Breeding Science 61: 208–211 (2011) doi:10.1270/jsbbs.61.208
Note
Development of a simple and rapid extraction method of glucosinolates from radish roots Masahiko Ishida*1), Tomohiro Kakizaki1), Takayoshi Ohara1) and Yasujiro Morimitsu2) 1) 2)
National Institute of Vegetable and Tea Science (NIVTS), 360 Ano-Kusawa, Tsu, Mie 514-2392, Japan Faculty of Human Life and Environmental Sciences, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo, Tokyo 112-8610, Japan
A simple and rapid method for the extraction of crude glucosinolates from powder of freeze-dried radish roots was developed using a common solvent, methanol/water = 80 : 20 (v/v) at room temperature. Hot methanol is not required for the extraction procedure of this new method. Both 4-methylthio-3-butenyl glucosinolate, which is a major glucosinolate in radish root, and total glucosinolates of Japanese radish cv. ‘Karami 199’ were quantified using HPLC. The amounts of glucosinolate extracted using this new method were 4– 6% higher than those obtained using the conventional method with hot methanol. To compare the new method with the conventional method, the amounts of glucosinolate extracted from roots of eight cultivars in Japanese radish and five breeding lines were estimated. A high positive correlation was found between them. This simple and rapid method 1) obviates hot methanol for glucosinolate extraction and 2) requires only onethird of the extraction time of the conventional method, enabling glucosinolate extraction from about 150 samples by one person in a single day. Key Words: glucosinolates, extraction, radish, daikon, Raphanus sativus.
Introduction Glucosinolates, secondary metabolites found in Brassicaceae and related families, have three moieties: a beta-thioglucose moiety, a sulfonated oxime moiety, and a variable aglycone side chain derived from an alpha-amino acid. Based on the distinct side chain, more than 120 glucosinolates are classified into three groups of so-called aliphatic, aromatic, and indolic glucosinolates (Fahey et al. 2001, Mithen et al. 2000). The composition and contents of glucosinolates differ completely among plant genera and different organs (Rosa et al. 1997). Radish (Raphanus sativus) is an important crucifer vegetable in Japan. Radish roots contain 4-methylthio-3-butenyl glucosinolate (4MTB-GSL) as a characteristic glucosinolate with the common name of glucoraphasatin (Carlson et al. 1985). By the action of myrosinase (EC 3.2.3.1), 4MTBGSL is hydrolyzed quickly and converted into 4-methylthio3-butenyl isothiocyanate (4MTB-ITC), commonly called raphasatin, which is a specific pungent compound in radish (Friis and Kjaer 1966) that affects the taste and odor of grated radish (daikon-oroshi), salads, and so on. Some isothiocyanates in cruciferous vegetables reportedCommunicated by T. Nishio Received October 27, 2010. Accepted February 15, 2011. *Corresponding author (e-mail:
[email protected])
ly provide physiological benefits through activities such as cancer chemoprevention and pylori growth inhibition in the stomach of rodents (Fahey et al. 1997, 1999, 2002). Therefore, glucosinolate, a precursor to isothiocyanate, is also in the spotlight. In general, the contents and compositions of glucosinolates might play an important role in the bioavailability and bioactivity of some crucifer vegetables. Regarding radish, many reports have described 4MTB-GSL contents in different cultivars (Carlson et al. 1985, Nakamura et al. 2008). A well-known analytical method used for glucosinolates is desulfonation of glucosinolates with sulfatase, followed by analysis using a reversed phase HPLC gradient system (Bjerg and Sørensen 1987, Bjorkqvist and Hase 1988). In the procedure of glucosinolate extraction, myrosinase should be inactivated to prevent the loss of glucosinolates. From this perspective, mixed alcohol and water or boiling water at almost its boiling point (around 60–100°C) was selected as the glucosinolate extraction solution. Furthermore, to prevent the loss of glucosinolate during the process of crushing or smashing, freezing with N2 liquid or freezedrying was adopted (Clarke 2010). For the extraction of glucosinolates from radish root, samples should be freeze-dried or cut into small cubes, with subsequent extraction using hot methanol (70–80% in water, at temperatures higher than 70°C) or using boiling water to prevent the hydrolysis reaction of glucosinolates by internal myrosinase (Barillari et al.
