Anti Cancer Potential of Brassicas

 

Anti-cancer Potential of Asian Brassicas

Glucosinolates & Chemoprevention

 

  

 

A report for the Rural Industries Research and Development Corporation

 

by Tim O'Hare, Lesleigh Force, Lung Wong and Donald Irving

 

  

 

 

December 2006


RIRDC Publication No 05/…
RIRDC Project No DAQ-307A

 

 

© 2005  Rural Industries Research and Development Corporation.

All rights reserved.  

  

ISBN 0 642 (…RIRDC to assign)
ISSN 1440-6845

 

Anti-cancer Potential of Asian Brassicas - Glucosinolates and Chemoprevention

Publication No. 05/

Project No. DAQ307A 

The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document.

 

This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.  

 

Researcher Contact Details

Dr Tim O'Hare

DPI&F, Gatton Research Station

LMB 7, MS437, Gatton Qld 4343

07-5466 2257

07-54662208

 

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.

 

RIRDC Contact Details

Rural Industries Research and Development Corporation
Level 1, AMA House
42 Macquarie Street
BARTON   ACT   2600

PO Box 4776 
KINGSTON   ACT   2604                   

 

Phone:   02 6272 4819
Fax:         02 6272 5877
Email:    rirdc@rirdc.gov.au.
Website:                http://www.rirdc.gov.au

 

Published in  2006
 

Foreword

 

Consumption of vegetables belonging to the Brassica family has been associated with a decreased incidence of various cancers, particularly colorectal cancer.  This has been attributed, at least partly, to the glucosinolates which they contain.  Asian vegetables contribute a large number of horticultural species to this family, and along with western vegetables such as broccoli, have potential to provide anti-cancer benefit. 

 

The present project characterised the glucosinolate profiles of a wide range of Asian vegetables, as well as defining at what stage of growth these glucosinolates were present in highest amount.  It became apparent early on that sprouted seed provided the most potent food-source.

 

With this information, it was possible to provide a theoretical estimate of the anti-cancer potential for each vegetable, whether as a sprout or mature. In conjunction with this, the project included trials on the growing conditions of the most promising sprouting species, as well as postharvest stability of glucosinolates once sprouts are placed in refrigeration. Additionally, industry feedback was presented in regard to other issues likely to affect the growing and marketing of sprouts with anti-cancer potential, and which would ultimately impact on the success of a 'new' product. 

 

Finally, the report has provided relevant advice in the area of addressing regulatory criteria associated with health claims for food.  This area is currently under change, and the report presents the most up to date advice available at the time of publishing.

 

This project was funded from RIRDC Core Funds which are provided by the Australian Government and from HAL (from industry revenue which is matched by funds provided by the Australian Government).

 

This report, an addition to RIRDC's diverse range of over 1200 research publications, forms part of our Asian Foods R&D program, which aims to foster the development of a viable Asian Foods industry in Australia.

 

        purchases atwww.rirdc.gov.au/eshop

 

Peter O'Brien

Managing Director

Rural Industries Research and Development Corporation

 

Acknowledgments

 

This project was operated in collaboration with industry partners 'Green Sprouts' (Sprout Farms Pty Ltd), 'Parilla Fresh' (Parilla Holdings Pty Ltd), and Opti-Grow Pty Ltd.  Wasabi seeds, sprouts and stems were supplied as gifts by Mr Stephen Welsh and Ms Angela Sparrow (Tasmania).

 

HPLC technical assistance was provided by Mr Graham Kerven and Ms Kath Raymont at the University of Queensland.

 

Contents

 

 

Foreword                                                                                                                                 iv

Acknowledgements                                                                                                                     v

Executive Summary                                                                                                                   viii

 

Introduction                                                                                                                             1

Objectives                                                                                                                                2

Methodology

              Approach to determining anti-cancer potential                                                                3

              Other factors of importance                                                                                          3

 

1.     Chapter One - Glucosinolate measurements

         Development of HPLC method for identification of glucosinolates                                         5

         Optimisation of glucosinolate extraction method                                                                 6

 

2.     Chapter Two - Glucosinolate composition of seeds

          Introduction                                                                                                                     8

          Materials and methods                                                                                                      8

                     Glucosinolate extraction and analysis                                                                      8

                     Determination of anti-cancer potential                                                                    8

          Results                                                                                                                            9

          Discussion                                                                                                                        11

          Acknowledgements                                                                                                           12

          Literature cited                                                                                                               12

 

3.      Chapter Three - Glucosinolate composition of sprouted-seed

          Introduction                                                                                                                     14

          Materials and methods

                     Plant material and sprouting conditions                                                                    14

                     Glucosinolate extraction and analysis                                                                       14

                     Determination of anti-cancer potential                                                                    14

          Results 15

          Discussion                                                                                                                        17

          Acknowledgements                                                                                                           17

          Literature cited                                                                                                               18

 

4.      Chapter Four - Glucosinolate composition of mature tissue versus seeds and sprouts

          Introduction                                                                                                                     19

          Materials and methods

                     Plant material                                                                                                       19

                     Glucosinolate extraction and analysis                                                                       19

                     Determination of anti-cancer potential                                                                    19

          Results and Discussion                                                                                                       20

          Literature cited                                                                                                               22

 

5.      Chapter Five - Effect of sprout growth, growing temperature and cultivar on glucosinolate composition using daikon sprouts as a model

          Introduction                                                                                                                     24

          Materials and methods

                     Plant material, sprouting conditions and growing temperature                                  24

                     Glucosinolate extraction and analysis                                                                       24

          Results and Discussion                                                                                                       24

          Acknowledgements                                                                                                           28

          Literature cited                                                                                                               28

 

6.      Chapter Six - Effect of refrigeration on glucosinolate levels in sprouts

          Introduction                                                                                                                     29

          Materials and methods

                     Plant material, sprouting conditions and cold storage                                               29

                     Glucosinolate extraction and analysis                                                                       29

          Results and Discussion                                                                                                      29

          Acknowledgements                                                                                                           32

          Literature cited                                                                                                                32

 

7.      Chapter Seven - Industry assessment of sprouts

          Radish or daikon sprouts                                                                                                   33

          Broccoli sprouts                                                                                                               33

          Kohl rabi sprouts                                                                                                              34

          Rocket sprouts & rocket leaves                                                                                          34

          Wasabi sprouts                                                                                                                 34

          Kale sprouts                                                                                                                     34

          Garden cress sprouts                                                                                                         35

          Chinese broccoli sprouts                                                                                                   35

          Other brassicaceous sprouts                                                                                               35

 

8.      Chapter Eight - Addressing regulatory issues relating to anti-cancer claims

          General level claims                                                                                                         36

          High level health claims                                                                                                    36

          General media & point of sale education material                                                               37

          Conclusions                                                                                                                      37

 

Recommendations                                                                                                                   38

 

Executive Summary

 

The aim of the current project was to investigate the anti-cancer potential of a range of Asian vegetables belonging to the brassica family.  There is increasing evidence that glucosinolates, or more specifically their isothiocyanate hydrolysis products are associated with a lower incidence of certain cancers, particularly those of the gastrointestinal tract (eg. colorectal cancer). 

 

Glucosinolates, of which there are over 120 types, are almost exclusively found in members of the brassica family (or Brassicaceae).  Asian vegetables represent a large number of horticultural brassicaceous species, and therefore hold particular potential as anti-cancer vehicles.  Examples include both members of the Brassica genus (pak choy, tatsoi, choy sum, Chinese broccoli, Chinese mustard, komatsuna, mizuna, Japanese turnip) as well as rocket (Eruca), daikon (Raphanus) and wasabi (Wasabia).

 

Over the last 10-15 years, considerable research has been conducted in relation to the anti-cancer potential of broccoli.  From a range of mature vegetables, broccoli appeared to be one of the best inducers of mammalian detoxification enzymes, also known as phase 2 enzymes.  Phase 2 enzymes act by binding with or inactivating certain carcinogens, making them more readily excretable from the body.

 

The current project optimised an HPLC technique for extraction and identification of glucosinolates, followed by identification of 25 glucosinolates in significant amounts.  Identification was tentatively based on compound molecular weight and published scientific literature, and in the case of 11 glucosinolates, confirmed with standards.

 

It has been already established that different glucosinolates vary in the ability to induce phase 2 enzymes, with the potency of a glucosinolate gauged by its in vitro ability to induce quinone reductase.  Using this information and the concentration of glucosinolates present in various plant tissues, we were able to calculate an anti-cancer index, by which we could estimate the relative potential of different species to induce phase 2 enzymes.

 

Seeds are by far the most concentrated source of glucosinolates, although the presence of erucic acid in brassicaceous seeds has potential health issues in regard to cardiovascular disease.  On the other hand, mature vegetables (which vary greatly in form) tended to have mainly low levels of glucosinolates, increasing the amount required to be consumed to have a chemopreventive effect. Sprouts by comparison have a high level of glucosinolates (however lower than seeds), but the level of erucic acid is negligible making them a good candidate as an anti-cancer product.

 

Sprouts which had the highest anti-cancer potential included daikon, radish, broccoli, rocket, kohl rabi, wasabi, kale, garden cress, and Chinese broccoli.  This varied slightly with mature vegetables, as the plant part varied, as did the mature glucosinolate profiles.  Mature vegetables with highest anti-cancer potential included rocket, broccoli, daikon and wasabi.

 

A complicating issue affecting anti-cancer potential was the question as to how much glucosinolate present in plants gets converted to anti-cancer isothiocyanates when it is eaten.  Unfortunately, cooking is often an effective way of stopping conversion, because the enzymes responsible for conversion is inactivated by heat.  Most of the sprouts studied in the current project would be eaten raw though, so this is less of an issue than for mature vegetables such as broccoli.  Many brassicaceous species however have a secondary issue relating to the conversion of glucosinolates to nitriles, rather than isothiocyanates.  The compound responsible for this is known as epithiospecifier protein (ESP), which can reduce anti-cancer potential by 50-80%.  Fortunately, ESP is not present in all species, including radish and daikon sprouts.

In a closer examination of daikon sprouts, sprout growth stage was found to significantly influence glucosinolate level, with younger sprouts being the most potent.  Growing temperature was found to have less impact on glucosinolate profile, although this varied between cultivar.

 

As sprouts are normally purchased and then stored in a domestic refrigerator, the impact of low temperature on glucosinolate levels for a range of sprouts was examined.  In most cases, glucosinolate levels remained stable at 4°C for up to 4 weeks, although glucosinolate levels in rocket sprouts declined significantly after one week.

 

Issues other than anti-cancer potential were also investigated in relation to commercially growing and marketing different species of sprouts.  Important issues included public awareness of a species (how many people are familiar with kohl rabi), seed price and availability, flavour, and growing characteristics.  Of the sprouts identified, the two most promising species were daikon (radish) sprouts and broccoli sprouts, the latter of which is not an 'Asian' vegetable.  Daikon sprouts (which are a form of radish sprouts) are presently on the market and are actually sold as 'radish' sprouts.

 

The final issue addressed by the current project was regulatory restrictions in regard to what can be claimed on food labelling in regard to health.  Although the regulatory standard is currently under review, producers are likely to be restricted to content claims.  Producers can say that a product is a natural source of glucosinolates, and then list the amount present on a nutrient content label, but they will not be able to say a product is a 'good' source of glucosinolates.  This is because the exact amount of glucosinolate required for a physiological effect has not been unequivocally established. Similarly, claims relating to reducing cancer risk are not yet able to be made on food labelling in Australia.


