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1. Wuhan Zhongnong South Science and Technology Co., Ltd., Wuhan 430064, China; 2. Institute of Economic Crop, Hubei Academy of Agricultural Science, Wuhan 430070, China; 3. Vegetable office of Hubei Province, Wuhan 430070, China

Supported by Wuhan City Huanghe Talents Plan; Second Batch Project for Modern Agricultural Industrial Technology System of Hubei Province.

1 Introduction

Glucosinolates (GSL) are important bioactive substances in cruciferous vegetables. This substance is hydrolyzed by endogenous myrosinase to produce a variety of biologically active degradation products, isothiocyanates, while isothiocyanates are the best bioactive substances found in vegetables so far[1]and they have antioxidant, anti-aging, anti-bacterial, fungi, viruses and aphids functions, and play an important role in human health[2-4].

Radish is an important medicinal vegetable. At present, more than 120 kinds of glucosinolates have been isolated. In radish, there are mainly three kinds: 4-methyl sulfinyl-butyl glucosinolates, 4-methyl sulfinyl-3-butyl glucosinolates, and 4-methyl thio-3- butenyl glucosinolates. Among these, 4-methyl sulfinyl-3-butyl glucosinolates are main glucosinolates in radish seeds, while 4-methyl thio-3- butenyl glucosinolates are main glucosinolates in succulent roots of radish[5]. Zhang Lietal.[6-7]studied the composition and content of glucosinolates in six radish varieties, and identified five types of glucosinolates. The total content of glucosinolates in different radish varieties differed greatly, mainly 4-methyl thio-3- butenyl glucosinolates, accounting for 70.5%-87.5% of the total content. The glucosinolates in the seedlings of different types of radish varieties were also different. Besides, the degradation product phenyl-isothiocyanate of 4-methyl thio-3-butenyl glucosinolates was found in radish[8-9].

In recent years, the components and content of glucosinolates in cruciferous vegetables have become a hot spot of research[10-12]. Previous studies have shown that there are differences in the composition and content of glucosinolates between different varieties of the same vegetable and in different parts of the same plant[6,13]. These studies provide a basis for breeding vegetable with high content of glucosinolates and high effective planting. However, there are few studies about glucosinolate content and components in different parts of radish. In this experiment, three different genotypes of radishes were used as test materials, the components and content of glucosinolates in the succulent roots and leaves of radish were analyzed and determined, to provide a theoretical basis for improving the radish varieties and breeding new radish varieties containing rich glucosinolates.

2 Materials and methods

2.1ExperimentalmaterialsIn this experiment, three different genotypes of radish varieties, Xinlimei, Super Zhengyan and Shawo were used as experimental materials. Among them, Xinmimei and Super Zhengyan were provided by Zhengyan Seedling Center of Zhengzhou Vegetable Research Institute, and Shawo was provided by Minghui Vegetable Purchase and Sales Center in Xiqing District of Tianjin City. Xinlimei radish is a local variety of Beijing, its upper surface is light green, lower is yellow-white, and the flesh is purple; Shawo radish is a local variety of Tianjin with green surface and green flesh; Super Zhengyan radish is developed by Zhengzhou Vegetable Research Institute with green surface and green flesh. The experimental materials were sown on the September 18th, 2016 in the experimental garden of Hubei Jinrun Agricultural Development Co., Ltd. in Jiayu County of Xianning City, Hubei Province. The seeds were directly sown in open field, each variety was set with three repetitions. The plot area was about 20 m2, the plant spacing was 25 cm and row spacing was 20 cm. The radishes were harvested on November 30, 2016. In the breeding period, the radishes were managed according to the conventional methods. In the expansion period, succulent roots and leaves were taken to detect glucosinolates.

2.2Measurementmethod

2.2.1Extraction of glucosinolates. For the extraction of glucosinolates, it could refer to methods of He Hongju[14]with slight modification. First, took succulent roots and leaves of fresh radishes, froze and dried. Weighed 1 g of sample and placed in a 150 mL flask. To inactivate the enzyme, added 45 mL of boiled methanol to a constant temperature water bath at 80℃ for 15 min. Filtered the extract and extracted with 80% methanol for 10 min. Mixed the filtrate of two times of extraction, concentrated to 2 mL using the rotary evaporator, fixed the volume with 10 mL volumetric flask, added 1 mL of 0.4 mol/L guanidine acetate, centrifuged at 7 000 r/min for 10 min. The supernatant was filtered through a 0.45 μm filter membrane before HPLC analysis.