Simple extraction method of glucosinolates from radish roots
2005, Van Etten et al. 1976, Visentin et al. 1992). A hightemperature condition must be maintained during these extraction processes. Moreover, their complicated procedures require much working time. This study revealed that extraction of glucosinolates from radish root can be accomplished without using hot methanol. A simple and rapid method for the extraction of crude glucosinolates from the powder of freeze-dried radish roots was developed for use at room temperature using a common solvent system: methanol/water = 80 : 20 (v/v). This simple method requires only one-third of the extraction time of the conventional method.
Materials and Methods Plant materials Nine cultivars of Japanese radish and five breeding lines were selected from a local variety of Chinese radish with red skin and red flesh in root. They were cultivated in the field of National Institute of Vegetable and Tea Science (NIVTS) during autumn–winter in 2009 in Tsu, Mie, Japan. Sample preparation The cultivars and breeding lines were harvested when the plants were at the first stage of the harvest period. Each radish root was washed with water and cut vertically into equally sized pieces. Each piece was frozen immediately with liquid N2 and then freeze-dried. The freeze-dried samples were ground (Multi-Beads Shocker MB831; Yasui Kikai Corp., Osaka, Japan). Each powder (0.1 g) was transferred to a 10-mL test tube. Extraction of glucosinolates (conventional method) The glucosinolates in radish roots were extracted according to the official EU method (ISO 9167-1 1992). Each test tube containing 0.1 g powder was pre-incubated in a hot water bath at 75°C for 1 min; then 1.5 mL of 80% methanol in water and 0.2 mL of 5 mM sinigrin solution were poured into each test tube. The suspension was incubated at 75°C for 10 min. After cooling, each test tube was centrifuged at 1,600 × g for 10 min. Each supernatant was collected. Subsequently, the same extraction procedure was repeated twice. The combined supernatants were filled up to 5 mL. Developing a novel method for extraction of glucosinolate Into each test tube, 80% methanol in water (4.8 mL) and 5 mM sinigrin solution (0.2 mL) were poured. Then various extraction times and temperatures were tested as follows. Some samples of the reaction mixture were kept for 60 min. Others were kept for 30 min and then shaken (120 r/min) for 30 min with a reciprocal shaker (SR-2s; Taitec Co., Saitama, Japan) at room temperature. As extraction temperatures, 25°C, 40°C, and 75°C were tested. After the extraction procedure, supernatants were collected and combined, then filled to 5 mL.
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Preparation of desulfoglucosinolate and HPLC analysis Preparation of desulfoglucosinolate with sulfatase digestion was conducted according to the method described by Bjerg and Sørensen (1987) for HPLC analysis. Then HPLC was performed using a class-VP chromatograph (Shimadzu Corp., Kyoto, Japan) fitted with a column (5C 18-MS-II, particle size 5 µm, 150 mm × 4.6 mm I.D.; Nacalai Tesque Inc., Kyoto, Japan). The amount of glucosinolate was determined using sinigrin as the internal standard, considering the relative response factors. Statistical analysis Data were analyzed using ANOVA followed by Turkey’s HSD multiple comparison test.