 

 

Introduction 

 

Members of the Brassica family include a wide range of both 'Asian' and 'Western' horticultural crops (eg. cabbage, mustard, rocket, pak choy, daikon, broccoli), all of which contain compounds known as glucosinolates (Fenwick et al., 1983).  Upon mastication or other forms of 'injury', glucosinolates mix freely with the plant enzyme myrosinase to form a number of potential products, including isothiocyanates with anti-cancer potential (Mithen et al., 2000).  The particular isothiocyanates that are formed are dependent on the glucosinolate profile of the plant species, with commonly 3-4 glucosinolates present in any significant degree (Fenwick et al., 1983). 

 

Consumption of Brassica vegetables has been linked to a reduction in the prevalence of certain types of cancer (eg. colorectal cancer).  Both in vitro and in vivo studies have shown that certain isothiocyanates (hydrolysis products of glucosinolates) are potent inducers of phase 2 detoxification enzymes in mammals (Zhang et al., 1992; Faulkner et al., 1998).  Phase 2 enzymes such as glutathione-S-transferase and quinone reductase promote the metabolism and excretion of potential carcinogens (Johnson et al., 1994). 

 

Glucosinolate concentration is usually highest in the seed (Mithen et al., 2000), with sprouted-seed of several horticultural Brassicaceae found to have 10-100 times the inducer potency of mature field-grown plants (Fahey et al., 1997).  One isothiocyanate that has recently received much publicity for its anti-cancer action is sulphoraphane, a compound that is derived from glucoraphanin, a glucosinolate of high abundance in broccoli (Zhang et al., 1992; Zhang et al., 1994; Nestle, 1997; Brooks et al., 2001).  Both sulphoraphane and broccoli sprouts have been identified as potent inducers of phase 2 enzymes (Zhang et al., 1992; Fahey et al., 1997), with sprout-potency declining exponentially (from a maximum in the seed) over a period of 15 days to the level of mature broccoli heads (Fahey et al., 1997). 

 

Many other isothiocyanates exist with anti-cancer potential (Talalay et al., 1988; Zhang et al., 1992; Rose et al. 2000), located within different members of the Brassica family.   Although there has been a tendency to favour sulphoraphane (and broccoli) research, it is now recognised that it is not the ingestion of sulphoraphane that is important for anti-cancer activity, but the total amount of anti-cancer isothiocyanates accumulating in cells (Zhang and Talalay, 1998).  Put simply, eating twice the amount of an isothiocyanate possessing half the anti-cancer potential of sulphoraphane will provide a similar response to the latter.  With this in mind, a broad range of Brassicaceae vegetables containing glucosinolates other than glucoraphanin may have anti-cancer potential.

 

Asian vegetables comprise of a surprisingly large number of brassicaceous species, some of which may have glucosinolate profiles capable of inducing phase 2 detoxification enzymes.  The current project sought to establish what glucosinolates are present in Asian vegetables available in Australia, and through this to estimate which species may have anti-cancer potential.  Most Asian vegetables purchased in Australia are bought on culinary reasons alone (ie. flavour).  Identifying additional reasons for purchase, such as for health reasons, offers potential to expand sales to consumers who would otherwise have not purchased Asian vegetables.

 

 

Objectives

 

The objective of this project was to identify members of the brassica family (of which Asian vegetables comprise a large number) which could form the basis of industry sales on health rather than culinary issues (ie. flavour), similar to broccoli sprouts in the United States.

 

The emphasis was on identifying species with anti-cancer properties, specifically in relation to chemoprevention, or in other words, identifying species containing compounds that would protect against cancer-causing carcinogens.  The specific compounds of note in the brassica family are the glucosinolates, comprising over 120 types, of which only a relatively small proportion have been screened for anti-cancer potential.

 

Although the scope of this project did not allow us to identify new cancer-fighting compounds, it did allow us to identify the range and levels of compounds with known chemo-preventive properties in Asian vegetables, and in some cases to postulate if a compound was likely to have chemo-preventive activity.  Using this information, we were able to provide a guide as to the anti-cancer potential of most commonly seen Asian vegetables available in Australia.

 

It became apparent early on that seed-sprouts were potentially by far the most potent inducers of anti-cancer compounds (phase 2 enzymes), and hence the project has concentrated in this largely uninvestigated area.

 

Finally, we have tried to provide advice or recommendations on addressing the regulatory issues relating to anti-cancer claims.  This area is presently under change, but the issues refer to health-claims in general in relation to food.  

 

Methodology

Approach to determining anti-cancer potential

 

The current project has been designed to specifically investigate the potential for different brassicaceous species to induce mammalian detoxification enzymes thought to be involved in a chemopreventive role by aiding in the rapid excretion (or inactivation) of carcinogens from the body.  In this regard, a moderate amount of information exists in the scientific literature concerning the relative potencies of different glucosinolates.  The most common method that has been used involves the use of a murine hepatoma cell assay (Hepa1c1c7 murine hepatoma cell assay, Prochaska et al., 1992). This assay compares different glucosinolates by determining the concentration of an individual glucosinolate to double the activity of quinone reductase, a phase 2 enzyme.  Hence, glucosinolates that are required in lower concentration to achieve this are 'potent' inducers.  It is from these studies that we have collated quantitative data to rank the various glucosinolates found in Asian vegetables.  For certain glucosinolates, no data exits, and therefore we have had to estimate (based on known similarities between glucosinolates) or omit any anti-cancer potential owing to these glucosinolates when determining overall anti-cancer potential of a species.

 

The ability to induce phase 2 enzymes is consequently based not only on the type of glucosinolate present, but also on its concentration in a given food matrix.  From the scientific literature, it appears that greater consumption of a less potent inducer can have a comparable effect to consuming a lesser amount of a more potent inducer.  Consequently, we have developed a crude 'Anti-cancer Index' which takes into account both potency of a glucosinolate, and its concentration.

 

Anti-cancer index = glucosinolate concentration / CD value*

 

*CD value: Concentration of a glucosinolate required to Double the activity of quinone reductase

 

 

As species tend to contain more than one glucosinolate (anything from 1 to 10 at measurable levels, but most often 3 to 4), the assessment of anti-cancer potential becomes slightly more complex.   Studies indicate however that effects of individual glucosinolates are additive, and that there is little synergy acting between individual glucosinolates, hence the calculation becomes:  

 

Anti-cancer index = (G1 / CD1) + (G2/CD2) + (G3/CD3) + etc.

 

In order to calculate the anti-cancer indices for different members of the brassica family, identification and quantification of individual glucosinolates was firstly necessary.  Development of a suitable extraction technique and HPLC quantification method are discussed in Chapter 1.

 

Other factors of importance

 

In general, the active inducer of detoxification enzymes is not the glucosinolate itself, but its hydrolysis product, which is usually an isothiocyanate.  In many cancer studies, it is commonly assumed that 100% of glucosinolates are converted to isothiocyanates, which in many cases is not correct.  In many species, much of the glucosinolate (50-80%) is actually converted to nitriles, which have very little phase 2 enzyme induction activity.  This is because many species contain an enzyme co-factor known as epithiospecifier protein (ESP).  Consequently, the anti-cancer potential of some products can be over-estimated.  In the current report, we have tried to specify wherever possible where this may be occurring.

 

Cooking can also impact on isothiocyanate yield.  Boiling for instance is used to deactivate myrosinase, the enzyme responsible for converting glucosinolate to isothiocyanate.  As a result, many cooked vegetables (as opposed to raw) are less able to produce isothiocyanates as their myrosinase has been heat-inactivated.  Although our gut microflora can convert glucosinolates to isothiocyanates, efficiency is considerably less, in the order of 10-20% that of the plant enzyme.

Another issue to take into account is that some glucosinolates may have potentially negative impacts on health.  For instance, excess progoitrin is associated with inducing goitre, due to interfering with iodine uptake, and an excess of indole glucosinolates may potentially activate phase 1 enzymes and trigger cancer growth.  In the rare occasions where we have found high concentrations of 'anti-nutritional' glucosinolates we have specified this.

 

Chapter 1 

 

Glucosinolate measurements

 

Development of HPLC method for identification of glucosinolates

A number of methods have been used in the past to analyse glucosinolate composition of plant material.  Each of these appears to have its own drawback, including loss of glucosinolates during extraction, poor resolution of peaks, difficult extraction methodology, and different columns required for polar and non-polar glucosinolates. 

 

The method used in the current project was based on a HPLC method developed by West et al. (2002), and was the same method chosen for use in the Horticulture Australia project 'Vital Vegetables 2003-2007'.  The method is a single column approach appropriate to both polar and non-polar glucosinolates, involving reversed-phase separation using hydrophilic endcapped C18-bonded silica and a 50 mM ammonium acetate-methanol gradient mobile phase.  

 

Identification of individual glucosinolates was achieved by HPLC-mass spectrometry to identify molecular weight of compounds reflecting UV at 235 nm.  Plant extracts were passed through HPLC-MS, generating a number of peaks at different elution times.  Each peak corresponded to a different glucosinolate, which was tentatively identified using a combination of molecular weight, previously reported composition, and pure glucosinolate standards where available. 

 

 

Table 1.  Observed glucosinolate elution times using the method of West et al. (2002).

 

Common name

Chemical name

Elution time (min)

Molecular weight

glucoiberin

3-methylsulphinylpropyl

4.4

422

progoitrin

2-hydroxy-3-butenyl

4.8

388

sinigrin

2-propenyl (allyl)

5.2

358

epiprogoitrin

2-hydroxy-3-butenyl

5.7

388

glucoraphanin

4-methylsulphinylbutyl

6.9

436

glucoraphenin

4-methylsulphinyl-3-butenyl

7.3

434

gluconapolieferin

2-hydroxy-4-pentenyl

8.8

402

gluconapin

3-butenyl

9.8

372

glucosinalbin

p-hydroxybenzyl

10.6

424

glucoalyssin

5-methylsulphinylpentyl

11.6

450

glucoiberverin

3-methylthiopropyl

14.0

406

4-hydroxyglucobrassicin

4-hydroxy-3-indolylmethyl

14.7

463

glucocheirolin

3-methylsulphonylpropyl

16.6

438

glucobrassicanapin

4-pentenyl

17.2

386

glucohesperalin

6-methylsulphinylhexyl

17.8

464

glucotropaeolin

benzyl

18.6

408

(not a glucosinolate)

(unknown)

19.9

223&339

glucoerucin

4-methylthiobutyl

20.8

420

glucodehydroerucin

4-methylthio-3-butenyl

21.1

418

glucobrassicin

3-indolylmethyl

24.3

447

glucosiberin

7-methylsulphinylseptyl

27.7

478

gluconasturtiin

2-phenylethyl

28.3

422

4-mercaptobutyl

4-mercaptobutyl

29.4

406

glucoberteroin

5-methylthiopentyl

31.4

434

neoglucobrassicin

1-methoxy-3-indolylmethyl

35.5

477

glucohirsutin

8-methylsulphinyloctyl

38.5

492

Table 1 indicates the elution times for 25 glucosinolates to the best of our knowledge and one unknown compound based on data obtained for over 20 species (pure standards were available for glucosinolates shown in bold).

 

Apart from sinigrin, pure standards were both difficult to attain and expensive, and consequently the level of individual glucosinolates was usually expressed as μmol sinigrin-equivalent per gram.  In order to determine the actual concentration of an individual glucosinolate, equimolar concentration of available standards were compared and conversion factors calculated (Table 2). 

 

Table 2.  Conversion factors for conversion of glucosinolate concentration expressed as sinigrin equivalents into actual glucosinolate concentrations based on integrated absorbance areas for equimolar concentrations (250g/ml) of individual glucosinolate standards.

