Correlation of Quinone Reductase Activity and Allyl Isothiocyanate

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Correlation of Quinone Reductase Activity and Allyl Isothiocyanate Formation Among Different Genotypes and Grades of Horseradish Roots Kang-Mo Ku, Elizabeth H. Jeffery, John A. Juvik, and Mosbah M Kushad J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505591z • Publication Date (Web): 14 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

Correlation of Quinone Reductase Activity and Allyl Isothiocyanate Formation Among Different Genotypes and Grades of Horseradish Roots

Kang-Mo Ku†, Elizabeth H. Jeffery‡, John A. Juvik†, and Mosbah M. Kushad†* †

Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL

61801-3838 ‡

Department of Food Science and Human Nutrition, University of Illinois at Urbana-

Champaign, Urbana, IL 61801-3838

*

To whom correspondence should be addressed

Mosbah M. Kushad Department of Crop Science, University of Illinois at Urbana-Champaign, 1019 Plant Sciences Lab 1201 S. Dorner Dr. Urbana, IL, 61801

Tel: 217-244-5691; Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT 1

Horseradish (Armoracia rusticana) is a perennial crop and its ground root tissue is used in

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condiments due to the pungency of the glucosinolates (GS)-hydrolysis products allyl

3

isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC) derived from sinigrin and

4

gluconasturtiin, respectively. Horseradish roots are sold in three grades: “U.S. Fancy”, “U.S. No.

5

1”, and “U.S. No. 2” according to the USDA standards. These grading standards are primarily

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based on root diameter and length. There is little information on whether root grades vary in their

7

phytochemical content or potential health promoting properties. This study measured GS, GS-

8

hydrolysis products, potential anti-cancer activity (as quinone reductase-inducing activity), total

9

phenolic content, and antioxidant activities from different grades of horseradish accessions. “U.S.

10

Fancy” showed significantly higher sinigrin and AITC concentrations than “U.S. No. 1” whereas

11

“U.S. No. 1” showed significantly higher 1-cyano 2,3-epithiopropane, the epithionitrile

12

hydrolysis product of sinigrin, and total phenolic concentrations than “U.S. Fancy”.

13 14

KEYWORDS:

15

isothiocyanate; quinone reductase; sinigrin; epithiospecifier protein

Allyl

isothiocyanate;

Armoracia

rusticana;

glucosinolate;

phenethyl

16 17 18 19 20


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INTRODUCTION

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Horseradish (Armoracia rusticana G. Gaertn., B. Mey. & Scherb.) is a perennial root crop

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belonging to the Brassicaceae family, which includes mustard, broccoli and other cruciferous

24

crops. Horseradish is an important horticultural crop in Illinois, providing over 60% of the total

25

U.S. production.1 Horseradish is asexually propagated by planting root sections collected from

26

the previous year’s crop.1 Typically, horseradish harvest in Illinois starts once the foliage has

27

been killed by frost (late October–November).1 Due to the intense pungent flavor of horseradish

28

root, it is used as a condiment, in sauces and other food ingredients, especially in ethnic dishes.

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Pungency is largely caused by allyl isothiocyanate (AITC) resulting from the hydrolysis of

30

sinigrin by the endogenous enzyme, myrosinase. Li and Kushad2 found that sinigrin accounts for

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about 80 and 90% of total glucosinolate (GS) content in horseradish roots and leaves,

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respectively. AITC is a mechanism of insect and other herbivore defense in the plant.3 AITC has

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been shown to induce phase II enzymes, including glutathione-S-transferase and quinone

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reductase (QR) which supports detoxification and elimination of carcinogens and other

35

xenobiotics in mammals.4 Specifically, QR can detoxify reactive quinones by converting them

36

into stable and non-toxic compounds.5 Moreover, induction of QR activity has been correlated

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with induction of other phase II enzymes, thus providing a biomarker for improved protection

38

against cancer initiation and proliferation.6

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The endogenous enzyme myrosinase is responsible for conversion of GS glucoraphanin to the

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isothiocyanate sulforaphane.7,8 Hydrolysis of GS by myrosinase is influenced by the

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epithiospecifier protein (ESP), and other co-factors including ascorbic acid and metal ions.8

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Abundant ESP promotes the formation of nitriles (the simple nitrile from gluconasturtiin; the

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epithionitrile from sinigrin), as alternative products to isothiocyanates during myrosinase-


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mediated GS hydrolysis.9 Our previous research reported that ESP activity in broccoli leads to

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the conversion of glucoraphanin to the nitrile form of sulforaphane, which has extremely weak

46

QR inducing activity compared to the isothiocyanate sulforaphane.10 It has been reported that

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sinigrin in horseradish is primarily hydrolyzed to bioactive AITC.11, 12 Like horseradish, cabbage

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contains high percentage of sinigrin, although it is reported to produce substantial amounts of 1-

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cyano 2,3-epithiopropane (CETP), the epithionitrile hydrolysis product of sinigrin, that has less

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health promoting effects (hypothetically due to ESP activity).11 Li and Kushad evaluated sinigrin

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concentrations in various horseradish genotypes and different tissues, but did not estimate

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sinigrin hydrolysis products that have been reported as potential cancer preventing agent.13, 14

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According to US Department of Agriculture (USDA) standards, horseradish roots are classified

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“U.S. Fancy”, “U.S. No. 1”, and “U.S. No. 2” based on the diameter and length of the root.19 For

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fresh-market grocery sales, “U.S. Fancy” and “U.S. No.1” roots are preferred and sold at

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premium price (http://pubs.ext.vt.edu/438/438-104/438-104.html). Thus, “U.S. Fancy” and “U.S.

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No.1” are major available grades in the market. However, there is no information whether the

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USDA grade of the horseradish root is associated not only with food quality, but also with cancer

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chemopreventive activity.

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Excess production of free radicals is associated with oxidative damage to biomolecules,

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including lipids, proteins, and DNA, eventually leading to many chronic diseases, such as

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atherosclerosis, cancer, and other degenerative diseases in humans.15 Plant-based dietary

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polyphenols and flavonoids, have been used to estimate antioxidant activity in vitro, and have

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been associated with the prevention of cancer, type 2 diabetes, and cardiovascular diseases.16-18

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However, there is little information on the polyphenol and flavonoid levels or the potential

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cancer prevention activity of horseradish roots. Since these phytochemicals and bioactivities


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have been associated with reduced disease incidence, quantitation of horseradish root polyphenol

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and antioxidant activity will be useful information.

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The objectives of this study were to determine the variation of AITC formation and

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corresponding nitrile formation from several horseradish accessions including accessions from

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the Li and Kushad study.2 Another objective of this study was to evaluate the potential cancer

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prevention properties of horseradish roots by assessing QR inducing bioactivity, as well as

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antioxidant activity, using two different commercial grades (“U.S. Fancy” vs “U.S. No. 1”) in

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four different genotypes of horseradish. As a result, significant variation of AITC and CETP

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formation was observed among genotypes and grades. To our knowledge, this is the first

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detection and measurement of CETP from horseradish. There were significant correlations

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between sinigrin and AITC as well as AITC and QR bioactivity that can be useful information

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for horseradish quality. “U.S. Fancy” grade showed significantly higher sinigrin and AITC

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concentrations than “U.S. No. 1” whereas “U.S. No. 1” showed significantly higher CETP and

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total phenolic concentrations than “U.S. Fancy”.