Results and Discussion In radish roots, prevention of myrosinase activity is necessary during the glucosinolate extraction procedure. For this reason, several extraction methods with inactivation of myrosinase have been used. The extraction method according to ISO 9167-1 (1992) using a sample powdered after freezedrying has been used (Barillari et al. 2005). The method, for which a sample is cut into 1–3 cm cubes from roots chilled below 5°C and extracted with 70–80% hot methanol, has also been used (Carlson et al. 1985, Ishii et al. 1989). The microwave method has been used (Nakamura et al. 2008). However, these processes were restricted to extraction of a few samples because of time-consuming sample preparation, moreover hot methanol vapor is poisonous and flammable. To breed radish, which is based on glucosinolate components, efficiently, development of a simple and rapid method for the extraction of glucosinolates from many radish root samples has been necessary for breeding programs. A radish cultivar that we have used as a standard cultivar for glucosinolate analysis, cv. ‘Karami 199’ (Kaneko Seed Co. Ltd., Maebashi, Japan), contains high contents of 4MTB GSL and total glucosinolates. Its traits are stable interannually and among individual plants. To develop a novel method for extraction, extraction efficiency was compared for three temperature conditions and among different procedures using this cultivar (Table 1). When kept at 60 min at 25°C, 40°C and 75°C, the amounts of 4MTB-GSL in extracts determined using HPLC analysis ranged from 85.5 µmol/g DW to 89.2 µmol/g DW. The extraction efficiency of the tested method was 76.0–79.3% of the conventional method. The amounts of total glucosinolates in the extract were 94.1–97.6 µmol/g DW, which were 76.7–79.5% of the efficiency of the conventional method. The extraction efficiency when kept for 60 min to extract was revealed as about 77% of the amount of 4MTB-GSL and total glucosinolates using the conventional method (significance, p < 0.01) at all tested temperatures. However, by holding 30 min, followed by shaking (120 r/min) for 30 min with a reciprocal shaker, the amounts of 4MTB-GSL in extracts were 117.2 µmol/g DW at 25°C,
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Ishida, Kakizaki, Ohara and Morimitsu
Table 1. Comparison of glucosinolate content in the simple extraction method and conventional method of Japanese radish cv. ‘Karami 199’ Simple extraction method GSL (µmol/g DW)
Conventional method
25°C Staying
4MTB-GSL Total-GSL
112.5 122.7
85.5** 94.1**
40°C Shaking
Staying
117.2** 127.1*
85.9** 94.4**
75°C Shaking
Staying
119.2** 128.6**
89.2** 97.6**
Shaking 110.6 119.4
Staying: Extraction by holding at 25°C, 40°C, 75°C for 30 min. Shaking: Extraction by holding at 25°C , 40°C, 75°C for 30 min and shaking at room temperature for 30 min. GSL: Contents of total and 4-methylthio-3-butenyl glucosinolate (4MTB-GSL) in the mature root of radish cv. ‘Karami 199’ were mean value of 10 levels, repetitions. *,**: Significant at the 5% and 1% probability level respectively. Table 2. Mean contents of 4MTB-GSL and total GSL with conventional and simple extraction method in eight Japanese radish cultivars and five breeding lines Method Cultivars and lines
Conventionala
Simple extractionb
4MTB-GSL (µmol/g DW) WNRD1* Tenan Koushin 2-1* Koushin 2* Oibane Hayabutori Ookura Fuyudori Shougoin Kenka 37 Fuyudori Ookura Wakakoma Ryuugin miura Hayabutori Shougoin D37H D26H
6.1 ± 0.2 14.2 ± 0.2 16.7 ± 0.4 22.4 ± 0.4 29.5 ± 0.2 32.1 ± 0.8 33.9 ± 1.5 35.9 ± 0.6 42.0 ± 1.4 50.4 ± 1.0 54.4 ± 0.2 81.6 ± 0.9 103.7 ± 2.7
6.5 ± 0.2 15.2 ± 0.2 17.4 ± 0.3 23.7 ± 0.4 30.6 ± 0.5 34.3 ± 0.5 36.6 ± 0.4 38.3 ± 1.0 44.6 ± 0.5 52.5 ± 1.0 59.4 ± 0.4 85.5 ± 1.2 111.4 ± 3.5
% of a/b 106.8 107.0 104.2 106.0 103.7 106.9 107.8 106.9 106.2 104.3 109.2 104.8 107.5
Conventionala
Simple extractionb
Total GSL (µmol/g DW) 7.6 ± 0.2 18.3 ± 0.3 22.3 ± 0.5 26.4 ± 0.5 30.8 ± 0.2 35.5 ± 0.8 36.9 ± 1.4 37.9 ± 0.6 46.3 ± 1.1 52.6 ± 1.1 59.8 ± 0.2 85.1 ± 0.8 110.1 ± 2.4
7.9 ± 0.2 18.7 ± 0.0 22.8 ± 0.4 27.8 ± 0.4 31.8 ± 0.5 37.9 ± 0.4 39.9 ± 0.4 40.4 ± 0.9 48.8 ± 0.5 54.7 ± 1.1 64.8 ± 0.3 89.2 ± 1.2 117.6 ± 3.5
% of a/b 103.9 102.7 102.2 105.5 103.3 106.7 108.1 106.5 105.3 104.1 108.4 104.9 106.9
The data represent means of three replicates ± SD. *: Breeding lines from Chinese radish cultivar with red skin and red flesh in root. a Three times extraction at 75°C for 10 min with 80% methanol. b Extraction at 25°C for 30 min and shaking at room temparature for 30 min.