 

Glucosinolate

Peak Area

Conversion Factor

sinigrin

36073

1.00

glucoiberin

16571

2.18

progoitrin

22876

1.58

epiprogoitrin

17096

2.11

glucoraphanin

11065

3.26

glucoraphenin

11219

3.22

gluconapin

21956

1.64

glucosinalbin

42539

0.85

glucocheirolin

19831

1.82

glucotropeolin

42092

0.86

glucoerucin

23702

1.52

gluconasturtiin

20625

1.75

 

  

Optimisation of glucosinolate extraction method

Boiling-water versus Triple-solvent method

The literature commonly cites two methods of glucosinolate extraction from plant material: triple solvent method and boiling water.   The triple solvent method involves homogenising plant material in a mixture of equal volumes of dimethylsulphoxide, dimethylformamide and acetonitrile at about minus 50°C.  Both methods have been described as yielding essentially identical chromatograms (West et al., 2002).  In the current project, we used the boiling water method for extraction of glucosinolates.

 

Inactivation of myrosinase

In order to prevent hydrolysis of glucosinolates, plant material is boiled to inactivate the endogenous myrosinase enzyme.  Early trials using plastic test-tubes inserted into a boiling water bath, indicated that the plastic tended to insulate the extract solution from the bath, resulting in lower extract temperatures and incomplete inactivation of myrosinase.  Use of glass test-tubes was found to be superior in this regard.  For larger samples (eg. sprouts), direct boiling of beakers on hot-plates was also an efficient means of inactivation.

 

Effect of boiling time on glucosinolate yield was also assessed.  Optimum boiling times were in the vicinity of 5-6 minutes, with less or greater times decreasing glucosinolate yield (Figure 1).  It was found that bulky tissues such as roots (eg. radish, turnip) required rapid grating prior to boiling, due to slow heat-transfer within intact material.

 

  

Figure 1. Effect of boiling time on glucosinolate yield from broccoli seeds (cv. Marathon) (top: 10 minutes; middle: 6 minutes; bottom: 3 minutes).  Glucosinolate peaks (left to right): glucoiberin, glucoraphanin, 4-hydroxyglucobrassicin, glucoerucin.  N.B.: later elution times were due to decreased pressure.

 

Chapter 2  

Glucosinolate composition of seeds

 

Introduction

The following chapter presents data outlining the spectrum of glucosinolates and the individual levels of specific glucosinolates found in the seeds of widely consumed Asian and Western horticultural species in the Brassica family.  Seeds were used as an easily replicable plant organ, with high glucosinolate concentration. Based on this data, the seeds have been ranked for their anti-cancer potential, calculated from reported concentrations of their respective isothiocyanate derivatives to double the activity of the mammalian detoxification phase 2 enzyme, quinone reductase, in Hepa 1c1c7 murine hepatoma cells (Prochaska et al., 1992).  The data provides preliminary indications as to which species may have anti-cancer potential, particularly as sprouted seeds.

           

Materials and methods

Glucosinolate extraction and analysis

Seed samples were taken from a range of 'Asian' and 'Western' commercially available seeds of the Brassica family.  'Asian' crops included: pak choy (B. rapa var. chinensis), choy sum (B. rapa var. parachinensis), tatsoi (B. rapa var. rosularis), senposai (B. rapa var. perviridis x B. rapa var. pekinensis), komatsuna (B. rapa var. perviridis), Chinese mustard (B. juncea), Chinese broccoli (B. alboglabra), Japanese turnip (B. rapa var. rapifera), kale (B. oleracea var. acephala), Chinese cabbage (B. rapa var. pekinensis), mizuna (B. rapa var. nipposinica), garden cress (Lepidium sativum), rocket (Eruca sativa), and daikon (R. sativus). 'Western' crops included: broccoli (Brassica oleraceae var. italica), white cabbage (B. oleracea var. capitata), kohl rabi (B. oleracea var. gongylodes), black mustard (B. nigra), broccoli raab (B. ruvo), watercress (Nasturtium officinalis), red radish (Raphanus sativus), and white mustard (Sinapsis alba).

 

Approximately 0.5 g seed was weighed and boiled in 10 ml water for 5 minutes to inactivate myrosinase-induced degradation of glucosinolates.  Seed was homogenised with an Ultra-Turrax for two minutes and centrifuged for 15 minutes at 13 000 rpm.  The supernatant was collected and filtered through Whatmans No.1 filter paper.  The filtrate was made up to 20 ml with distilled water and refiltered through a 0.2 m syringe filter.

 

Supernatants were analysed for glucosinolates by HPLC-UV and HPLC-MS using a Prevail C18 column as described by West et al. (2002).  The mobile phase was a linear gradient from 100% 50 mM ammonium acetate to 50 mM ammonium acetate-methanol (80:20) in 40 minutes.  The column was operated at ambient temperature with a flow-rate of 1 ml/min.  Preliminary peak identification was based on the molecular weight of the daughter [M-H]- anion released during FAB (fast atom bombardment).  Quantification of individual glucosinolates was based on commercially available high-purity (99.3%) sinigrin.

 

Determination of anti-cancer potential

A crude determination of 'Anti-cancer potential' was made based on the 'CD values' of the isothiocyanate derivative of the parent glucosinolates present in a species, and the concentration of the individual glucosinolate.  The 'CD value' for an isothiocyanate is defined as the concentration (M) of an isothiocyanate required to double quinone reductase (QR) activity (Talalay et al., 1995).  Anti-cancer potential of a species was quantified by dividing the concentration of the isothiocyanate present by its respective CD value.  For example, in broccoli seed, a concentration of 102 mol/gFW glucoraphanin divided by a CD value of 0.4 M for sulphoraphane (derived from glucoraphanin) equates to an anti-cancer potential rating of 255.  When a number of glucosinolates were present in a sample, these values were added together.  The calculation does not take into account potential synergistic interactions between glucosinolate derivatives (Staack et al., 1998) or differences in the relative integrated absorbance areas for glucosinolates of an equimolar concentration of sinigrin (Fahey et al., 1997).

 

CD Values were taken from published studies (Talalay et al., 1988; Zhang et al., 1992; Posner et al., 1994; Tawfiq et al., 1995; Fahey et al., 1997; Faulkner et al., 1998; Zhang and Talalay, 1998; Rose et al., 2000).  Where CD values differed between studies, an average value was taken.  CD values for glucodehydroerucin, gluconapolieferin and epiprogoitrin were estimated to be similar to glucoerucin, glucobrassicanapin, and progoitrin, respectively. 

 

Results

Glucosinolate composition within seeds of individual species is shown in Table 1.  The number of glucosinolates present at a detectable level (>0.1umol/g) ranged from 1 to 10, with a median value of 4. In total, 21 glucosinolates were tentatively identified in the seeds of species studied.  Glucosinolates fell into seven groups: methylsulphinylalkyl (glucosiberin, glucoraphanin, glucohirsutin, glucoalyssin, glucoiberin); methylsulphinylalkenyl (glucoraphenin); methylthioalkyl (glucoerucin, glucoberteroin, glucoiberverin); methylthioalkenyl (glucodehydroerucin); aromatic (glucotropaeolin, gluconasturtiin, glucosinalbin); olefin (sinigrin, gluconapin, glucobrassicanapin, gluconapolieferin, progoitrin, epiprogoitrin) and; indole (4-hydroxyglucobrassicin, glucobrassicin).  Glucoraphanin concentration for our broccoli cultivar (cv. Green Dragon) was 102 mol/gFW, similar to that of cultivars reported to have high glucoraphanin concentration (West et al., 2004).  

 

A ranking of anti-cancer potential for each crop is shown in Figure 1.  Based on constituent glucosinolates and the level present, red radish, daikon, and broccoli had the highest anti-cancer potential of all seeds tested.  Anti-cancer potential of these three vegetables was of a similar level, but more than three times the level of the next highest-ranked vegetable (kohl rabi).  Those with moderate anti-cancer potential included garden cress, rocket, kale, and watercress.  The remaining vegetables had moderate to low anti-cancer potential. 

Figure 1. Estimated anti-cancer potential of seed of Asian and Western vegetables based on 'CD-values' of isothiocyanates derived from glucosinolates identified in seed.  Higher values indicate higher anti-cancer potential (ranking without ESP: □; adjusted ranking with ESP: ■).

 

 

Table 1.  Glucosinolate composition and concentration (mol/g) of seeds of Asian and Western vegetables.  (glucosinolates in italics are presented as mol sinigrin equivalent/gFW).  CD value corresponds to the cited concentration of isothiocyanate derivatives required to double the activity of the phase 2 enzyme, quinone reductase, in Hepa 1c1c7 murine hepatoma cells.

Glucosinolate

 

CD value

 

red radish

daikon

broccoli

kohl rabi

garden cress

rocket

kale

water cress

Chinese broccoli

cabbage

choy sum

mizuna

senposai

red giant mustard

pak choy

black mustard

Japanese turnip

broccoli raab

tatsoi

Chinese cabbage

komatsuna

white mustard

wasabi

 

glucosiberin

0.2

 

 

 

 

 

 

 

2.7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.8

glucoraphenin

0.4

377.9

351.6

 

 

 

4.5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

glucoraphanin

0.2-0.8

 

6.5

333.6

86.0

 

10.4

31.9

 

 

 

3.2

5.3

5.4

 

8.9

 

 

 

 

3.7

 

 

 

glucohirsutin

0.5

 

 

 

 

 

 

 

1.3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

glucohesperalin

0.5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4.9

glucoalyssin

0.95

 

 

 

 

 

 

 

 

 

 

2.2

1.0

 

 

 

 

1.0

3.2

1.0

0.7

 

 

1.0

glucoberteroin

1.7

 

 

2.6

1.9

 

 

1.9

 

 

3.1

1.8

2.8

 

 

 

 

 

1.6

 

1.1

 

 

1.2

glucoiberin

1.8-2.4

 

 

 

30.4

 

 

36.6

 

 

46.9

 

 

 

 

 

 

 

 

 

 

 

 

 

glucoerucin

2.3

 

 

44.9

12.3

 

110.7

4.9

 

 

 

 

7.1

 

 

 

 

1.7

 

 

 

 

 

 

glucodehydroerucin

2.3

 

8.1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

glucotropaeolin

2.0-3.0

 

 

 

 

117.7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

glucoiberverin

3.5

 

 

 

1.7

 

 

3.1

 

1.5

7.4

 

 

 

 

 

 

 

 

 

 

 

 

 

gluconasturtiin

5

 

 

 

 

 

 

 

106.4

 

 

 

 

 

 

 

 

 

4.3

 

 

 

 

 

sinigrin

6.1-8.3

 

 

 

2.5

 

 

25.1

 

42.7

22.2

 

 

20.4

84.3

 

71.0

 

1.4

 

 

 

 

33.1

Gluconapin

7.4

 

 

 

1.4

 

 

7.6

1.3

161.1

 

106.9

66.1

61.0

4.6

62.2

 

97.8

50.0

87.9

26.7

60.9

 

1.2

glucobrassicanapin

>15

 

 

 

 

 

 

 

 

 

 

9.1

4.0

 

 

1.4

 

11.9

16.1

5.2

1.1

4.0

 

1.6

Gluconapolieferin

>15

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.4

 

 

 

 

 

Glucosinalbin

>15

 

 

 

 

 

 

 

 

 

0.4

 

 

 

 

 

 

 

1.0

 

 

 

212.1

 

4-hydroxyglucobrassicin

>15

11.0

9.6

8.9

7.0

 

 

10.6

 

6.3

10.5

5.8

6.4

11.2

6.0

5.2

4.1

6.4

7.7

6.0

9.7

6.0

 

 

progoitrin

47.7

 

 

 

6.5

 

 

18.5

 

 

 

14.5

2.9

97.6

 

9.2

 

2.8

3.4

1.7

10.6

1.1

 

 

epiprogoitrin

47.7

 

 

 

 

 

 

 

 

 

 

 

 

1.3

 

 

 

 

 

 

 

 

10.4

 

glucobrassicin

76.8

 

 

 

 

 

 

 

 

 

0.6

 

 

 

 

 

 

 

 

 

1.2

 

 

 

Neoglucobrassicin

Unknown

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MercaptobutylGS

unknown

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Discussion

The data from the present trial indicates that apart from broccoli, a number of horticultural species within the Brassica family have anti-cancer potential.  In the past, research has concentrated largely on broccoli, as extracts (especially from seed and young sprouts) appeared to have high phase 2 enzyme inducer activity, and broccoli was already consumed in substantial quantities in the Western world (Talalay et al., 1995).  The present data however, indicates that red radish and daikon (white radish) may have similar potential to broccoli, while kohl rabi, garden cress, rocket, kale and watercress have a moderate anti-cancer potential. 

