was first identified in this study. The concentrations of special alkaloids in rhizomes from some growing regions were under the detection limit because lotus rhizomes generally have lower levels of alkaloids. There were some unknown peaks in the HPLC chromatograms that need further analysis. In conclusion, the antioxidant activity and total phenolic and flavonoid content in lotus seeds and rhizomes varied characteristically depending on the growing region and dryness. Total phenolic content in the LE fraction positively correlated with the antioxidant activity. In addition, the LE fractions of lotus seeds and rhizomes contained several specific flavonoids and alkaloids, and their concentrations also varied depending on the growing region and dryness. The HPLC analysis showed that

the LE fractions of lotus seeds from Vietnam, raw rhizomes from Korea (Siheung), and dried rhizomes from Japan have the highest flavonoid content, and they were 2.25, 3.34, and 10.07 times more than the flavonoid content in samples with the lowest levels, that is, the seeds from China (Hubei), raw rhizomes from Korea (Haman), and dried rhizomes from Vietnam, respectively. However, the LE fractions of seeds from China (Hubei), raw rhizomes from Japan, and dried rhizomes from Korea (Siheung) had the highest alkaloid content. The HPLC analysis indicated that these samples contained 2.08, 6.15, and 6.41 times more alkaloid content than the samples with the lowest alkaloid levels which were the seeds and raw and dried rhizomes from Korea (Muan), respectively. Therefore, these results indicated that the conditions in the growing region may significantly affect the antioxidant activity and concentration of functional components in the extracts of lotus seeds and rhizomes. The antioxidant activity and concentration of functional components in rhizomes were also sensitive to dryness. Moreover, this study is the first to detect the presence of astragaline, rutin, isoquercetin, dauricine, nuciferine, isoliensinine, and neferine in lotus rhizomes and to provide novel data regarding these functional components. Therefore, the lotus part, i.e., seeds or rhizomes, obtained from different growing regions can be selected based on the antioxidant activity and concentration of functional components for specific applications as functional foods.

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ASSOCIATED CONTENT

S Supporting Information *

Correlation coefficients among EC50 values and total phenolic and flavonoid content of lotus seeds (Table S1) and raw (Table S2) and dried rhizomes (Table S3). This material is available free of charge via the Internet at http://pubs.acs.org. 6233



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +82-02-450-3420. Fax: +82-02-3436-5382. Funding

This work was supported by Konkuk University in 2011. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We express warm thanks to Dr. Sung Wha Seok of K&S Medical Center for his kind comments on this manuscript.

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