which was 4.2% higher than those using the conventional method, 119.2 µmol/g DW at 40°C, which was 6.0% higher than those using the conventional method, and 110.6 µmol/g DW at 75°C, which was 98.3% of the conventional method. The amounts of total glucosinolates were 127.1 µmol/g DW at 25°C (3.6% higher than the conventional method), 128.6 µmol/g DW at 40°C (4.8% higher than the conventional method), and 119.4 µmol/g DW at 75°C (97.3% of the conventional method). At temperatures of 25°C and 40°C, the glucosinolate concentrations in the extracts obtained using this method were significantly higher than those obtained using the conventional method (p < 0.05 or 0.01). At 75°C, no significant difference was found between this method and the conventional method. Based on these results, we report the following simple and rapid method to extract glucosinolates from radish roots. In this method, 80% methanol solution is added directly to freeze-dried powder as an extraction solvent at room temperature. Then it is centrifuged to obtain supernatant of crude extracts. The protocol of glucosinolate extraction de-
veloped in this study is presented as follows. 1. Transfer 100 mg of the prepared sample powder into the tube. 2. Add 4.8 ml of 80% MeOH and 0.2 ml of 5 mM internal standard sinigrin. 3. Hold at 25°C for 30 min. 4. Shake the tube at 120 r/min for 30 min at room temperature. 5. Centrifuge at 1,600 × g for 10 min. 6. Transfer the supernatant liquid. 7. Keep it at −20°C until used for glucosinolate desulfatation. These results (using cv. ‘Karami 199’) show that the extraction efficiency of the simple method is higher than that of the conventional method. Therefore, to confirm the analytical accuracy of the simple method using eight cultivars and five breeding lines having different genetic background, extraction efficiencies of this method were compared with those of the conventional method. As Table 2 shows, the amounts of 4MTB-GSL and total glucosinolates in extracts
Simple extraction method of glucosinolates from radish roots
obtained using the simple method were 3.7%–9.2% (6.3% on average) higher and 2.2%–8.4% (5.3% on average) higher, respectively, than those obtained using the conventional method. Significant differences in the amounts of 4MTBGSL and total glucosinolates were found neither between the simple method and the conventional method nor among the extraction efficiencies of different cultivars/lines. Differences of glucosinolate contents and red color pigments of the crude extracts of 13 cultivars and breeding lines did not affect the extraction efficiency. Based on results of this study, use of 80% methanol as the extraction solvent at 25°C is sufficient for inactivation of myrosinase. Kiddle et al. (2001) reported that GLS was poorly hydrolyzed by myrosinases in the presence of denaturants such as methanol. Considering that report, it is possible that the extraction solvent of 80% methanol penetrates freeze-dried radish root tissues instantly to inhibit hydrolysis by myrosinases, even at room temperature. The glucosinolate extraction efficiency achieved using this simple method was better than that of the conventional method: the yield of glucosinolates was approximately 5% higher. Use of a reciprocal shaker for 30 min was necessary to increase the extraction efficiency of the simple method. Although the reason that no significant difference was found between this simple method and the conventional method at 75°C remains unclear, the extraction efficiency was greater at temperatures of 25°C and 40°C. Few quantitative data documenting the stability of glucosinolates during processing and extraction are available. Particularly, there is a lack of clear validation data to assess the effects of avoiding high temperatures and to determine the significance of myrosinase inactivation (Clarke 2010). Further research is needed on those points. In this study, we developed a novel simple method. The conditions of glucosinolate extraction require neither hot methanol nor a repetitive extraction procedure. The simple method requires only one-third of the extraction time of the conventional method. Some breeding lines of radish having characterized contents and/or compositions of glucosinolate are being developed using this simple method. Further studies are underway to develop an efficient method of extracting glucosinolates from leaves, seeds, and other organs of Brassicaceae.
Acknowledgments This research was partly supported by the Program for Promotion of Basic and Applied Researchers for Innovations in Bio-oriented Industry (BRAIN).
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