 

Certainly, recent research has indicated the potential for watercress (Rose et al., 2000) and garden cress (Kassie et al., 2002), although little positive mention seems to have been made of radish, daikon, rocket, or kale.  The present study would indicate that the anti-cancer potential of these vegetables warrants further investigation, especially if the glucosinolate profiles in the seed are indicative of profiles that may be attained in sprouted-seed or mature vegetables.  Considering that both the glucosinolate profile and concentration can change or dilute with growth, as well as with the plant organ being studied (Fenwick et al., 1983), it is possible the rank-order of anti-cancer potential could change, and the anti-cancer potential to decline, as is seen in broccoli (Fahey et al., 1997).

 

The current estimates of anti-cancer potential were also based on the assumption that glucosinolates are converted exclusively to isothiocyanates.  This appears to be a widespread assumption, but is apparently not the case for all of the vegetables studied, due to the presence of an enzyme cofactor in some species known as epithiospecifier protein (ESP).  At least in some of the vegetables tested (broccoli, garden cress, cabbage) glucosinolates are autolytically degraded to a large degree (50-80%) by myrosinase and ESP to isothiocyanate nitrile, a compound with no anti-cancer potential (Matusheski, 2004).  By contrast, glucosinolates of daikon and white mustard are converted solely by myrosinase to isothiocyanates.  Considering this, an adjusted rank order of the seeds with highest anti-cancer potential would actually be radish, daikon, broccoli (reduced potency), rocket, kohl rabi and wasabi (Figure 1).  At this stage, we have not confirmed the presence of ESP in the remaining vegetables, but it would undoubtedly further adjust the rank order of anti-cancer potential.  Whatever the case, vegetables containing only low levels of glucosinolates (particularly of sulphinyl and thio-containing glucosinolates) would tend to have limited anti-cancer potential.

 

An additional 'anti-nutritional' factor to be considered is the presence of the indole glucosinolates, glucobrassicin and 4-hydroxyglucobrassicin.  Indole compounds such as indole-3-carbinol and indole-3-acetonitrile are formed from the hydrolysis of indole glucosinolates, and these can form mutagenic N-nitroso compounds (Wakabayashi et al., 1985).  In the current study, indole glucosinolates were confined to the Brassica and Raphanus genera (Table 1).  A similar finding has recently been reported by Bennett and co-workers (2004). Also, certain breakdown products from aliphatic glucosinolates containing a -hydroxyl group  (progoitrin, epiprogoitrin and gluconapolieferin) can be goitrogenic (Mithen et al., 2000).  In the current study, excessive progoitrin was found in senposai, and to a lesser degree in kale and choy sum (Table 1).  These factors need to be considered in addition to the anti-cancer potential of isothiocyanates, especially when taken at higher concentration than 'normal'.

 

The current study indicates that a number of horticultural species apart from broccoli have anti-cancer potential.  Particular vegetables warranting further investigation include red radish and daikon (white radish), seeds from both of which rated similarly to broccoli.  Seed from other vegetables indicating moderate potential included garden cress, rocket, kale, and watercress.  The presence of ESP in some vegetables should be considered as a negative modifier of the anti-cancer potential of certain vegetables, particularly where autolytic degradation is probable, as would be the case with the consumption of uncooked vegetables.  The other issue to consider is the potential 'anti-nutritional' effects associated with some glucosinolates, which may accompany the more beneficial glucosinolates present.  Further evaluation of plant extracts is required to confirm anti-cancer potential, with the current study providing a crude estimate of which vegetables are more likely to be beneficial.

Acknowledgements

This study was presented as an oral presentation to the 'International Symposium on Harnessing the Potential of Horticulture in the Asian-Pacific Region' held at Coolum, Queensland, 1st-3rd September 2004.  The paper has been published within the conference proceedings as follows: 

O'Hare, TJ, Wong LS and Irving DE, 2005.  Asian and Western horticultural species of the brassica family with anti-cancer potential.  Acta Horticulturae, 694:457-462.

 

Literature cited

Bennett, R.N., Mellon, F.A. and Kroon, P.A. 2004.  Screening crucifer seeds as sources of specific intact glucosinolates using ion-pair high-performance liquid chromatography negative ion electrospray mass spectrometry.  J. Agric. Food Chem. 52: 428-438.

Brooks, J, Paton, V. and Vidanes, G. 2001.  Potent induction of phase 2 enzymes in prostate cells by sulforaphane.  Cancer Epidemiol. Biomarkers Prev. 10: 949-954.

Fahey, J.W., Zhang, Y.S. and Talalay, P. 1997.  Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Natl. Acad. Sci. USA 94: 10367-10372.

Faulkner, K., Mithen, R. and Williamson, G. 1998.  Selective increase of the potential anticarcinogen 4-methylsulphinylbutyl glucosinolate in broccoli.  Carcinogenesis 19: 605-609.

Fenwick, G.R., Heaney, R.K. and Mullin W.J. 1983.  Glucosinolates and their breakdown products in food and food plants.  Crit. Rev. Food Sci. Nutr. 18: 123-201.

Johnson, I.T., Williamson, G., and Musk, S.S.R. 1994.  Anticarcinogenic factors in plant foods: a new class of nutrients.  Nutr. Res. Rev. 7: 175-204.

Kassie, F., Rabot, S., Uhl. M., Huber, W., Qin, H.M., Helma, C., Schulte-Hermann, R. and Knasmuller, S. 2002.  Chemoprotective effects of garden cress (Lepidium sativum) and its constituents towards 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ)-induced genotoxic effects and colonic preneoplastic lesions.  Carcinogenesis 23: 1155-1161.

Matusheski, N.V., Juvik, J.A. and Jeffery, E.H. 2004.  Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli.  Phytochemistry 65: 1273-1281.

Mithen, R., Dekker, M., Verkerk, R., Rabot, S. and Johnson, I.T. 2000.  The nutritional significance, biosynthesis and bioavailability of glucosinolates in human foods.  J. Sci. Food Agric. 80: 967-984.

Nestle, M. 1997. Broccoli sprouts as inducers of carcinogen-detoxifying enzyme systems; clinical, dietary, and policy implications.  Proc. Natl. Acad. Sci. USA 94: 11149-11151.

Posner, G.H., Cho, C.G., Green, J.V., Zhang, Y.S. and Talalay, P. 1994.  Design and synthesis of bifunctional isothiocyanate analogs of sulforaphane: correlation between structure and potency as inducers of anticarcinogenic detoxification enzymes.  J. Med. Chem. 37: 170-176.

Prochaska, H.J., Santamaria, A.B. and Talalay, P. 1992.  Rapid detection of inducers of enzymes that protect against carcinogens.  Proc. Natl. Acad. Sci. USA 89: 2394-2398.

Rose, P., Faulkner, K., Williamson, G. and Mithen, R. 2000.  7-methylsulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes.  Carcinogenesis 21: 1983-1988.

Staack, R., Kingston, S., Wallig, M.A. and Jeffery, E.H. 1998.  A comparison of the individual and collective effects of four glucosinolate breakdown products from Brussels sprouts on induction of detoxification enzymes.  Toxic. Applied Pharmacol. 149: 17-23.

Talalay, P., De Long, M.J. and Prochasta, H.J. 1988.  Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis.  Proc. Natl. Acad. Sci. USA 85: 8261-8265.

Talalay, P., Fahey, J.W., Holtzclaw, W.D., Prestera, T. and Zhang, Y.S. 1995.  Chemoprotection against cancer by phase 2 enzyme induction.  Toxicol. Lett. 82/83: 173-179.

Tawfiq, N., Heaney, R.K., Plumb, J.A., Fenwick, G.R., Musk, S.R.R. and Williamson, G. 1995.  Dietary glucosinolates as blocking agents against carcinogenesis: glucosinolate breakdown products assessed by induction of quinone reductase activity in murine hepa1c1c7 cells.  Carcinogenesis 16: 1191-1194.

Wakabayashi, K., Nagao, M., Ochiai, Tahira, T., Yamaizumi, Z. and Sugimura, T. 1985.  A mutagen precursor in Chinese cabbage, indole-3-acetonitrile, which becomes mutagenic on nitrite treatment.  Mutation Research 143: 17-21.

West, L., Tsui, I. and Haas, G. 2002.  Single column approach for the liquid chromatographic separation of polar and non-polar glucosinolates from broccoli sprouts and seeds.  J. Chromatography A 966: 227-232.

West, L.G., Meyer, K.A., Balch, B.A., Rossi, F.J., Schultz, M.R. and Haas, G.W. 2004.  Glucoraphanin and 4-hydroxyglucobrassicin contents in seeds of 59 cultivars of broccoli, raab, kohlrabi, radish, cauliflower, Brussels sprouts, kale, and cabbage.  J. Agric. Food Chem. 52: 916-926.

Zhang, Y.S., Kensler, T.W., Cho, C.G., Posner, G.H. and Talalay, P. 1994.  Anticarcinogenic activities of sulforaphane and structurally related synthetic norboryl isothiocyanates.  Proc. Natl. Acad. Sci. USA 91: 3147-3150.

Zhang, Y.S. and Talalay, P. 1998.  Mechanism of differential potencies of isothiocyanates as inducers of anticarcinogenic phase 2 enzymes.  Cancer Res. 58: 4632-4639.

Zhang, Y.S., Talalay, P., Cho, C.G. and Posner, G.H. 1992.  A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure.  Proc. Natl. Acad. Sci. USA 89: 2399-2403.

 

Chapter 3 

Glucosinolate composition of sprouted-seed

 

Introduction

The current chapter investigates the glucosinolate profile of a wide range of sprouts (sprouted seed) of species belonging to the Brassicaceae.  Based on the isothiocyanate derivatives of these glucosinolates, and the concentration required for a particular isothiocyanate to induce a known increase in the phase 2 enzyme, quinone reductase, the sprouts have been rated for their anti-cancer potential.  

 

Materials and methods

Plant Material and Sprouting Conditions

Commercially-available seed were sprouted at 20ºC on water-soaked tissue under fluorescent light (70 mol.m-2.s-1) to induce chlorophyll formation.  Water was purified via a reverse osmosis system.  Prior to sprouting, seed were soaked for 2 hours to induce imbibition.  Seed were rinsed daily and allowed to drain.   Seed-sprouts were harvested 7-8 days after imbibition.  Species included: broccoli (Brassica oleraceae var. italica), kale (B. oleracea var. acephala), white cabbage (B. oleracea var. capitata), kohl rabi (B. oleracea var. gongylodes), pak choy (B. rapa var. chinensis), choy sum (B. rapa var. parachinensis), tatsoi (B. rapa var. rosularis), senposai (B. rapa var. perviridis x B. rapa var. pekinensis), komatsuna (B. rapa var. perviridis), Japanese turnip (B. rapa var. rapifera), Chinese cabbage (B. rapa var. pekinensis), mizuna (B. rapa var. nipposinica), Chinese mustard (B. juncea), Chinese broccoli (B. alboglabra), black mustard (B. nigra), broccoli raab (B. ruvo), garden cress (Lepidium sativum), rocket (Eruca sativa), daikon and red radish (Raphanus sativus).