81 82

METHODS AND MATERIALS

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Chemicals. Benzyl GS was purchased from POS Pilot Plant Corp (Saskatoon, SK, Canada).

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DEAE Sephadex A-25 resin was purchased from GE Healthcare (Piscataway, NJ, USA). Lead

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acetate, barium acetate, AITC, benzyl isothiocyanate (BITC), 2-penethyl isothiocyanate (PEITC),

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2-phenylpropionitrile (PPNT), benzyl cyanide, alpha-minimum essential medium (α-MEM) high

87

performance liquid chromatography (HPLC) grade acetonitrile, ACS grade hexane, 2,2'-azino-

88

bis(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS), Folin-Ciocalteu, potassium persulfate,


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2,4,6-tripyridyl-s-triazine, FeCl3·6H2O, and Trolox were purchased from Sigma (St. Louis, MO,

90

USA).

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Cultivation of Horseradish. Six accessions of horseradish were selected for this study: IL196,

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IL706, IL785, IL813, IL1091, and IL1573. Horseradish accessions IL813 and IL196 previously

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showed high sinigrin content while IL1573 showed comparatively low levels.2 In addition, IL785

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and IL1091 accessions were selected as new high root sinigrin containing horseradish accessions

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as well as IL706 with low levels. These accessions are part of a germplasm collection housed at

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the Vegetable Research Farm, University of Illinois, Urbana-Champaign, IL. Vegetative

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propagation of two plants in three replications of each accession were established in a completely

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randomized block design on the Vegetable Research Farm on 4 April, 2012 from root cuttings

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(sets) approximately 35-40 cm long and 1.0-1.5 cm in diameter. Sets were hand-planted in rows

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spaced at 90 cm between rows and 56 cm within each row. The ends of the sets that would send

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up a shoot were planted facing the same direction (for ease of harvest) in furrows, then hilled-

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over into ridges approximately 18 cm high and 30 cm wide. According to the US Department of

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Agriculture standard, horseradish roots are classified “U.S. Fancy”, “U.S. No. 1”, and “U.S. No.

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2” based on diameter of horseradish and length of root.19 Specifically, in order to meet “U.S.

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Fancy” grade, the roots ‘shall be not less than 20.32 cm in length when the diameter is 3.81 cm

106

or more’. For “U.S. No. 1” grade, the roots ‘shall be not less than 15.24 cm in length when the

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diameter is 3.18 cm or more’. For “U.S. No. 2” grade, the roots shall be not less than 10.16 cm in

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length and not less than 1.27 cm in diameter’. The two primary horseradish roots harvested from

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each three field replications of each accession (n = 6) that met the “U.S. Fancy” grade standard,19

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were cleaned with tap water, and dried with paper towels. Two primary main roots from “U.S.

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Fancy” grade plants were pooled for each three field replications for four accessions and an


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additional two root samples from the same plants that met the “U.S. No. 1” requirements were

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collected as this manners. Since “U.S. No. 2” grade root is not commercially preferred, replicated

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samples were collected from only one accession that has low sinigrin concentration for better

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correlation analysis. Harvest was done on October 20th 2013. Samples were frozen in liquid

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nitrogen and stored at -20 °C prior to freeze-drying (less than a week). Freeze-dried tissues were

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ground into a fine powder using a coffee grinder and stored at -20 °C prior to analyses (less than

118

a month).

119 120

Determination of GS Concentrations. Extraction and quantitation of GS using HPLC was

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performed using a protocol described by Brown et al.20 with some modifications. Freeze-dried,

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powdered horseradish (0.2 g) and 2 mL of 70% methanol were placed in 10 mL centrifuge tubes

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(Nalgene, Rochester, NY) and heated on a heating block at 95 °C for 10 min. After cooling on

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ice for 5 min, 0.5 mL benzyl GS (1 mM) was added as internal standard (POS Pilot Plant Corp,

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Saskatoon, SK, Canada), mixed, and centrifuged at 3,000 × g for 15 min at 4 °C using a

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Beckman L7-65 Ultracentrifuge (Beckman Coulter, Brea, CA). The supernatant was saved and

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the pellet was re-extracted with 2 mL 70% methanol at 95 °C for 10 min and the two extracts

128

were combined. A subsample (1 mL) from each pooled extract was transferred into a 2 mL

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microcentrifuge tube (Fisher Scientific, Waltham, MA). Protein in the extract was precipitated

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with 0.15 mL of a 1:1 mixture of 1 M lead acetate and 1 M barium acetate. After centrifugation

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at 12,000 × g for 1 min using an Eppendorf 5415 benchtop centrifuge (Hamburg, Germany),

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each sample was then loaded onto a column containing DEAE Sephadex A-25 resin for

133

desulfation with arylsulfatase (Helix pomatia Type-1, Sigma-Aldrich, St. Louis, MO) for 18 h.

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The desulfated GS samples were eluted off the column using 2 mL distilled water. A 100 µL


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sample of each was filtered through a 0.2 µm nylon filter and injected into an HPLC system

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under the previously reported conditions.21 For individual GS quantitation (sinigrin,

137

gluconasturtiin, gluconapin glucobrassicin, and 4-methoxy glucobrassicin), relative response

138

factors was used as previously published.21 The concentration of GS was expressed as µmol/g

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dry weight (DW).

140 141

Measurement of GS Hydrolysis Products. Freeze-dried horseradish tissue (50 mg) was

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suspended in 1 mL distilled water and 1 mL hexane in a 2 mL microcentrifuge tube (Fisher

143

Scientific, Waltham, MA). Hydrolysis products were generated naturally by endogenous

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myrosinase in the absence of light at room temperature for 24 h. The samples were then

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centrifuged at 12,000 × g for 2 min using a Eppendorf 5415 benchtop centrifuge (Hamburg,

146

Germany) and the supernatant was collected for analysis using a Hewlett Packard HP 5890

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Series-I gas chromatography (GC) system equipped with a single flame ionization detector

148

(Agilent Technologies, Santa Clara, CA), an Agilent model 7683B series auto sampler, and a 30

149

m × 0.32 mm J&W HP-5 capillary column (Agilent Technologies). A 1 µL sample of the hexane

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extract was injected onto the GC. After an initial temperature hold at 40 °C for 2 min, the oven

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temperature was increased by 10 °C/min to 260 °C and held for 10 min.8 Injector and detector

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temperatures were set at 200 °C; and 280 °C, respectively. The flow rate of the helium carrier

153

gas was set at 25 mL/min. Results were quantitated based on external standards of AITC, BITC,

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PEITC, PPNT, and benzyl cyanide. 1-Cyano 2,3-epithiopropane (CETP) was quantitated based

155

on effective carbon number concept.22 Each peak from the horseradish sample extracts were

156

further identified using a 6890N GC coupled to an HP-5973N MS Detector, according to a


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previously published study.11 The concentration of GS hydrolysis products were expressed as

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µmol/g dry weight (DW). Samples were assayed in triplicate.