2.2.2HPLC analysis. Instruments used included SP8450 high-performance liquid chromatography system, 510 gradient pump, 717 automatic sampler, 2487 UV detector, and Novapak C18column. The detection was carried out at the wavelength of 230 nm and injection volume of 20 μL. Mobile phase A was 0.05% tetramethylammonium chloride; mobile phase B was 0.05% tetramethylammonium chloride. The flow rate of mobile phase was 1 mL/min. Gradient elution was carried out according to Table 1, and all glucosides can be isolated within 30 min.

Table1Gradientofmobilephase

Time∥minMobilephaseA∥%MobilephaseB∥%0100011000200100251000301000

The glucosinolate content was determined on the basis of retention time and peak area using benzyl thioglycoside as an internal standard. The glucosinolate content was calculated using internal standard and response factors.

2.3DataanalysisThe experimental results were expressed as the mean value and standard deviation of three replicates measured in each time. The experimental data were statistically analyzed using SPSS10.0 software.

3 Results and analyses

3.1ComponentsandcontentofglucosinolatesinsucculentrootsoffruityradishesAs can be seen from Table 2, nine kinds of glucosinolate components were detected in succulent roots of the three kinds of fruity radish. There were six kinds of aliphatic glucosinolates (4-methyl sulfinyl-3-butenyl glucosinolates, 2-allyl glucosinolates, 4-methyl sulfinyl butyl glucosinolates, 5-methyl sulfinyl amyl glucosinolates, 4-methyl n-butyl glucosinolates, 4-methyl thio-3- butenyl glucosinolates) and three kinds of indole glucosinolates (4-methoxy methyl indole glucosinolates, 3- methyl indole glucosinolates, 1-methoxy methyl indole glucosinolates).

There were significant differences in aliphatic glucosinolates between succulent roots of three kinds of fruity radish (Table 2), Shaowo radish had the highest content (27 002.97 μg/g DM), followed by Xinlimei, and the lowest was Super Zhengyan. However, the relative content of total aliphatic glucosinolates in all three radish varieties was above 95%. The content and relative content of six kinds of aliphatic glucosinolates in the succulent roots of three kinds of fruity radishes also showed significant differences. Among them, the content of 4-methyl thio-3- butenyl glucosinolates was the highest, accounting for 90.11%-93.92% of the total glucosinolates. The highest content came from Shawo radish (26 145.06 μg/g DM), followed by Xinlimei, and the lowest was Super Zhengyan (only 12 304.20 μg/g DM).

Besides, there were also great differences in the content of indole glucosinolates between succulent roots of three kinds of fruity radishes, Shawo radishes had the highest content of total indole glucosinolates (1 197.73 μg/g DM), followed by Xinlimei, and the lowest was Super Zhengyan (572.06 μg/g DM). The relative content of total indole glucosinolates in three kinds of radishes was 4.25%, 3.53%, and 4.21%.

3.2ComponentsandcontentofglucosinolateinleavesoffruityradishesFrom Table 3, it can be seen that the content and relative content of total aliphatic glucosinolates in the leaves of Xinlimei and Super Zhengyan were relatively close to each other, the content was 4 425.38 μg/g DM and 4 423.63 μg/g DM respectively, and the relative content was close to 90%. The total content of aliphatic glucosinolates in leaves of Shawo radish was 4 990.59 μg/g DM, but its relative content was only 80.59%. In aliphatic glucosinolates, the relative content of 4-methyl thio-3-butenyl glucosinolates in three kinds of radishes was above 60%, so 4-methyl thio-3-butenyl glucosinolates were the main aliphatic glucosinolates.

In leaves of Xinlimei, Super Zhengyan, and Shawo radishes, the content of 4-methyl sulfinyl-3-butenyl glucosinolates was also high, only second to 4-methyl thio-3-butenyl glucosinolates. Besides, the content of 2-allyl glucosinolates was relatively high in Shaowo radish, up to 575.22 μg/g DM, so 2-allyl glucosinolates were the secondary aliphatic glucosinolates in Shawo radish.

The content of the total indole glucosinolates of the three kinds of fruity radishes also showed significant differences (Table 3). The total content of indole glucosinolates of Shawo radish was 1 202.10 μg/g DM, which was two times of that of Xinlimei and Super Zhengyan, and the relative content was up to 19.41%. In leaves of three kinds of fruity radishes, the content of 3-methyl indole glucosinolates was 481.46-1 152.01 μg/g DM, it was highest in Shawo radish, about two times of other two varieties; the relative content of 3-methyl indole glucosinolates in Shawo radish was up to 18.60%, and other two varieties showed 10% difference. The content of 1-methoxy methyl indole glucosinolates and 4-methoxy methyl indole glucosinolates was relatively low, and the sum of both was only 0.52%-0.90%.