 

Glucosinolate extraction and analysis

Intact seed-sprouts (root and shoot, excluding seed-coat) were sampled at 7-8 days.  Approximately 0.4 g sprout was weighed and boiled in 10 ml water for 6 minutes to inactivate myrosinase-induced degradation of glucosinolates.  Sprouts were homogenised with an Ultra-Turrax for two minutes and centrifuged for 15 minutes at 14000 rpm.  The supernatant was collected and filtered through Whatmans No.1 filter paper.  The filtrate was made up to 20 ml with distilled water and refiltered through a 0.2 m syringe filter.

 

Supernatants were analysed for glucosinolates by HPLC-UV and HPLC-MS using a Prevail C18 column as described by West et al. (2002).  The mobile phase was a linear gradient from 100% 50 mM ammonium acetate to 50 mM ammonium acetate-methanol (80:20) in 40 minutes.  The column was operated at ambient temperature with a flow-rate of 1 ml/min.  Preliminary peak identification was based on the molecular weight of the daughter [M-H]- anion released during FAB (fast atom bombardment).  Quantification of individual glucosinolates was initially based on commercially available high-purity (99.3%) sinigrin.  Differences in the relative integrated absorbance areas for glucosinolates of an equimolar concentration of sinigrin were calculated for 12 glucosinolate standards.

 

Determination of anti-cancer potential

A crude determination of 'Anti-cancer potential' was made based on the 'CD values' of the isothiocyanate derivative of the parent glucosinolates present in a species, and the concentration of the individual glucosinolate.  The 'CD value' for an isothiocyanate is defined as the concentration (M) of an isothiocyanate required to double quinone reductase (QR) activity (Talalay et al., 1995).  Anti-cancer potential of a species was quantified by dividing the concentration of the isothiocyanate present by its respective CD value.  For example, in broccoli sprouts, a concentration of 32 mol/gFW glucoraphanin divided by a CD value of 0.4 M for sulphoraphane (derived from glucoraphanin) equates to an anti-cancer potential rating of 80.  When a number of glucosinolates were present in a sample, these values were added together.  The calculation does not take into account potential synergistic interactions between glucosinolate derivatives (Staack et al., 1998), and is based on 100% conversion of glucosinolates to isothiocyanates.

 

CD Values were taken from published studies (Talalay et al., 1988; Zhang et al., 1992; Posner et al., 1994; Tawfiq et al., 1995; Fahey et al., 1997; Faulkner et al., 1998; Zhang and Talalay, 1998; Rose et al., 2000).  Where CD values differed between studies, an average value was taken.  CD values for glucodehydroerucin, gluconapolieferin and epiprogoitrin were estimated to be similar to glucoerucin, glucobrassicanapin, and progoitrin, respectively. 

 

Results

Approximately 17 glucosinolates were identified in 7-8 day old sprouts within the species tested.  The number of glucosinolates present at concentrations greater than 0.1 mol/gFW in any one species ranged from 1-7, with 3 most common (Table 1).  Glucosinolates were divided into eight groups: methylsulphinylalkyl (glucoraphanin, glucoalyssin, glucoiberin); methylsulphinylalkenyl (glucoraphenin); methylthioalkyl (glucoerucin, glucoberteroin, glucoiberverin); methylthioalkenyl (glucodehydroerucin); mercaptoalkyl (4-mercaptobutyl glucosinolate); aromatic (glucotropaeolin); olefin (sinigrin, gluconapin, glucobrassicanapin, gluconapolieferin, progoitrin, epiprogoitrin) and; indole (4-hydroxyglucobrassicin).  An unidentified peak (m/z 223 and 339) was also produced in 10 of the species tested (all B. rapa sprouts, Raphanus sativus, and Lepidium sativum). 

 

 

Figure 1. Estimated anti-cancer potential of sprouts based on 'CD-values' of isothiocyanates derived from identified glucosinolates.  Higher values indicate higher anti-cancer potential (ranking without ESP: □; adjusted ranking with ESP: ■).

 

Anti-cancer potential based on constituent glucosinolates and the level present for sprouts is shown in Figure 1.  Broccoli and red radish had highest anti-cancer potential (score > 50); kohl rabi and daikon had moderate to high potential (score 20-50), and; kale, rocket, Chinese broccoli, cabbage, garden cress and wasabi had moderate potential (score 5-11).   The remaining species had low potential (score < 2). 

 

Table 1.Glucosinolate composition and concentration (mol/gFW) of seed-sprouts (glucosinolates in italics are presented as mol sinigrin equivalent/gFW).  CD value corresponds to the cited concentration of isothiocyanate derivatives required to double the activity of the phase 2 enzyme, quinone reductase, in Hepa 1c1c7 murine hepatoma cells.

 

 

Glucosinolate

 

CD value

 

red radish

daikon

Broccoli

(Green Dragon)

Broccoli (BroccoSprouts)

kohl rabi

garden cress

rocket

kale

Chinese broccoli

cabbage

choy sum

mizuna

senposai

red giant mustard

pak choy

black mustard

Japanese turnip

broccoli raab

tatsoi

Chinese cabbage

komatsuna

wasabi

glucosiberin

0.2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.3

glucoraphenin

0.4

24.5

6.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

glucoraphanin

0.2-0.8

 

 

33.2

18.8

15.7

 

3.7

3.3

1.3

 

 

 

 

 

 

 

 

 

 

 

 

 

glucohesparalin

0.5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.7

glucoalyssin

0.95

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.1

0.3

0.1

 

 

 

glucoberteroin

1.7

 

 

 

 

 

 

 

 

 

 

 

0.4

 

 

 

 

 

 

 

 

 

0.2

glucoiberin

1.8-2.4

 

 

 

4.0

4.0

 

 

4.0

 

10.7

 

 

 

 

 

 

 

 

 

 

 

 

glucoerucin

2.3

 

 

6.3

2.6

 

 

 

0.5

 

 

 

1.1

 

 

 

 

 

 

 

 

 

 

glucodehydroerucin

2.3

9.0

21.6

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

glucotropaeolin

2.0-3.0

 

 

 

 

 

12.3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

glucoiberverin

3.5

 

 

 

 

 

 

 

0.5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

sinigrin

6.1-8.3

 

 

 

 

 

 

 

2.4

6.3

3.3

 

 

3.6

11.9

 

7.7

 

 

 

 

 

4.5

gluconapin

7.4

 

 

 

 

 

 

 

 

23.2

 

9.7

8.1

3.5

0.6

7.2

 

9.9

4.2

8.3

1.3

12.8

 

glucobrassicanapin

>15

 

 

 

 

 

 

 

 

 

 

1.2

0.8

 

 

0.3

 

1.4

1.7

0.6

0.2

0.8

 

gluconapolieferin

>15

 

 

 

 

 

 

 

 

 

 

 

0.8

 

 

 

 

0.3

0.6

 

 

 

 

4-hydroxyglucobrassicin

>15

0.4

0.3

 

0.5

0.3

 

 

0.9

0.4

0.9

 

 

0.6

0.2

 

 

 

 

 

 

 

 

Progoitrin

47.7

 

 

 

 

 

 

 

1.3

 

 

4.5

1.6

11.2

 

1.7

 

2.4

2.0

1.7

1.7

1.2

 

epiprogoitrin

47.7

 

 

 

 

 

 

 

 

 

 

 

 

0.5

 

 

 

 

 

 

 

 

 

mercaptobutylglucosinolate

unknown

 

 

 

 

 

 

4.1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fragment 223/339

unknown

1.1

1.0

 

 

 

4.1

 

 

 

 

2.9

5.9

1.4

 

3.1

 

3.6

 

3.0

1.0

4.1

 


 

Discussion

The predictions from the present trial support existing evidence that broccoli sprouts are a potent inducer of phase 2 enzymes (Fahey et al., 1997), but also raise the possibility that sprouts of radish and kohl rabi may have high potency.  Previous experiments with mature vegetable tissue of radish and kohl rabi have indicated less potency in comparison to mature broccoli (Prochaska et al., 1992), although comparison between sprouts do not appear to have been made.  Differences in the physiology between mature vegetables make comparison difficult (eg. a swollen stem versus a floral head), but nevertheless are useful for consumers of mainstream vegetables.   Evidence also exists for 'mature' cabbage and kale as being able to induce phase 2 enzymes, but again to a lesser degree.

 

Interestingly, the rank-order of anti-cancer potential for sprouts varied slightly to that found previously for seeds (O'Hare et al., 2004).  Although sprouts generally had a considerably lower concentration of glucosinolates than seeds, particular species and particular glucosinolates were affected more than others.  Daikon sprouts, for example, showed an increase in glucodehydroerucin (moderate inducer) and a concominant decline in glucoraphenin (strong inducer) when compared to seeds.  Rocket sprouts showed a disappearance of glucoerucin (moderate inducer) and the appearance of 4-mercaptobutyl glucosinolate (unknown induction capacity). These differences may indicate down-regulation of certain enzymes during the early stages of germination, but also indicate that evaluation of seed-potency may not give an accurate indication of sprout-potency.  Having said this, the five species identified with highest potential in seed (O'Hare et al., 2004) remained the same species identified in sprouts.

 

It is also of note that the concentration of the indole glucosinolate, 4-hydroxyglucobrassicin, declined to very low levels or disappeared in many 7-8 day old sprouts, in stark comparison to seeds of Brassica and Raphanus species. The presence of indole glucosinolates has been considered unfavourable due to their ability to form mutagenic degradation products, such as indole-3-carbinol and indole-3-acetonitrile (Wakabayashi et al., 1985).  Consequently, the reduction of 4-hydroxyglucobrassicin seen in sprouts may be seen as a positive factor.  By contrast, progoitrin and gluconapolieferin (both reputedly goitrogenic olefin glucosinolates) (Mithen et al., 2000) remained as high as in seeds in many sprouts (Table 1).  This however appeared to be confined to species with low anti-cancer potential (Figure 1).  Despite this, this 'anti-nutritional' factor should be taken into consideration when determining the overall health benefit of particular sprouts.  Sprouts with highest progoitrin levels (in decreasing order) were senposai, choy sum, and Japanese turnip. 

 

The current estimates of anti-cancer potential were also based on the assumption that glucosinolates are converted exclusively to isothiocyanates.  For certain species however, a large percentage (50-80%) of glucosinolates may be converted to isothiocyanate nitriles or epinitriles (Lambrix, 2001; Matusheski, 2004), compounds of no anti-cancer potential.  This is due to the presence of an enzyme cofactor known as epithiospecifier protein (ESP), which is present in at least broccoli, garden cress and cabbage, but absent in daikon.  Although ESP is heat-labile, sprouts from broccoli are generally eaten raw, which may impact on the yield of anti-cancer isothiocyanates.  Consequently, if the effect of ESP is taken into account, the top-ranking sprouts for 'anti-cancer potential' would be similar to that found for seed (Chapter 2): red radish, daikon, broccoli, rocket, kohl rabi and wasabi (Figure 1).

 

Acknowledgements

This study was presented as an oral presentation to the 'International Symposium on Human Health Effects of Fruits and Vegetables' held at Quebec City, Canada, 17th-20th August 2005.  The paper will be published within the conference proceedings:

O'Hare, TJ, Wong LS, Force LE and Irving DE, 2006.  Glucosinolate Composition and Anti-cancer Potential of Seed-sprouts from Horticultural Members of the Brassicaceae.  Acta Horticulturae, (in press).