159 160

Quinone Reductase (QR) Activity. Freeze-dried powdered horseradish root (75 mg) was

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suspended in 1.5 mL water in the dark for 24 h at room temperature in a sealed 2 mL

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microcentrifuge tube (Fisher Scientific, Waltham, MA) to allow for GS hydrolysis by

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endogenous myrosinase. GS hydrolysis extracts were then centrifuged at 12,000 × g for 5 min

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using an Eppendorf 5415 benchtop centrifuge (Hamburg, Germany) and the supernatant was

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used for the QR assay. Hepa1c1c7 murine hepatoma cells (ATCC, Manassas, VA) were grown

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in α-MEM, enriched with 10% fetal bovine serum and maintained at 37 °C in ambient air

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enriched with 5% CO2. The cells were divided every three days with a split ratio of 7. Cells with

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80-90% confluence were plated into 96-well plates (Costar 3595, Corning Inc., Corning, NY), 1

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× 104 cells per well, and incubated for 24 h in antibiotic-enriched media (100 units/mL penicillin,

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100 µg/mL streptomycin). The QR induction activities of different samples were determined by

171

means of the protocol described by Prochaska & Santamaria.23 Samples were assayed in

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triplicate.

173 174

Estimation of Myrosinase Activity Using Glucose Appearance. Total myrosinase activity was

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measured by using the glucose release assay.24 Briefly, freeze-dried plant material (50 mg) was

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weighed in duplicate (for adjusting background glucose) into 2 mL tubes and one mL sinigrin

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(10 mM) was added to each tube. After 10 sec of vigorous vortexing, one of the paired samples

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was put directly into a heating block (95 ˚C) for 10 min to inactivate the myrosinase enzyme


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(zero time blank). The second sample was incubated at 40˚C for 30 min and then inactivated as

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outlined above. After inactivation, samples were cooled on ice for 5 min then centrifuged at

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16,000 g for 2 min using an Eppendorf 5415 benchtop centrifuge (Hamburg, Germany). The

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supernatants were diluted 96-fold with water and aliquots of 30 µL were added in a 96 well plate,

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followed by adding 200 µL of an ABTS-glucose solution (2.7 mM ABTS, 10,000 units

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peroxidase, and 4,000 units glucose oxidase). The mixtures were incubated for 20 min and their

185

absorbances were measured at 630 nm in a µQuant plate reader (Bio-Tek instruments, Winooski,

186

VT). Glucose standards were also prepared similarly, as described above, and their absorbance

187

used to develop a standard curve (7.8-250 µg/mL). One unit defined as 1 µmole glucose release

188

per min. Samples were assayed in triplicate.

189 190

Determination of Nitrile and Epithionitrile Formation from Different GSs. Freeze-dried

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horseradish root (75 mg) was suspended in 0.75 mL of 1 mM benzyl GS. After 20 s vigorous

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vortexing, 0.75 mL hexane was added and incubated for 20 min in the absence of light. The

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mixture was vigorously vortexed for 10 s then centrifuged at 16,000 g for 1 min using an

194

Eppendorf 5415 benchtop centrifuge (Hamburg, Germany). The supernatant was transferred to

195

GC vials and injected onto the GC as described above. Nitrile formation (the simple nitrile PPNT

196

from gluconasturtiin, the epithionitrile CEPT from sinigrin) is presented as percentage formed

197

relative to total hydrolysis products of individual endogenous and exogenous GS, as previously

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described.9 Samples were assayed in triplicate.

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Determination of Total Polyphenol Content (TPC). Analysis of TPC was conducted as

201

previously described.25 The extraction procedure was identical as that described above for the

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QR activity measurement. The assay conditions were as follows: a 10 µL sample was added to

203

0.2 N Folin-Ciocalteu’s phenol reagent (100 µL) in 96 well plates. After 3 min, 90 µL of 7.5%

204

sodium carbonate solution was added to the mixture and subsequently incubated at room

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temperature for 1 h. The absorbance of the mixture was measured at 630 nm using a BioTek EL

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808 microplate reader (Biotek Instruments Inc., Power Wave XS, Winooski, VT). Total

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polyphenol content was calculated on the basis of a standard curve using gallic acid

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(concentration range 31.25 to 500 µg/mL). Results are expressed in mg gallic acid equivalents

209

(GAE) per g of dried horseradish. Samples were assayed in triplicate.

210 211

Determination of ABTS Radical Scavenging Activity and Ferric Reducing/Antioxidant

212

Power (FRAP) activity. The ABTS assay was conducted as previously described.25 Briefly, 7

213

mM ABTS ammonium salt was dissolved in a 2.45 mM potassium persulfate solution. The

214

mixture was then allowed to stand at room temperature for 12-16 h for full color development

215

(dark blue). The solution was then diluted with potassium phosphate buffer (pH 7.4) until

216

absorbance reached 1.0 ± 0.02 at 630 nm using a BioTek EL 808 microplate reader (Biotek

217

Instruments Inc., Power Wave XS, Winooski, VT). Subsequently, 190 µL of this solution was

218

mixed with 10 µL of the sample. The absorbance was recorded at room temperature after 6 min.

219

Results are expressed as percent of radical scavenging activity compared to controls as extraction

220

solvent. Samples were evaluated in triplicate.

221

The FRAP reagent, freshly prepared and kept at room temperature, contained 2.5 mL of a 10

222

mM 2,4,6-tripyridyl-s-triazine solution in 40 mM HCl plus 2.5 mL of 20 mM FeCl3·6H2O and


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25 mL of 300 mM acetate buffer (pH 3.6). 25 The FRAP reagent (190 µL), was mixed with 10 µL

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of sample and absorbance was measured at 593 nm after 4 min, using a BioTek EL 808

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microplate reader. The FRAP values were calculated from a standard curve of Trolox

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concentrations ranging from 62.5-500 µmols.

227 228

Statistical Analysis. Horseradish accessions were considered a fixed factor with blocks a

229

random factor. Analysis of variance (ANOVA), Student’s T-test, paired T-test, F-test, and

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Pearson’s correlation coefficient analysis were conducted using JMP 10 (SAS institute Inc., Cary,

231

NC). All sample analyses were conducted in triplicate. The results are presented as means ±

232

standard deviation (SD). Coefficients of variation (CV) are presented in the tables.