3.3ComparisonoftotalcontentofglucosinolatesbetweendifferentfruityradishesFrom Table 2 and Table 3, it can be seen that succulent roots and leaves of Xinlimei, Super Zhengyan, and Shawo radishes contained the same components of glucosinolates: the main aliphatic glucosinolates were 4-methyl sulfinyl-3-butenyl glucosinolates, and the main indole glucosinolates were 3-methyl indole glucosinolates. The total content of glucosinolates in succulent roots of three kinds of fruity radish was 22 472.84, 13 585.86 and 28 200.70 μg/g DM, respectively; the total content of glucosinolates in leaves of fruity radish was 4 932.68, 5 010.08 and 6 192.69 μg/g DM, respectively, showing that the content of glucosinolates in succulent roots was 2.71-4.56 times of that in leaves (Fig.1).

There were also significant differences in the content and relative content of glucosinolates and total aliphatic and indole glucosinolates in the succulent roots and leaves (Table 2 and Table 3). The total content of aliphatic glucosinolates in succulent roots was 13 013.80-27 002.97 μg/g DM, while it was only 4 423.63-4 990.59 μg/g DM in leaves. The relative content of the total aliphatic glucosinolates in the succulent roots of three kinds of fruity radish was higher than 95%, while that in leaves was only 80%-90%. There was little difference in content of total indole glucosinolates between succulent roots and leaves, but the differences were significant between different varieties. The content of total indole glucosinolates in succulent roots of Xinlimei was 1.56 times of that in leaves, while Super Zhengyan and Shawo were only 0.97-0.99 times. The relative content of total indole glucosinolates in succulent roots of three kinds of fruity radish was 3.53%-4.25%, while it was up to 10.28%-19.41% in leaves.

Fig.1Totalcontentofglucosinolatesinsucculentrootsandleavesoffruityradish

Table2Componentsandcontentofglucosinolatesinsucculentrootsoffruityradishes

ComponentsofglucosinolatesXinlimeiContentμg/gDMPercenttototalglucosinolates∥%SuperZhengyanContentμg/gDMPercenttototalglucosinolates∥%ShawoContentμg/gDMPercenttototalglucosinolates∥%4?methylsulfinyl?3?butenylglucosino?lates127．02±1．20e0．57±0．0218．10±0．31e0．13±0．01136．11±1．82c0．25±0．022?allylglucosinolates703．11±2．54b3．13±0．03492．25±2．62b3．62±0．02273．54±0．52b0．51±0．014?methylsulfinylbutylglucosinolates13．20±0．22f0．06±0．0115．50±0．30e0．11±0．0118．16±0．24d0．03±0．015?methylsulfinylamylglucosinolates371．23±4．63c1．65±0．0229．30±0．42d0．22±0．01132．10±0．39c0．25±0．024?methyln?butylglucosinolates215．07±3．23d0．96±0．01154．45±1．72c1．14±0．02296．00±1．70b0．79±0．054?methylthio?3?butenylglucosinolates20249．37±61．03a90．11±0．0912304．20±0．39a90．57±0．0226145．06±94．08a93．92±0．04Totalaliphaticglucosinolates21679．00±32．1796．47±0．0313013．8±0．4795．79±0．0227002．97±95．0595．75±0．034?methoxymethylindoleglucosinolates38．29±0．19b0．17±0．016．37±0．04c0．05±0．015．52±0．08c0．01±0．003?methylindoleglucosinolates702．36±12．37a3．13±0．08528．01±5．05a3．89±0．021146．03±1．40a2．14±0．051?methoxymethylindoleglucosinolates53．19±0．04b0．24±0．0337．68±0．41b0．28±0．0246．18±0．44b0．09±0．01Totalindoleglucosinolates793．84±11．043．53±0．05572．06±4．024．21±0．161197．73±1．124．25±0．06

Note: The data in the table were the mean±standard deviation. Different small letters in the same column indicated that the difference was 5% significant. The same as below.

4 Conclusions and discussions

The glucosinolates are widely found in cruciferous vegetables such as broccoli, radish, cabbage, mustard, turnip, rape,etc., and are important secondary metabolites of cruciferous plants. The glucosinolates and their degradation products play an important role in the resistance to insect pests and diseases, special flavor composition and human health[15-16]. At present, there are many studies on the glucosinolates of cruciferous plants, which mainly focus on the biological activities and functions of glucosinolates and their degradation products. In particular, the beneficial role of glucosinolates in anti-cancer has attracted increasing attention[17-18]. According to research of Talayapetal.[17], the degradation products of 4-methyl sulfinyl-3-butenyl glucosinolates and indole-3-carbinol and indole-3-carbonitrile of indole glucosinolates have anti-cancer function. The degradation product isothiocyanates of 4-methyl thio-3-butenyl glucosinolates are the volatile components contained in radish leaves that has a strong activity of attracting Myzus persicae (Sulzer)[19]. Most of the aliphatic glucosinolates such as 2-allyl glucosinolates are special flavor substances[20], and their degradation products can inhibit the proliferation of colorectal cancer cells and accelerate their programmed cell death[21]. The antioxidant properties of radish sprouts have been confirmed in mice[22].