 

Literature cited

Fahey, J.W., Zhang, Y.S. and Talalay, P. 1997.  Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Natl. Acad. Sci. USA 94: 10367-10372.

Faulkner, K., Mithen, R. and Williamson, G. 1998.  Selective increase of the potential anticarcinogen 4-methylsulphinylbutyl glucosinolate in broccoli.  Carcinogenesis 19: 605-609.

Fenwick, G.R., Heaney, R.K. and Mullin W.J. 1983.  Glucosinolates and their breakdown products in food and food plants.  Crit. Rev. Food Sci. Nutr. 18: 123-201.

Lambrix, V., Reichelt, M., Mitchell-Olds, T., Klubenstein, D.J., and Gershenzou, J. (2001). The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory. Plant Cell 13, 2793-2807.

Matusheski, N.V., Juvik, J.A. and Jeffery, E.H. 2004.  Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli.  Phytochemistry 65: 1273-1281.

Mithen, R., Dekker, M., Verkerk, R., Rabot, S. and Johnson, I.T. 2000.  The nutritional significance, biosynthesis and bioavailability of glucosinolates in human foods.  J. Sci. Food Agric. 80: 967-984.

O'Hare, TJ, Wong LS and Irving DE, 2005.  Asian and Western horticultural species of the brassica family with anti-cancer potential.  Acta Horticulturae, 694:457-462.

Posner, G.H., Cho, C.G., Green, J.V., Zhang, Y.S. and Talalay, P. 1994.  Design and synthesis of bifunctional isothiocyanate analogs of sulforaphane: correlation between structure and potency as inducers of anticarcinogenic detoxification enzymes.  J. Med. Chem. 37: 170-176.

Prochaska, H.J., Santamaria, A.B. and Talalay, P. 1992.  Rapid detection of inducers of enzymes that protect against carcinogens.  Proc. Natl. Acad. Sci. USA 89: 2394-2398.

Rose, P., Faulkner, K., Williamson, G. and Mithen, R. 2000.  7-methylsulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes.  Carcinogenesis 21: 1983-1988.

Staack, R., Kingston, S., Wallig, M.A. and Jeffery, E.H. 1998.  A comparison of the individual and collective effects of four glucosinolate breakdown products from Brussels sprouts on induction of detoxification enzymes.  Toxic. Applied Pharmacol. 149: 17-23.

Talalay, P., De Long, M.J. and Prochasta, H.J. 1988.  Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis.  Proc. Natl. Acad. Sci. USA 85: 8261-8265.

Talalay, P., Fahey, J.W., Holtzclaw, W.D., Prestera, T. and Zhang, Y.S. 1995.  Chemoprotection against cancer by phase 2 enzyme induction.  Toxicol. Lett. 82/83: 173-179.

Tawfiq, N., Heaney, R.K., Plumb, J.A., Fenwick, G.R., Musk, S.R.R. and Williamson, G. 1995.  Dietary glucosinolates as blocking agents against carcinogenesis: glucosinolate breakdown products assessed by induction of quinone reductase activity in murine hepa1c1c7 cells.  Carcinogenesis 16: 1191-1194.

Wakabayashi, K., Nagao, M., Ochiai, Tahira, T., Yamaizumi, Z. and Sugimura, T. 1985.  A mutagen precursor in Chinese cabbage, indole-3-acetonitrile, which becomes mutagenic on nitrite treatment.  Mutation Research 143: 17-21.

West, L., Tsui, I. and Haas, G. 2002.  Single column approach for the liquid chromatographic separation of polar and non-polar glucosinolates from broccoli sprouts and seeds.  J. Chromatography A 966: 227-232.

Zhang, Y.S. and Talalay, P. 1998.  Mechanism of differential potencies of isothiocyanates as inducers of anticarcinogenic phase 2 enzymes.  Cancer Res. 58: 4632-4639.

Zhang, Y.S., Talalay, P., Cho, C.G. and Posner, G.H. 1992.  A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure.  Proc. Natl. Acad. Sci. USA 89: 2399-2403.

 

Chapter 4 

Glucosinolate composition of mature tissue versus seeds and sprouts

 

Introduction

A preliminary report by Fahey et al. (1997) indicated that the anticancer activity (phase 2 enzyme inducer potencies) of extracts of young sprouts from 15 species of brassicaceae were 10 to 100 times those of mature field-grown plants.  The current chapter investigates the changes in glucosinolate profile with growth, and its impact on the anticancer potential, in mature vegetables of 13 species of the brassicaceae, in comparison to that observed in seed and sprouts.    

 

Materials and methods

Plant Material

Commercially available seed used in the seed and sprout studies (Chapters 2 and 3), were sown in soil and grown to maturity using standard farming practices.  Species included: broccoli (Brassica oleraceae var. italica), white cabbage (B. oleracea var. capitata), kohl rabi (B. oleracea var. gongylodes), choy sum (B. rapa var. parachinensis), komatsuna (B. rapa var. perviridis), Japanese turnip (B. rapa var. rapifera), mizuna (B. rapa var. nipposinica), Chinese broccoli (B. alboglabra), garden cress (Lepidium sativum), rocket (Eruca sativa), wasabi (Wasabia japonica), daikon and red radish (Raphanus sativus).

 

Glucosinolate extraction and analysis

At maturity, tissue was quickly chopped or grated, weighed and immediately added to boiling water for 6 minutes to inactivate myrosinase-induced degradation of glucosinolates.  Samples were homogenised with an Ultra-Turrax for two minutes and centrifuged for 15 minutes at 14000 rpm.  The supernatant was collected and filtered through Whatmans No.1 filter paper.  The filtrate was made up to 20-300 ml (depending on initial weights and volumes) with distilled water and refiltered through a 0.2 m syringe filter.  The commonly edible tissues used in the study were root tissue for radish, daikon and turnip; leaves for rocket, cabbage, mizuna, choy sum and komatsuna; inflorescence for broccoli; stem for wasabi and kohl rabi; shoots for garden cress; and floral shoots for Chinese broccoli.

 

Supernatants were analysed for glucosinolates by HPLC-UV and HPLC-MS using a Prevail C18 column as described by West et al. (2002).  The mobile phase was a linear gradient from 100% 50 mM ammonium acetate to 50 mM ammonium acetate-methanol (80:20) in 40 minutes.  The column was operated at ambient temperature with a flow-rate of 1 ml/min.  Preliminary peak identification was based on the molecular weight of the daughter [M-H]- anion released during FAB (fast atom bombardment).  Quantification of individual glucosinolates was initially based on commercially available high-purity (99.3%) sinigrin.  Differences in the relative integrated absorbance areas for glucosinolates of an equimolar concentration of sinigrin were calculated for 12 glucosinolate standards.

 

Determination of anti-cancer potential

A crude determination of 'Anti-cancer potential' was made based on the 'CD values' of the isothiocyanate derivative of the parent glucosinolates present in a species, and the concentration of the individual glucosinolate.  The 'CD value' for an isothiocyanate is defined as the concentration (M) of an isothiocyanate required to double quinone reductase (QR) activity (Talalay et al., 1995).  Anti-cancer potential of a species was quantified by dividing the concentration of the isothiocyanate present by its respective CD value. CD Values were taken from published studies (Talalay et al., 1988; Zhang et al., 1992; Posner et al., 1994; Tawfiq et al., 1995; Fahey et al., 1997; Faulkner et al., 1998; Zhang and Talalay, 1998; Rose et al., 2000).  Where CD values differed between studies, an average value was taken.  CD values for glucodehydroerucin, gluconapolieferin and epiprogoitrin were estimated to be similar to glucoerucin, glucobrassicanapin, and progoitrin, respectively. 

 

Results and Discussion

The glucosinolate profile of mature tissue differed to that seen in sprouts and seed.  In Table 1, bold values represent the appearance of glucosinolates not detected in either sprouts or seeds, whereas dashed spaces indicate a disappearance of glucosinolates that were present in both the sprouts and seeds for the various species tested.

 

Table 1 - Glucosinolate composition and concentration (mol/gFW) of mature tissues (glucosinolates in italics are presented as mol sinigrin equivalent/gFW).  CD value corresponds to the cited concentration of isothiocyanate derivatives required to double the activity of the phase 2 enzyme, quinone reductase, in Hepa 1c1c7 murine hepatoma cells.

 

Glucosinolate

 

CD value

 

red radish

daikon

broccoli

kohl rabi

garden cress

rocket

Chinese broccoli

cabbage

choy sum

mizuna

Japanese turnip

komatsuna

wasabi

glucosiberin

0.2

 

 

 

 

 

 

 

 

 

 

 

 

---

glucoraphenin

0.4

0.1

0.6

 

 

 

 

 

 

 

 

 

 

 

glucoraphanin

0.2-0.8

 

 

5.2

0.6

 

3.8

1.2

0.5

0.3

 

 

 

 

glucohesperalin

0.5

 

 

 

 

 

 

 

 

 

 

 

 

---

glucoalyssin

0.95

 

 

 

 

 

 

 

 

0.1

0.1

<0.1

<0.1

 

glucoberteroin

1.7

 

 

 

 

 

 

 

 

 

0.1

0.1

 

---

glucoiberin

1.8-2.4

 

 

 

---

 

 

 

0.9

 

 

 

 

 

glucoerucin

2.3

 

 

---

0.4

 

---

0.2

0.1

 

<0.1

 

 

 

glucodehydroerucin

2.3

1.3

3.4

 

 

 

 

 

 

 

 

 

 

 

glucotropaeolin

2.0-3.0

 

 

 

 

6.5

 

 

 

 

<0.1

 

 

 

glucoiberverin

3.5

 

 

 

 

 

 

 

<0.1

 

 

 

 

 

gluconasturtiin

5

 

 

 

 

 

 

 

 

0.1

0.1

1.1

<0.1

 

sinigrin

6.1-8.3

 

 

 

 

 

 

0.6

0.3

 

 

 

 

19.7

gluconapin

7.4

 

 

 

 

 

 

2.9

0.1

0.1

0.4

0.5

0.4

0.2

glucobrassicanapin

>15

 

 

 

 

 

 

 

 

0.4

1.1

0.2

0.1

0.3

gluconapolieferin

>15

 

 

 

 

 

 

 

 

0.2

 

 

 

 

4-hydroxyglucobrassicin

>15

---

---

---

---

 

 

<0.1

---

 

 

 

 

 

progoitrin

47.7

 

 

 

 

 

 

 

 

0.1

---

---

0.4

 

glucobrassicin

76.8

 

 

0.2

 

 

 

 

0.2

 

0.1

 

0.1

 

neoglucobrassicin

unknown

 

 

 

 

 

 

 

0.1

 

 

 

 

0.5

mercaptobutylGS

unknown

 

 

 

 

 

2.8

 

 

 

 

 

 

 

 

Of particular interest in mature tissue are the indole-type glucosinolates, with glucobrassicin (broccoli, mizuna and komatsuna) and neoglucobrassicin (wasabi) showing an appearance while 4-hydroxyglucobrassicin is no longer detectable (red radish, daikon, broccoli, kohl rabi and cabbage) (Table 1).  The appearance of glucobrassicin and neoglucobrassicin in the mature tissue of broccoli was also observed by Fahey et al. (1997).  Gluconasturtiin is another glucosinolate that was detected in mature tissue but not in seed or sprouts for a number of species (choy sum, mizuna, turnip and komatsuna).