233 234

RESULTS AND DISCUSSION

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Concentration of GS in Different Accessions and Grades of Horseradish Root. There is

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significant variation in sinigrin concentration among the “U.S. Fancy” horseradish accession

237

extracts (F5,12=110, P < 0.001); the lowest sinigrin concentration was found in accession IL706

238

extract (41.0 µmol/g dry weight) while highest one was found in IL1091 (80.3 µmol/g DW;

239

Table 1). Among accessions of “U.S. Fancy” grade, the maximum to minimum ratio is

240

approximately 2 fold, suggesting that the genotype plays a major role in determining sinigrin

241

concentration. Gluconasturtiin concentrations from six “U.S. Fancy” grade accession root

242

extracts ranged from 3.77 to 10.0 µmol/g DW. Average glucobrassicin concentrations from six

243

“U.S. Fancy” accession root extracts ranged from 0.16 to 2.07 µmol/g DW, which is a 13-fold


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difference in minimum to maximum values. Aside from these, the only other GS found was

245

gluconapin in trace amounts (less then 0.05 µmol/g DW). According to a previous study26 using

246

168 Nordic horseradish accessions, sinigrin levels varied between 10 and 45, gluconasturtiin

247

between 1.3 and 7.4 and glucobrassicin between 0.1 and 2.6 µmol/g DW. A recent paper

248

reported that sulfate supply was closely related with GS accumulation in horseradish.27 The

249

different GS concentration between US Midwest horseradish and Nordic horseradish accessions

250

may be related with different weather and/or soil conditions as well as genetic backgrounds,

251

which require further research. Another study28 detected 11 different GS from horseradish roots

252

using LC-electrospray ionization-hybrid linear ion trap with Fourier transform ion cyclotron

253

resonance mass spectrometry and infrared multiphoton dissociation but we only identified five

254

GS by HPLC as also reported in previous studies.2, 26

255

Horseradish root diameter (grades) was another source of variation in sinigrin concentration.

256

Partitioning variance component analysis based on ANOVA estimated that differences among

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root diameters accounted for 44% of sinigrin variation displayed in Table 1. Selected accessions

258

of horseradish of “U.S. Fancy” grade (IL196, IL1573, IL813, and IL785) contained an average

259

of 60.9 µmol/g DW of sinigrin while corresponding “U.S. No. 1” grade samples contained 38.1

260

µmol/g DW of sinigrin. Across the 4 accessions tested, the “U.S. Fancy” grade root extracts

261

exhibited significantly greater sinigrin concentrations than the “U.S. No. 1” grade roots extracts

262

by paired T-test (P = 0.003; Table 1). Previous research also reported that the total amount of GS

263

as well as sinigrin was higher in the shoulder zone than in the middle and tip zones of

264

horseradish roots.26 4-Methoxyglucobrassicin was not present in the “U.S. Fancy” grade, while

265

the “U.S. No. 1” and “U.S. No. 2” grade root extracts had measurable concentrations of this

266

compound (0.24-2.21 µmol/g DW; data not shown).


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Variation in Content of GS Hydrolysis Products among Horseradish Accessions and

269

Grades. Generation of isothiocyanate (AITC) or epithionitrile (CETP) concentrations from

270

sinigrin varied among the horseradish accessions. Average of AITC concentration from the six

271

“U.S. Fancy” grade horseradish root extracts was 42.4 µmol/g DW and ranged from 37.4 to 47.1

272

µmol/g DW. Even though sinigrin showed about 2-fold ratio between maximum to minimum

273

concentrations, the variation in AITC (CV = 9.2%) was smaller than that of its precursor GS (CV

274

= 23.2%). The reduced variation in AITC may be related its volatility. Average CETP

275

concentration from root extracts of the six “U.S. Fancy” grade horseradish accessions was 3.73

276

µmol/g DW and ranged from 0 to 11.4 µmol/g DW. PEITC showed significant variation with a

277

3.4-fold difference between minimum (3.9 µmol/g DW) to maximum (13.4 µmol/g DW) values

278

(F5,12 = 135, P < 0.001). Regardless of genotypes, PPNT concentrations were less than 1.0

279

µmol/g DW. Allyl cyanide was not detected because of its extreme volatility and loss during

280

sample preparation.12

281 282

Horseradish root diameter (grade) was another source of variation in AITC and CETP

283

concentrations. Selected horseradish accessions of “U.S. Fancy” grade (IL196, IL1573, IL813,

284

and IL785) contained 43.0 µmol/g DW of AITC while corresponding “U.S. No. 1” grade roots

285

generated only 19.5 µmol/g DW of AITC which was significantly lower than “U.S. Fancy”

286

based on the paired T-test (P = 0.013; Table 2). These same accessions of “U.S. Fancy” grade

287

contained 2.75 µmol/g DW of CETP while their corresponding “U.S. No. 1” grade roots

288

contained significantly greater amounts averaging 6.95 µmol/g DW (P = 0.022; Table 2). In

289

general, AITC levels fell and CETP levels rose in the lower root grades including “U.S. No. 1”


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290

and “U.S. No. 2”, compared to the “U.S. Fancy” graded roots. PEITC and PPNT concentrations

291

were not significantly different between “U.S. Fancy” and “U.S. No. 1” grade although PPNT

292

levels rose in the lower grades (Table 2, Figure 1). Horseradish accession IL785 is good example

293

how phytochemical composition changes depending on the grade. Past reports suggest that

294

horseradish roots contain about 79% AITC and 18% PEITC, whereas wasabi, another GS-rich

295

plant root contains 95% AITC.29 Thus, the PEITC in horseradish provides a distinct flavor

296

difference compared to wasabi.29 Most horseradish roots produced AITC as the major hydrolysis

297

product (Table 2). However, “U.S. No. 1” grade roots of accession IL813 produced more CETP

298

and PPNT than AITC and PEITC (Table 2). The “U.S. Fancy” grade root of accession IL785

299

produced a greater proportion of AITC than “U.S. No. 1” or “U.S. No. 2” grades (Table 2, Figure

300

1). It has been reported that horseradish produces only AITC and no CETP due to a lack of ESP

301

in horseradish.11, 12 However, this study reports on the presence of CETP (the epithionitrile form

302

of sinigrin) in certain horseradish accessions, suggesting that there may be ESP activity in

303

horseradish or the activity of some other co-factors including ascorbic acid and metal ions to

304

form nitriles.7 Reduction in CETP levels as root grade improves suggests that less ESP or other

305

myrosinase co-factors may be present in larger, more mature roots.

306 307

According to previous research, AITC has about a 300-fold stronger odor than CETP.30 AITC-

308

rich horseradish is therefore suitable material for the food industry; for example high quality

309

horseradish sauce. In addition to the flavor of AITC, the compound has potential cancer

310

prevention bioactivity, which can be quantitated by its capacity for the induction of QR. In

311

contrast CETP has comparatively reduced QR inducting activity.5, 31 AITC may be a good dietary

312

anticarcinogen, because bioavailability of AITC is extremely high, with nearly 90% absorption


15


Page 16 of 36

313

of an oral dose.14 In addition AITC, even at high doses, exhibits a relatively low degree of

314

cytotoxicity and genotoxicity in animal studies.14 Significant variation in AITC levels across the

315

accessions was observed within the “U.S. No. 1” grade samples (F3,8 = 71.0, P < 0.001; Table 2):

316

“U.S. No. 1” grade IL785 accession has about 9 fold higher AITC concentration than IL813

317

accession. These chemical analyses indicate that the USDA grading system is a good indicator

318

for pungency.

319

Averaged AITC and PEITC concentrations determined that the genotype and root grade

320

described 66% and 17% of the total variation (calculated from the Table 2), respectively which is

321

comparable to a previous study that reported these variables accounted for 64-82% and 4-18% of

322

the variation.32 However, this study reported a broader range of AITC and PEITC concentrations.