Table3Componentsandcontentofglucosinolateinleavesoffruityradishes

ComponentsofglucosinolatesXinlimeiContentμg/gDMPercenttototalglucosinolates∥%SuperZhengyanContentμg/gDMPercenttototalglucosinolates∥%ShawoContentμg/gDMPercenttototalglucosinolates∥%4?methylsulfinyl?3?butenylglucosino?lates582．01±0．05b11．80±0．03379．68±0．75b7．58±0．04262．83±8．25c4．24±0．112?allylglucosinolates91．35±0．14c1．85±0．02196．12±2．78c3．91±0．03575．22±0．81b9．29±0．044?methylsulfinylbutylglucosinolates65．94±1．37cd1．34±0．0255．02±0．53d1．10±0．0553．09±0．96e0．86±0．055?methylsulfinylamylglucosinolates13．25±0．06e0．27±0．0316．68±0．54e0．33±0．0329．27±0．37e0．47±0．064?methyln?butylglucosinolates36．65±0．47d0．74±0．03154．70±2．55c3．09±0．05167．11±1．43d2．70±0．044?methylthio?3?butenylglucosinolates3636．18±2．80a73．72±0．093621．43±6．32a72．28±0．123903．07±11．38a63．03±0．16Totalaliphaticglucosinolates4425．38±1．4889．72±0．084423．63±0．6388．29±0．074990．59±3．4980．59±0．054?methoxymethylindoleglucosinolates7．02±0．15c0．14±0．036．56±0．04c0．13±0．036．37±0．36c0．10±0．053?methylindoleglucosinolates481．46±2．63a9．76±0．08541．51±4．90a10．81±0．091152．01±1．45a18．60±0．071?methoxymethylindoleglucosinolates18．82±0．45b0．38±0．0338．38±0．61b0．77±0．0543．72±0．35b0．71±0．08Totalindoleglucosinolates507．30±5．8210．28±0．05586．45±3．0211．71±0．081202．10±1．5319．41±0．04

In this experiment, six kinds of aliphatic glucosinolates and three kinds of indole glucosinolates were detected in both the succulent roots and leaves of three kinds of fruity radishes, no aromatic glucosinolates were found. Main glucosinolates of succulent roots of all these three kinds of fruity radish were 4-methyl thio-3- butenyl glucosinolates, each accounting for more than 90% of total glucosinolates. This is similar to the components and content of glucosinolates detected by Yang Lijuanetal.[23]from succulent roots and leaves of local varieties of radishes. However, this is different from results of Zhang Lietal.[7]. In this experiment, only 2-allyl glucosinolates, 5-methyl sulfinyl amyl glucosinolates, and 4-methoxy methyl indole glucosinolates were detected, while 2-phenylethyl glucosinolate was not detected, possibly connected with difference in varieties.

With the improvement of people’s living standards, fruity radish is increasingly favored by consumers. According to studies of Li Qiuyunetal.[24], the main glucosinolate component in leaves of Xinlimei was 3-methyl indole glucosinolates. However, in this experiment, the main glucosinolate component in leaves of Xinlimei, Super Zhengyan and Shawo was 4-methyl thio-3-butenyl glucosinolates, accounting for 63.03%-73.72% of the total glucosinolates, while the relative content of 3-methyl indole glucosinolates was only 9.76%-18.60% and belonged to secondary glucosinolates. In addition, the glucosinolate content in succulent roots of the three kinds of fruity radish were significantly higher than that in the leaves (2.71-4.56 times higher), showing that the content of glucosinolates is different in different genotypes, and the glucosinolate content in different organs of the same genotype radish is also different.

In recent years, the incidence of cancer has been increasing year by year, and the degradation product isothiocyanate of glucosinolates is by far the best biologically active substance found in vegetables having anti-cancer effect. Besides, these degradation products also have anti-insect, antibacterial and other functions, the selection and breeding of high glucosinolate content of medicinal vegetable varieties gradually get the attention of breeding experts. In Japan, scholars are currently working on the selection and breeding of new radish varieties with high content of glucosinolates. Research and optimization of the composition and content of glucosinolates and their hydrolysates in fruity radish has made it become a very significant research filed to maximize its role in human health and plant defense. In this experiment, three kinds of fruity radishes were used as test materials, and the components and content of glucosinolates were analyzed between the succulent roots and leaves. It was found that radish leaves and succulent roots are rich in glucosinolates. In conclusion, radish is a vegetable that has great use value and development potential, and the biological and health care value of its components of glucosinolates is to be further studied.

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