 

In situations where a particular glucosinolate is retained from seed to sprout to mature tissue: (glucoiberin in cabbage; progoitrin in choy sum and komatsuna; sinigrin in Chinese broccoli, cabbage and wasabi, glucoraphanin in broccoli, rocket and kohl rabi; glucoraphenin in daikon and radish; gluconapin in turnip, mizuna, komatsuna, Chinese broccoli and choy sum; glucobrassicanipan in turnip, mizuna, komatsuna, and choy sum; glucoalyssin in turnip; 4-hydroxyglucobrassicin in Chinese broccoli; glucotropaeolin in garden cress, glucoerucin in mizuna; glucodehydroerucin in daikon; and glucoberteroin in mizuna) the content generally declined (see also Table 1 in chapters 2 and 3), except for mizuna where there was an increase in glucobrassicanapin from sprout to mature tissue; in rocket where the glucoraphanin level in the sprout was similar to that in rocket leaf; in komatsuna were the seed value for progoitrin was similar to that for the sprout; and for wasabi where the sinigrin content increased from sprout to mature tissue.  In other studies, Rangkadilok et al. (2002) observed no significant difference in the sinigrin content of seeds and 7-day-old seedlings for five cultivars of mustard, however the sinigrin content declined from seedling to the early flowering stage of the mature mustard plants.  With respect to glucoraphanin and broccoli, our results are similar to those of Fahey et al. (1997) and Rangkadilok et al. (2002) who monitored declines in glucoraphanin content as broccoli seed sprouted and developed into mature plants. 

 

The trend for a decline in glucosinolate content can also be seen for total glucosinolate content as seeds continued to grow (Table 2).  The total glucosinolate content was observed to decline in all species from seed to sprout to mature tissue except for wasabi and rocket, where wasabi recorded an increase from sprout to stems, while rocket recorded similar values for sprout and leaves.  These exceptions reflect the change in individual glucosinolates described in the previous paragraph for these two species. 

 

The decline in total glucosinolate concentrations is reflected in the anticancer potential index for seed, sprout and mature tissue, with mature tissue having the lowest potentials while seed has the highest values (Table 2).  Fahey et al. (1997) commented that anticancer activity (inducer potencies) of extracts from young sprouts of broccoli, cabbage, Chinese cabbage, daikon, kohlrabi, turnip and cress ranged from 10 to 100 times those of mature field-grown plants, although data was not presented.

Table 2. Progressive change in glucosinolate concentration, anti-cancer potential, and the main glucosinolate present, with growth from seed to sprout to mature vegetable.

 

 

Total Glucosinolates

(mol·g-1 FW)

Anti-cancer

potential

Main glucosinolate

( % of total glucosinolates)

 

Seed

Sprout

Mature

Seed

Sprout

Mature

Seed

Sprout

Mature

Radish

389

34

1

945

65

1

GRE

97

GRE

72

GDE

100

Daikon

376

28

4

899

24

3

GRE

94

GDE

78

GDE

85

Broccoli*

390

40

5

855

(177)

86

(17)

13

(3)

GRA

86

GRA

84

GRA

96

Rocket

126

8

7

86

9

10

GE

88

GRA & 4MB

47 & 53

GRA & 4MB

58 & 42

Kohl rabi*

150

25

1

237

(47)

43

(9)

2

(<1)

GRA

57

GRA

64

GRA

60

Wasabi

44

6

20

20

4

3

Sin

76

Sin

79

Sin

98

Garden cress*

118

12

7

47

(9)

5

(1)

3

(1)

GTP

100

GTP

100

GTP

100

Cabbage*

91

15

2

29

(6)

6

(1)

2

(<1)

GI

51

GI

72

GI

45

Chinese broccoli*

212

31

5

28

(6)

7

(1)

4

(1)

GN

76

GN

75

GN

59

Mizuna*

96

13

2

28

(6)

2

(<1)

<1

(<1)

GN

69

GN

63

GBC

55

Choy sum*

144

15

1

26

(5)

1

(<1)

1

(<1)

GN

75

GN

62

GBC

31

Turnip*

122

14

2

15

(3)

2

(<1)

<1

(<1)

GN

80

GN

80

GNU

55

Komatsuna*

72

15

1

8

(2)

2

(<1)

<1

(<1)

GN

85

GN

86

GN & GI

36 & 36

 

Although the negative impact of epithiospecifier protein (ESP) (which diverts the glucosinolate hydrolysis product from isothiocyanates into impotent nitriles) is often overlooked in broccoli studies, we have included its impact in Table 2 as an 80% reduction in anti-cancer potential (bracketed values) for species thought to contain this cofactor (species with *).  The proposed presence of ESP significantly changes the ranking of species in the anti-cancer index with rocket moving up a place and wasabi moving up five places as shown in Table 2 (unbracketed values).  Taking into account the presence of ESP, the ranking from highest to lowest for the top seed or sprouts with anticancer potential are; radish, daikon, broccoli, rocket, kohl rabi and wasabi.  In mature tissue the ranking is different with the order being; rocket, daikon/broccoli/wasabi, radish/garden cress/Chinese broccoli.  

 

Apart from total glucosinolate content having an impact on anti-cancer potential, the change in glucosinolate profile also can have an effect.  Table 2 shows the main glucosinolate present in each species at the seed, sprout and mature tissue stage as well as what percentage that glucosinolate is of the total glucosinolate content.  In the case of broccoli, kohl rabi, wasabi, garden cress, cabbage, and chinese broccoli the main glucosinolate is the same in seed, sprout and mature tissue.  But in radish, daikon, rocket, mizuna, choy sum, turnip and komatsuna the main glucosinolate present changes in the sprout or mature tissue from what was present in the seed.

 

Interestingly, the anticancer activity of the main glucosinolate present in each species shows a correlation with the anticancer potential index rating for the various species.  The main glucosinolates present in the top five ranking species possess either a high (glucoraphanin GRA, glucoraphenin GRE) or moderate (glucodehydroerucin GDE) phase 2 enzyme activity.  Glucotropaeolin (GTP) found in garden cress and glucoiberin (GI) found in cabbage have moderate phase 2 enzyme activity, while the main glucosinolates (gluconapin GN, glucobrassicanapin GBC, gluconasturtiin GNU) found in the lower ranked species of Chinese broccoli, mizuna, choy sum, turnip and komatsuna, have low phase 2 enzyme activity.  Out of place appears to be the sixth ranked species wasabi containing mainly sinigrin, a low rating enzyme inducer.  However, in wasabi seed, 48% of the anticancer potential is coming from glucohesperalin (11% of the total glucosinolates) with only 23% of the potential coming from sinigrin.  In wasabi sprouts, 38% of the anticancer potential is coming from glucohesperalin (12% of the total glucosinolates), 44% from glucosiberin (5% of the total glucosinolates) with only 17% from sinigrin.  Glucohesperalin and glucosiberin are both high inducers of phase 2 enzyme activity (see CD values in table 1).

 

Unlike our result for mature broccoli where glucoraphanin constituted 96% of the total glucosinolate content, Fahey et al. (1997) found the main glucosinolate present in mature broccoli to be 50% glucobrassicin while glucoraphanin constituted only 32%.  Hansen et al. (1995) found the main glucosinolate to be glucoraphanin at 71% of the total glucosinolate content with glucobrassicin constituting just 16% of the total content.  Possibly shedding some light on conflicting results is the study by Rodrigues & Rosa (1999), which showed the main glucosinolate present in mature broccoli fluctuated depending on whether the primary (52% glucoraphanin and 26% glucobrassicin) or secondary inflorescences (39% glucobrassicin and 36% glucoraphanin) were monitored.

 

This study supports the report of Fahey et al. (1997) that sprouts have a greater anti-cancer potential for inducing phase 2 enzyme activity than mature vegetables.  The top four ranked species for anticancer potential in sprouts (radish, daikon, broccoli, rocket) were also represented in the top mature vegetable performers.  The potentially most potent mature vegetables were rocket, broccoli, daikon and wasabi, with mature rocket actually outperforming rocket sprouts.  

 

Literature cited

Fahey, J.W., Zhang, Y.S. and Talalay, P. 1997.  Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Natl. Acad. Sci. USA 94: 10367-10372.

Faulkner, K., Mithen, R. and Williamson, G. 1998.  Selective increase of the potential anticarcinogen 4-methylsulphinylbutyl glucosinolate in broccoli.  Carcinogenesis 19: 605-609.

Hansen, M., Moller, P. and Sorensen, H. 1995.  Glucosinolates in Broccoli Stored under Controlled Atmosphere.  J. Amer. Soc. Hort. Sci. 120: 1069-1074.

Posner, G.H., Cho, C.G., Green, J.V., Zhang, Y.S. and Talalay, P. 1994.  Design and synthesis of bifunctional isothiocyanate analogs of sulforaphane: correlation between structure and potency as inducers of anticarcinogenic detoxification enzymes.  J. Med. Chem. 37: 170-176.

Rangkadilok, N., Nicolas, M.E., Bennett, R.N., Premier, R.R., Eagling, D.R. and Taylor, P.W.J. 2002.  Developmental changes of sinigrin and glucoraphanin in three Brassica species (Brassica nigra, Brassica juncea and Brassica oleracea var. italica).  Scientia Horticulturae 96: 11-26.

Rodrigues, A.S. and Rosa, E.A.S. 1999.  Effect of post-harvest treatments on the level of glucosinolates in broccoli.  J. Sci. Food Agric. 79: 1028-1032.

Rose, P., Faulkner, K., Williamson, G. and Mithen, R. 2000.  7-methylsulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes.  Carcinogenesis 21: 1983-1988.

Talalay, P., De Long, M.J. and Prochasta, H.J. 1988.  Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis.  Proc. Natl. Acad. Sci. USA 85: 8261-8265.

Talalay, P., Fahey, J.W., Holtzclaw, W.D., Prestera, T. and Zhang, Y.S. 1995.  Chemoprotection against cancer by phase 2 enzyme induction.  Toxicol. Lett. 82/83: 173-179.

Tawfiq, N., Heaney, R.K., Plumb, J.A., Fenwick, G.R., Musk, S.R.R. and Williamson, G. 1995.  Dietary glucosinolates as blocking agents against carcinogenesis: glucosinolate breakdown products assessed by induction of quinone reductase activity in murine hepa1c1c7 cells.  Carcinogenesis 16: 1191-1194.

West, L., Tsui, I. and Haas, G. 2002.  Single column approach for the liquid chromatographic separation of polar and non-polar glucosinolates from broccoli sprouts and seeds.  J. Chromatography A 966: 227-232.

Zhang, Y.S. and Talalay, P. 1998.  Mechanism of differential potencies of isothiocyanates as inducers of anticarcinogenic phase 2 enzymes.  Cancer Res. 58: 4632-4639.

Zhang, Y.S., Talalay, P., Cho, C.G. and Posner, G.H. 1992.  A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure.  Proc. Natl. Acad. Sci. USA 89: 2399-2403.

 

Chapter 5 

Effect of sprout growth, growing temperature and cultivar on glucosinolate composition using daikon sprouts as a model

 

Introduction

Although we have determined glucosinolate levels in seed, sprout and mature tissue of many brassicaceous vegetables, other factors (genetic, physiological and environment) can impact on final glucosinolate level and subsequent anti-cancer potential.  As a good candidate for anti-cancer potential, we chose two different cultivars of white radish (daikon) as a model to determine the effect of growing temperature, growth stage and cultivar on the final glucosinolate composition of sprouts. 