323 324

Quinone Reductase (QR), Myrosinase, and Nitrile/Epithionitrile Formation (%) of

325

Different Accessions and Grades of Horseradish. The QR inducing activity of the “U.S. Fancy”

326

grade horseradish did not correlate with the variation in phytochemical concentrations (Table 3).

327

The mean value of QR inducing activity from extracts of all six of the “U.S. Fancy” grades

328

evaluated (expressed as induction ratio relative to the control) was 1.33 ± 0.04. QR inducing

329

activity ranged from 1.29 to 1.37, which was small compared to the variation in sinigrin and

330

AITC. Myrosinase activity of “U.S. Fancy” horseradish variation ranged from 0.25 to 1.72 U/g.

331

Nitrile generation from sinigrin (0.0-20.2%) was greater than gluconasturtiin (1.3-7.3%) and

332

benzyl GS (0.4-0.9%). There was no epithionitrile formation in accessions IL706 and IL1573.

333

These accessions could be good resources for a high AITC producing horseradish-breeding

334

program.

335


16

Page 17 of 36


336

QR inducing activity of horseradish roots varied among grades. In general, QR inducing activity

337

in the “U.S. Fancy” grade roots of accessions IL196, IL1573, IL813, and IL785 (Mean = 1.34,

338

Table 3) were not significantly higher than those from “U.S. No. 1” grade roots of the same

339

accessions (Mean = 1.19, P = 0.129, Table 3). However, “U.S. Fancy” grade roots of accessions

340

IL1573 and IL813 have significantly higher QR inducing activity than those from “U.S. No. 1”

341

grade roots of the same accessions, suggesting more further research is needed to confirm the

342

interaction between accessions and grades. QR inducing activity of “U.S. No. 1” grade roots

343

ranged from 1.05 to 1.33. Myrosinase activity in “U.S. Fancy” and “U.S. No. 1” grade roots were

344

similar (0.68 U/g, n = 4 and 0.57 U/g, n = 4 respectively, Table 3) and not significantly different.

345

Average of percent epithionitrile formation (expressed as percent nitriles formed over total

346

hydrolysis products) for endogenous sinigrin from the selected four “U.S. Fancy” grade

347

accessions was 5.7% (mean of all “U.S. Fancy” grade roots = 7.1%), whereas the corresponding

348

percent epithionitrile formation across the “U.S. No. 1” grade root samples was 32.9%. Thus

349

“U.S. No. 1” grade horseradish roots had about 4.6-fold higher percent epithionitrile formation

350

than the “U.S. Fancy” grade horseradish.

351

Average percent nitrile formation relative to endogenous gluconasturtiin from the selected four

352

“U.S. Fancy” grade horseradish accessions was 2.6% (mean of all “U.S. Fancy” = 3.2%),

353

whereas corresponding percent nitrile formation across the “U.S. No. 1” grade samples was 19.3%

354

(T-test P = 0.243) (Table 3). “U.S. No. 1” grade horseradish had about a 7-fold higher nitrile

355

formation than the “U.S. Fancy” grade (Table 3). Average percent nitrile formation for

356

exogenous benzyglucosinolate from selected four “U.S. Fancy” grade horseradish accessions

357

was 0.50% (mean of all “U.S. Fancy” = 0.55%), less than the percent nitrile formation across

358

“U.S. No. 1” grade samples of 1.60% (T-test P = 0.324) (Table 3). Thus “U.S. No. 1” grade


17


Page 18 of 36

359

horseradish had about a 3-fold greater percent nitrile formation than the “U.S. Fancy” grade

360

horseradish. The low percent nitrile formation from benzyl GS compared to two other substrates

361

has previously been reported.9 This indicates that ESP or other myrosinase co-factor activity may

362

be different for different GS and/or for endogenous and exogenous GS. The variation of

363

nitrile/epithionitrile formation observed here for different accessions and grades of horseradish

364

root could be due to differences in ESP abundance or different levels of co-factors such as

365

ferrous or magnesium metal ions or other protein co-factors.9

366 367

Total Phenolic Content and Antioxidant Activity in Different Accessions and Grades of

368

Horseradish. Total phenolic content of horseradish roots varied among accessions and grades.

369

Total phenolics present in “U.S. Fancy” grade horseradish ranged from 2.83 to 5.48 mg/g DW

370

(Table 4). ABTS and FRAP antioxidant activities showed significant variations in “U.S. Fancy”

371

grade horseradish ranged from 19.7 to 31.4 mmol/g DW (F5,12 = 145) and from 20.5 to 34.1

372

mmol/g DW (F5,12 = 83.3), respectively (Table 4).

373 374

Within the same accession, “U.S. No.1” grade horseradish roots have higher concentrations (4.63

375

mg/g DW) of total phenolics compared to the “U.S. Fancy” grade roots (3.68 mg/g DW),

376

implying that total phenolic accumulation may be related to diameter and length of horseradish

377

roots. Smaller diameter roots have more total phenolics (and less sinigrin and total GS, as

378

showed in Table 1) than thicker roots but the trend was not significant in a paired T-test (P =

379

0.073, Table 4).

380


18

Page 19 of 36


381

Correlations between Phytochemicals and Bioactivities in Different Accession and Grades

382

of Horseradish Roots. As expected, there were significant correlations between sinigrin and

383

AITC (r = 0.834, P = 0.001) and between gluconasturtiin and PEITC (r = 0.830, P = 0.002;

384

Table 5). Similarly, AITC also significantly correlated to QR activity (r = 0.727, P = 0.011;

385

Table 5). It is reported that AITC is relatively volatile and has low solubility in water (1.3

386

mg/mL) compared to sulforaphane (8.0 mg/mL).33 Although AITC was successfully extracted by

387

adding hexane in water and measured by GC, there would be some AITC loss after treatments in

388

cell media and incubation at 37˚C for 24 h for the QR assay. This probably attributed to the

389

relatively small QR inducing difference between “U.S. Fancy” and “U.S. No. 1” even though

390

there was a significant AITC concentration difference between “U.S. Fancy” and “U.S. No. 1”

391

(Table 2 and 3). AITC has high bioavailability when orally consumed.14 Considering the

392

significant correlation between AITC and QR activity, the QR activity difference between “U.S.

393

Fancy” and “U.S. No. 1” maybe underestimated due to the volatility of active compounds and

394

attributes of the QR assay system. This also indicates that a reliable QR assay system needs to be

395

be established for volatile compounds. Similarly, PEITC has been reported to induce QR

396

activity34 and was detected in our study when measured immediately after hydrolysis, the lack of

397

correlation between PEITC and QR may also have been due to evaporation of this volatile

398

compound (solubility in water: 0.051 mg/mL),33 during the 24 h cell treatment. Previous studies

399

have reported that PEITC was not detected following hydrolysis of watercress and broccoli.34, 35

400

Interestingly, QR activity was negatively correlated with concentrations of two different nitriles

401

(sinigrin-dependent epithionitrile formation: r = -0.682, P = 0.021; and gluconasturtiin-

402

dependent simple nitrile formation: r = -0.742, P = 0.009). AITC was also significantly

403

negatively correlated with epithionitrile formation (from sinigrin) and simple nitrile formation


19


Page 20 of 36

404

(from gluconasturtiin, Table 5). Our data show that higher grades of horseradish accumulate

405

more sinigrin and tend to have lower ESP or other myrosinase co-factor activity than lower

406

grades (Table 1 and 3), which may explain why epithionitrile formation correlates negatively

407

with AITC concentrations.

408

There were significant and positive correlations between TPC and FRAP (r = 0.750, P = 0.008)

409

as well as between FRAP and ABTS (r = 0.974, P < 0.001). These correlations are explained by

410

the fact that these two different antioxidant assays and TPC measurement are based on the

411

electron-transfer reaction mechanism.25,

412

event should result in correlations, otherwise one of the methods is not valid. There were

413

significant correlations between TPC and epithionitrile formation (from singirin; r = 0.682, P =

414

0.021) as well as between TPC and AITC (r = -0.689, P = 0.019). This relationship between TPC

415

and epithionitrile formation requires further investigation to determine if TPC impacts on GS

416

hydrolysis.

36

Thus, three different methods measuring the same

417

In conclusion, this study has examined for the first time the variation in health promoting

418

properties of different accessions and grades of horseradish roots, measured as induction of QR,

419

as well as estimates of AITC and nitrile formation. “U.S. Fancy” showed significantly higher

420

sinigrin and AITC concentrations than “U.S. No. 1” whereas “U.S. No. 1” showed significantly

421

higher CETP, the epithionitrile hydrolysis product of sinigrin, and total phenolic concentrations

422

than “U.S. Fancy”. The present results suggested that higher grades of horseradish roots display

423

better quality, with higher sinigrin and AITC that is responsible for chemopreventive bioactivity.

424 425 426


20

Page 21 of 36


427

Abbreviations Used

428

GS glucosinolate

429

AITC allyl isothiocyanate

430

PEITC phenethyl isothiocyanate

431

ESP epithionitrile specifier protein

432

BZITC benzyl isothiocyanate

433

CETP 1-cyano 2,3-epithiopropane

434

PPNT 2-phenylpropionitrile

435

TPC total phenolic content

436

ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

437

FRAP ferric reducing/antioxidant power


21


Page 22 of 36

438

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Li, X.; Kushad, M. M., Correlation of glucosinolate content to myrosinase activity in

Agrawal, A.; Kurashige, N., A role for isothiocyanates in plant resistance against the

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Munday, C. M., Selective induction of phase II enzymes in the urinary bladder of rats by

Zhang, Y., Allyl isothiocyanate as a cancer chemopreventive phytochemical. Mol. Nutr.

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sinensis L.). J. Agric. Food Chem. 2010, 58, 418-26.

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Scherb.). J. Sci. Food Agric. 2013, 93, 918-923.

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Ku, K. M.; Jeffery, E. H.; Juvik, J. A., Exogenous methyl jasmonate treatment increases

Scanlon, J. T.; Willis, D. E., Calculation of flame ionization detector relative response

Prochaska, H. J.; Santamaria, A. B., Direct measurement of NAD(P)H:quinone reductase

Dosz, E. B.; Ku, K. M.; Juvik, J. A.; Jeffery, E. H., Total myrosinase activity estimates in

Ku, K. M.; Choi, J. N.; Kim, J.; Kim, J. K.; Yoo, L. G.; Lee, S. J.; Hong, Y. S.; Lee, C.

Wedelsbäck Bladh, K.; Olsson Kerstin, M.; Yndgaard, F., Evaluation of glucosinolates in

Alnsour, M.; Kleinwächter, M.; Böhme, J.; Selmar, D., Sulfate determines the


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of glucosinolate profile and qualitative aspects in sprouts and roots of horseradish (Armoracia

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rusticana) using LC-ESI-hybrid linear ion trap with Fourier transform ion cyclotron resonance

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mass spectrometry and infrared multiphoton dissociation. J. Agric. Food Chem. 2012, 60, 7474-

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compounds in wasabi and horseradish. J. Food Agric. Environ. 2003, 1, 117-121.

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hydrolysis products in fresh cabbage. J. Food Sci. 1996, 61, 101-104.

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M.; Hayes, J. D., 1-Cyano-2,3-epithiopropane is a novel plant-derived chemopreventive agent

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which induces cytoprotective genes that afford resistance against the genotoxic α,β-unsaturated

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aldehyde acrolein. Carcinogenesis. 2009, 30, 1754-1762.

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of horseradish roots (Armoracia rusticana L.) depending on the genotype. In Proceedings of the

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Latvia University of Agriculture, 2013; Vol. 29, p 1.

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determination of isothiocyanates using high-temperature reversed-phase HPLC. J. Separation Sci.

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2012, 35, 2026-2031.

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34.

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methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes.

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Carcinogenesis 2000, 21, 1983-8.

Agneta, R.; Rivelli, A. R.; Ventrella, E.; Lelario, F.; Sarli, G.; Bufo, S. A., Investigation

Sultana, T.; Savage, G.; McNeil, D.; Porter, N.; Clark, B., Comparison of flavour

Chin, H. W.; Zeng, Q.; Lindsay, R. C., Occurrence and flavor properties of sinigrin

Kelleher, M. O.; McMahon, M.; Eggleston, I. M.; Dixon, M. J.; Taguchi, K.; Yamamoto,

Tomsone, L.; Kruma, Z.; Galoburda, R.; Talou, T., Composition of volatile compounds

Wilson, E. A.; Ennahar, S.; Marchioni, E.; Bergaentzlé, M.; Bindler, F., Improvement in

Rose, P.; Faulkner, K.; Williamson, G.; Mithen, R., 7-Methylsulfinylheptyl and 8-

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527

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jasmonate mediated induction of glucosinolate biosynthesis on quinone reductase activity in

529

broccoli florets. J. Agric. Food Chem. 2013, 61, 9623-31.

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36.

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Agric. Food Chem. 2005, 53, 1841-56.

Ku, K. M.; Jeffery, E. H.; Juvik, J. A., Influence of seasonal variation and methyl

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26

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532

Figure captions

533

Figure 1. Gas chromatograms of different USDA grades of horseradish accession IL785: “U.S.

534

Fancy” (A), “U.S. No. 1” (B), and “U.S. No. 2” (C). 1. AITC, 2. CETP, 3. BZCN, 4. PPNT, 5.

535

BZITC, and 6. PEITC.


27


Page 28 of 36

Table 1. Glucosinolate concentration (µmol/g DW) of different accessions and USDA grades of horseradish roots. Accession IL706 IL1091 IL196 IL196 IL1573 IL1573 IL813 IL813 IL785 IL785 IL785 Mean Mean Mean

Grade Sinigrin “U.S. Fancy” 41.0 ± 1.2 “U.S. Fancy” 80.3 ± 3.2 “U.S. Fancy”b 60.9 ± 2.1 “U.S. No. 1” 44.9 ± 4.5 “U.S. Fancy”b 55.1 ± 2.4 “U.S. No. 1” 32.4 ± 2.1 “U.S. Fancy”b 54.4 ± 2.3 “U.S. No. 1” 26.1 ± 2.2 73.2 ± 3.2 “U.S. Fancy”b “U.S. No. 1” 48.9 ± 4.1 “U.S. No. 2” 42.0 ± 3.2 “U.S. Fancy” (n=6) 60.8 ± 14.1 b “U.S. Fancy” marked (n=4) 60.9 ± 10.6 “U.S. No. 1” (n=4) 38.1 ± 8.7 b “U.S. Fancy” 0.003c P value vs “U.S. No. 1” F5,12d ; CV “U.S. Fancy” (n=6) 110**; 23.2e F3,8 ; CV “U.S. Fancy”b marked (n=4) 39.4**; 17.4 F3,8 ; CV “U.S. No. 1” (n=4) 32.5**; 22.8 a includes gluconapin and 4-methoxyglucobrassicin.

Gluconasturtiin 4.81 ± 0.26 5.82 ± 0.14 10.0 ± 0.24 14.5 ± 0.20 9.42 ± 0.25 9.33 ± 0.05 9.91 ± 0.04 5.35 ± 0.06 3.77 ± 0.07 9.56 ± 0.24 10.3 ± 0.33 7.91 ± 2.81 8.28 ± 3.01 9.67 ± 3.75

Glucobrassicin 0.52 ± 0.04 1.31 ± 0.11 0.41 ± 0.07 0.27 ± 0.05 0.16 ± 0.06 0.00 ± 0.00 0.31 ± 0.06 0.00 ± 0.00 2.07 ± 0.10 0.32 ± 0.05 0.25 ± 0.05 0.80 ± 0.74 0.74 ± 0.89 0.15 ± 0.17

Totala 46.4 ± 1.4 87.5 ± 3.6 71.4 ± 2.4 61.9 ± 4.8 64.7 ± 2.7 42.4 ± 2.4 64.6 ± 2.4 31.8 ± 2.4 79.1 ± 3.5 59.1 ± 4.2 53.5 ± 3.4 70.0 ± 14.1 70.0 ± 6.9 48.8 ± 14.2

0.591c

0.464c

0.021c

662**; 35.5 969**; 36.4 1834**; 38.8

652**; 92.5 1062**; 120 1836**; 113

103**; 20.1 355**; 9.9 55.5**; 29.1

b

indicates selected “U.S. Fancy” accession for comparing with corresponding “U.S. No. 1”.

c

was calculated by paired T-test. All data are expressed as mean ± SD, n=3.

d

indicates degree of freedom of numerator and denominator for F-test.

e

indicates coefficient of variation (%).

**

indicates highly significant variance by F-test at ≤ 0.01.


28

Page 29 of 36


Table 2. Hydrolysis products of GS from different genotypes and USDA grades of horseradish.

Accession IL706 IL1091 IL196 IL196 IL1573 IL1573 IL813 IL813 IL785 IL785 IL785 Mean Mean Mean

Sinigrin hydrolysis (µmol/g DW) AITC CETP 37.4 ± 5.3 0.00 ± 0.00 44.8 ± 3.1 11.4 ± 0.19 44.7 ± 4.4 5.8 ± 0.22 18.5 ± 2.1 10.3 ± 0.30 38.2 ± 1.9 0.00 ± 0.00 20.7 ± 3.0 2.8 ± 0.20 41.9 ± 6.5 3.3 ± 0.28 3.9 ± 0.1 10.1 ± 0.37 47.1 ± 6.2 1.09 ± 0.33 34.8 ± 3.3 4.6 ± 0.22 14.8 ± 1.8 10.0 ± 0.35 42.4 ± 3.9 3.73 ± 4.35 43.0 ± 12.7 2.75 ± 2.44 19.5 ± 3.8 6.95 ± 3.82

Gluconasturtiin hydrolysis (µmol/g DW) PEITC PPNT 4.30 ± 0.48 0.10 ± 0.18 4.30 ± 0.28 0.40 ± 0.18 13.4 ± 0.64 0.70 ± 0.04 10.9 ± 0.68 2.30 ± 0.08 7.30 ± 0.53 0.10 ± 0.13 8.80 ± 0.55 0.50 ± 0.13 7.70 ± 0.84 0.40 ± 0.24 3.50 ± 0.57 5.20 ± 0.17 3.90 ± 0.20 0.10 ± 0.10 7.70 ± 0.60 0.40 ± 0.20 8.10 ± 0.46 2.10 ± 0.16 6.82 ± 3.62 0.30 ± 0.26 8.10 ± 3.11 0.32 ± 0.31 7.73 ± 3.94 2.09 ± 2.22

Grade “U.S. Fancy” “U.S. Fancy” “U.S. Fancy”a “U.S. No. 1” “U.S. Fancy”a “U.S. No. 1” “U.S. Fancy”a “U.S. No. 1” “U.S. Fancy”a “U.S. No. 1” “U.S. No. 2” “U.S. Fancy” “U.S. Fancy”a marked “U.S. No. 1” (n=4) “U.S. Fancy”a P value vs “U.S. No. 1” 0.013b 0.022b 0.860b F5,12c ; CV “U.S. Fancy” (n=6) 5.53NS; 9.2d 280**; 117 135**; 53.1 b * ** F3,8 ; CV “U.S. Fancy” marked (n=4) 7.0 ; 29.5 875 ; 88.7 648**; 38.4 F3,8 ; CV “U.S. No. 1” (n=4) 71.0**; 19.5 334**; 55.0 80.2**; 51.0 a indicates selected “U.S. Fancy” accession for comparing corresponding “U.S. No. 1”. b


c


d


NS, *,

0.192b 0.919NS; 86.7 2.2**; 96.9 665**; 106

and ** indicate non-significant, significant, and highly significant variance by F-test at > 0.05, ≤ 0.05, and ≤ 0.01, respectively.


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Table 3. Quinone reductase (QR) inducing activity, myrosinase activity, and ESP activities on individual GS of different genotype and USDA grades of horseradish. Accession

Grade

QR

Myrosinase (U/g)

“U.S. Fancy” 1.37 ± 0.10a 0.25 ± 0.18b “U.S. Fancy” 1.29 ± 0.09 1.72 ± 0.18 e “U.S. Fancy” 1.35 ± 0.05 0.55 ± 0.04 “U.S. No. 1” 1.33 ± 0.03 0.53 ± 0.08 “U.S. Fancy”e 1.32 ± 0.04 0.51 ± 0.13 “U.S. No. 1” 1.14 ± 0.03 0.16 ± 0.13 “U.S. Fancy”e 1.37 ± 0.08 1.13 ± 0.24 “U.S. No. 1” 1.05 ± 0.07 0.82 ± 0.17 “U.S. Fancy”e 1.30 ± 0.06 0.53 ± 0.10 “U.S. No. 1” 1.25 ± 0.02 0.75 ± 0.20 “U.S. No. 2” 1.26 ± 0.05 0.88 ± 0.16 “U.S. Fancy” 1.33 ± 0.04 0.78 ± 0.54 “U.S. Fancy”e marked 1.34 ± 0.03 0.68 ± 0.30 “U.S. No. 1” (n=4) 1.19 ± 0.12 0.57 ± 0.30 “U.S. Fancy”e P value 0.453f vs “U.S. No. 1” 0.129f F5,12g ; CV “U.S. Fancy” (n=6) 0.685NS; 3.0h 35.2**; 69.2 b F3,8 ; CV “U.S. Fancy” marked (n=4) 9.94**; 2.2 38.5**; 44.1 F3,8 ; CV “U.S. No. 1” (n=4) 25.5**; 10.1 11.5**; 52.6 a indicates sample extract activity divided by the non-treated control cells. IL706 IL1091 IL196 IL196 IL1573 IL1573 IL813 IL813 IL785 IL785 IL785 Mean Mean Mean

Sinigrin Gluconasturtiin Benzyl GS (% of nitrile formation to individual GS ) 0.0 ± 0.0c 1.4 ± 0.4c 0.40 ± 0.10d 20.2 ± 1.1 7.3 ± 0.4 0.90 ± 0.10 11.5 ± 2.1 4.2 ± 0.9 0.70 ± 0.10 35.8 ± 3.2 14.3 ± 1.0 0.90 ± 0.10 0.0 ± 0.0 1.4 ± 0.2 0.40 ± 0.10 11.9 ± 1.7 4.6 ± 0.5 0.50 ± 0.20 7.3 ± 1.8 3.6 ± 0.8 0.50 ± 0.20 71.9 ± 1.0 54.2 ± 1.3 4.40 ± 0.20 3.8 ± 1.4 1.3 ± 0.4 0.40 ± 0.10 11.8 ± 2.1 3.9 ± 0.7 0.60 ± 0.10 40.3 ± 2.5 17.4 ± 1.1 1.80 ± 0.10 7.1 ± 7.8 3.2 ± 2.4 0.55 ± 0.21 5.7 ± 4.9 2.6 ± 1.5 0.50 ± 0.14 32.9 ± 28.4 19.3 ± 23.8 1.60 ± 1.87 0.126f 100**; 110 83.8**; 86.0 521**; 86.3

0.243f 51.3**; 75.0 61.2**; 57.7 5088**; 123

0.324f 8.61**; 38.2 24.0**; 28.0 421**; 117

b

unit defined as 1 µmole glucose release per min. Analysis was done in triplicates.

c

the amount of products are expressed as a percentage of nitrile form over the total amount of hydrolysis products measured [CETP

/(AITC+CETP) and PPNT/(PEITC+PPNT), respectively].


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d

exogenous benzyl glucosinolate was used for determination of nitrile formation (%) [benzyl cyanide/(benzyl cyanide + benzyl

isothiocyanate). e

indicates selected “U.S. Fancy” accession for comparing corresponding “U.S. No. 1”.

f


g


h


NS

and ** indicate non-significant and highly significant variance by F-test at > 0.05 and ≤ 0.01, respectively


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Table 4. Total phenolics content, ABTS, and FRAP antioxidant activity from different genotype and USDA grades of horseradish. Accession IL706 IL1091 IL196 IL196 IL1573 IL1573 IL813 IL813 IL785 IL785 IL785 Mean Mean Mean

Grade Total phenolics (mg/g) “U.S. Fancy” 2.83 ± 0.10a “U.S. Fancy” 4.46 ± 0.15 e “U.S. Fancy” 3.87 ± 0.09 “U.S. No. 1” 5.48 ± 0.09 e “U.S. Fancy” 3.80 ± 0.12 “U.S. No. 1” 4.75 ± 0.07 e 3.25 ± 0.29 “U.S. Fancy” “U.S. No. 1” 4.65 ± 0.11 e “U.S. Fancy” 3.66 ± 0.21 “U.S. No. 1” 3.64 ± 0.05 “U.S. No. 2” 5.20 ± 0.13 “U.S. Fancy” 3.67 ± 0.58 e “U.S. Fancy” marked 3.68 ± 0.26 “U.S. No. 1” (n=4) 4.63 ± 0.79 e “U.S. Fancy” P value vs “U.S. No. 1” 0.073d F5,12e ; CV 30.6**; 15.8f “U.S. Fancy” (n=6) F3,8 ; CV 28.1**; 7.1 “U.S. Fancy”b marked (n=4) F3,8 ; CV 248**; 17.1 “U.S. No. 1” (n=4) a the value expressed as gallic acid equivalent concentration.

ABTS (mmol/g) 19.7 ± 0.8b 31.4 ± 0.8 28.1 ± 0.2 30.1 ± 0.4 24.1 ± 0.7 24.3 ± 0.3 20.6 ± 0.7 32.0 ± 0.5 22.4 ± 0.5 20.5 ± 0.8 21.6 ± 0.4 24.4 ± 4.6 23.8 ± 3.3 26.7 ± 5.3

FRAP (mmol/g) 20.5 ± 0.5b 34.1 ± 0.6 30.3 ± 1.3 34.9 ± 1.3 26.8 ± 1.6 28.2 ± 0.4 22.5 ± 0.7 35.3 ± 0.1 25.2 ± 0.3 24.1 ± 0.4 26.3 ± 0.6 26.6 ± 5.0 26.2 ± 3.3 30.6 ± 5.4

0.392 145**; 18.9 551**; 13.9 294**; 19.9

0.240 83.3**; 18.8 93.0**; 12.6 175**; 17.6

b

the value expressed as Trolox equivalent concentration.

c

indicates selected “U.S. Fancy” accession for comparing corresponding “U.S. No. 1”.

d



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e


f


**

indicates highly significant variance by F-test at ≤ 0.01.


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Table 5. Correlations between phytochemicals and bioactivities of different genotype and USDA grades of horseradish. 2 -0.224

3 0.834 -0.255

4 -0.069 0.830 0.009

5 0.511 -0.111 0.180 -0.265

6 0.569 0.211 0.727 0.292 0.051

7 -0.110 -0.184 -0.224 -0.120 -0.081 -0.426

8 -0.177 -0.086 -0.106 0.057 -0.398 -0.121 0.974

9 -0.258 0.474 -0.689 0.269 0.117 -0.466 0.596 0.750

10 -0.495 0.076 -0.836 -0.120 0.240 -0.682 0.155 -0.137 0.682

11 1 Sinigrin -0.546 2 Gluconasturtiin -0.116 3 AITC -0.792 4 PEITC -0.272 5 Myrosinase 0.161 6 QR -0.742 7 ABTS 0.101 8 FRAP -0.178 9 TPC 0.480 10 Epithionitrile formation (Sinigrin) 0.952 11 Nitrile formation (Gluconasturtiin) 1.000 The numbers in the first row from 2 to 11 correspond to the phytochemicals and bioactivities listed in the left column. Pearson’s correlation was calculated based on P = 0.05. Bold values are significantly correlated at P = 0.05.


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Figure 1.

counts

A

solvent 1

80000

5 60000 40000

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20000

2 0 counts

5

B

3

10

15

20

25

30

35

min

25

30

35

min

25

30

35

min

solvent 1

80000

5

60000

6

40000 20000

2 0

counts

5

C

3

10

4 15

20

solvent

80000

5

60000

1

6

40000

2

20000

3 0

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TOC graphic


36

Correlation of Quinone Reductase Activity and Allyl Isothiocyanate

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