 

Materials and Methods

Plant Material, Sprouting Conditions and Growing Temperature

Commercially available seed of white radish (Raphanus sativus) was obtained: 'Minowase Summer Hybrid', and an un-named variety of winter daikon. Seed were rinsed in distilled water and then sanitised for 2 hours in 2.1g·L-1 Na2HClO3 .  50 seeds were dispensed into individual transparent and perforated 100ml plastic cups lined with thin dampened muslin cloth.  Seed-sprouts were grown at three temperature regimes (13, 20 and 27°C) in continuous 24hr lighting (30 mol·s-1·m-2) and washed with 10ml of distilled water twice a day.   At 2, 4, 6, 8, 10 and 12 days after sowing, a cup was randomly selected from each temperature regime and sprouts prepared for glucosinolate analysis.

 

Glucosinolate extraction and analysis

Approximately 0.5g of seed (30 and 60 seeds for summer and winter radish, respectively) or intact sprouts (between 3 to 25 sprouts depending on sprout age) were weighed and added to approximately 25ml boiling water for 6 minutes to inactivate myrosinase enzyme activity.  Samples were homogenised with an Ultra-Turrax (IKA Labortechnik) for two minutes and then centrifuged for 15 minutes at 14000rpm.  The supernatant was collected and filtered through a Whatmans No.1 filter paper.  The filtrate was made up to 20ml with distilled water and refiltered through a 0.2m syringe filter. Supernatants were analysed for glucosinolates by HPLC-UV and HPLC-MS as described by West et al. (2002).  Quantification of individual glucosinolates was initially based on commercially available high-purity (99.3%) sinigrin (Fluka) and expressed as sinigrin-equivalents.  Because of the difficulty in obtaining standards for individual glucosinolates in sufficient amount for routine standard curves, the relative integrated absorbance areas for equimolar concentrations of glucoraphanin, glucoraphenin and glucoerucin (Glucosinolate.com, Denmark) was calculated.  Conversion factors were then used to convert sinigrin-equivalents to actual glucosinolate concentrations.  In the case of glucodehydroerucin where no standard was available, the conversion factor for glucoerucin was used.

 

Results and Discussion

The main glucosinolates present in white radish seeds and sprouts were glucoraphenin and glucodehydroerucin, with trace amounts of glucoraphanin and 4-hydroxyglucobrassicin (data not shown).  These findings are in agreement with West et al. (2004) and Barillari et al. (2005). Isothiocyanate products from glucoraphenin and glucoraphanin are potent inducers of phase 2 enzymes, and although no data is available in the literature, glucodehydroerucin is estimated to have about one tenth the potency, and 4-hydroxyglucobrassicin no negligible effect. 

 

Both 'Minowase Summer Hybrid' and winter daikon sprouts showed a significant decline in glucoraphenin content with increasing sprout age, with both cultivars exhibiting similar levels of glucoraphenin with growth.  Decline was more rapid as growth temperature was increased from 13 to 27°C (Fig. 1A&B) which corresponded with rate of sprout growth.  Similarly, glucoraphanin and 4-hydroxyglucobrassicin fell significantly to less than 3% of their initial seed value, with decline more rapid at higher growth temperature (data not shown).  Decline in glucoraphenin content with sprout growth has also been observed in radish sprouts by Barillari et al. (2005).  In broccoli, Fahey et al. (1997) also reported a decline in glucosinolate content from seed to mature sprout and assigned this to cellular dilution. In the current trial, comparison of data on a dry weight basis (data not shown) indicated that decline was closely associated with decline in dry matter percentage due to cell expansion.

Figure 1. Influence of sprout age on glucosinolate content in summer (A&C) and winter (B&D) white radish sprouts grown at 13°C (), 20°C (), and 27°C ().  Data are means of 3 replicates. 

 

In contrast to glucoraphenin, glucodehydroerucin levels in radish sprouts increased with sprout growth, with the rise more rapid at the higher growth temperatures (Fig. 1C&D).  Barillari et al. (2005) also reported an increase in glucodehydroerucin with radish sprout growth.  'Minowase Summer Hybrid' daikon glucodehydroerucin content increased to a peak at day 4 and day 6 for 27 and 20°C, respectively, before falling to half these peak values by day 12 (Fig. 1C).  Glucodehydroerucin values for the 13°C 'Minowase Summer Hybrid' daikon treatment increased to a plateau (Fig. 1C): a trend seen for all three growth temperatures of the winter daikon variety (Fig. 1D).  Winter daikon glucodehydroerucin levels were generally lower than the 'Minowase Summer Hybrid' daikon levels at all sprout ages for each growth temperature (Fig. 1C&D). 

 

The increase in glucodehydroerucin content with sprout age appeared to be due to de novo synthesis of this glucosinolate.  The observed peaking and subsequent decline of glucodehydroerucin is likely to be due to a concomitant dilution of cellular contents due to cell expansion.  Comparison of dry weight and fresh weight concentration of glucodehydroerucin supports this supposition (data not shown). Although Barillari et al. (2005) have postulated that glucodehydroerucin may be being converted from glucoraphenin, further studies with other radish cultivars support de novo synthesis as the major source of glucodehydroerucin, rather than conversion. Furthermore, when data from the current trial is expressed on a per sprout basis (Fig 3) it can be seen that increase in glucodehydroerucin per sprout is greater than the concominant decrease in glucoraphenin.  Figure 2 also shows that in 'Minowase Summer Hybrid' daikon, higher growth temperature can increase the content of glucodehydroerucin with no apparent change in glucoraphenin content, suggesting in this instance an increase in de novo glucodehydroerucin synthesis. 

  

Figure 2. Influence of growth temperature on glucosinolate content in summer () and winter () white radish sprouts grown to a uniform size of 80mg.  Data are means of 3 replicates.

 

As sprouts grew at different rates depending upon temperature, effect of temperature on glucosinolate composition was compared for sprouts of uniform size (80mg).  'Minowase Summer Hybrid' sprouts reached 80mg at 9.5, 5.2 and 3.6 days and winter radish at 10.6, 6.0 and 2.8 days, when grown at temperatures of 13, 20 and 27°C, respectively.  Temperature and cultivar was found to have no significant effect on glucoraphenin content (Fig 2A), although other radish cultivars have been shown to vary in glucoraphenin concentration (data not shown).  While there was no effect of temperature on glucodehydroerucin in winter radish sprouts (Fig 2B), sprouts of 'Minowase Summer Hybrid' had higher glucodehydroerucin levels at 20 and 27°C than at 13°C. 

 

The above results show that growth temperature does have an impact on glucodehydroerucin content in 'Minowase Summer Hybrid' daikon.  A similar study with broccoli sprouts (Pereira et al., 2002) showed that growth temperature significantly lowered total methylthioalkyl glucosinolate content (glucoraphanin+glucoiberin+glucoerucin) when grown at 20°C rather than 13 or 33°C.  This study in combination with our results shows that growth temperature can have a significant effect on glucosinolate content, and this will be dependent upon the species and cultivar.

 

  

Figure 3. Influence of sprout age on glucosinolate content expressed on a per sprout basis in summer (A&C) and winter (B&D) white radish sprouts grown at 13°C (), 20°C (), and 27°C ().  Data are means of 3 replicates.

In conclusion, physiological age has a major impact on glucosinolate concentration in radish sprouts.  Higher growth temperatures can potentially alter glucosinolate concentration, although this appears to be cultivar-dependant.   The observed decline in glucoraphenin, glucoraphanin and 4-hydroxyglucobrassicin levels in both radish cultivars with increasing sprout age appears to be linked to cellular dilution as cells enlarge with growth and dry matter percentage declines.  In contrast, glucodehydroerucin increases, most likely due to de novo synthesis, rather than conversion from glucoraphenin, as has been previously suggested. Cultivar, sprout developmental stage and growing temperature appear to be critical factors determining the glucosinolate profile and potential anticancer activity of white radish sprouts.

 

In most of the species we have studied, glucosinolates have tended to decline with sprout growth.  In some circumstances, this may have positive health benefits - as in the case of reducing indole glucosinolates, such as 4-hydroxyglucobrassicin (potentially mutagenic), which is present in radish and most brassica species. Increases such as that seen in radish for glucodehydroerucin were less common, although we recorded (see Chapter 2) an increase in 4-mercaptobutyl glucosinolate (unknown induction capacity) in rocket sprouts.  Also worthy of note was the observation that progoitrin (reputedly goitrogenic) in some species did not decline (eg. komatsuna, tatsoi), despite cellular dilution of other glucosinolates.  This may indicate active synthesis of progoitrin in some species.

 

 

Acknowledgements

Preliminary findings from this chapter were presented as an oral presentation at the Australasian Postharvest Horticulture Conference held in Rotorua, New Zealand in 2005. 

 

Literature cited

Barillari, J., Cervellati, R., Paolini, M., Tatibouet, A., Rollin, P. and Iori, R. 2005.  Isolation of 4-methylthio-3-butenyl glucosinolate from Raphanus sativus sprouts (kaiware daikon) and its redox properties. J. Agric. Food Chem. 53: 9890-9896.

Fahey, J.W., Zhang, Y.S. and Talalay, P. 1997.  Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Natl. Acad. Sci. USA 94: 10367-10372.

Pereira, F. M. V., Rosa, E., Fahey, J. W., Stephenson, K. K., Carvalho, R., Aires, A., 2002. Influence of temperature and ontogeny on the levels of glucosinolates in broccoli (Brassica oleracea Var. italica) sprouts and their effect on the induction of mammalian phase 2 enzymes. J. Agric. Food Chem. 50: 6239-6244.

West, L., Tsui, I. and Haas, G. 2002.  Single column approach for the liquid chromatographic separation of polar and non-polar glucosinolates from broccoli sprouts and seeds.  J. Chromatography A 966: 227-232.

West, L.G., Meyer, K.A., Balch, B.A., Rossi, F.J., Schultz, M.R. and Haas, G.W. 2004.  Glucoraphanin and 4-hydroxyglucobrassicin contents in seeds of 59 cultivars of broccoli, raab, kohlrabi, radish, cauliflower, Brussels sprouts, kale, and cabbage.  J. Agric. Food Chem. 52: 916-926.

 

Chapter 6 

Effect of refrigeration on glucosinolate levels in sprouts

 

Introduction

Commercially, seed-sprouts are purchased in containers which are then kept in domestic refrigerators until consumed.  Although glucosinolate concentrations may be high at time of purchase, there appears to be no data documenting the stability of different glucosinolates during cold storage.  The present trial investigated changes in glucosinolate levels of four species of brassicaceous sprouts and the implications of domestic refrigeration on the content of bioactive glucosinolates.

 

Materials and methods

Plant Material, Sprouting Conditions and Cold Storage

Commercially available seed of broccoli (Brassica oleracea var. italica, BroccoSprout™), kohl rabi (B. oleracea var. gongylodes, 'Purple Vienna'), rocket (Eruca sativa, un-named variety), summer white radish (Raphanus sativus, 'Minowase Summer Hybrid') and winter white radish (Raphanus sativus, an un-named variety of winter daikon) were sanitised for 2 hours in 2500L·L-1 sodium hypochlorite, rinsed with tap water and sprouted for 7 days inside a growth chamber of a commercial hydroponic facility (24C, 24-hour lighting and a 3-hourly watering with a commercial nutrient solution [NZ Hydroponics]).   After 7 days, sprouts were cut from their root mats, divided into 8 lots weighing more than 4g each and placed in polypropylene bags (140mm x 200mm).  The bags were sealed, perforated for ventilation, weighed and placed at 4C in the dark simulating a domestic refrigerator (USDA 2005).  Bags were removed at 1, 4, 6, 7, 11(or 12), 14, 17, and 21 days for glucosinolate analysis (see below).  On removal, bags were re-weighed and the atmosphere within the bag (oxygen and carbon dioxide levels) monitored using a M.A.P. Test 4000 packaging atmosphere analyser (Hitech Instruments Ltd., Luton England) to ensure the bags had not developed modified atmospheres.  The entire experiment was replicated three times.

 

Glucosinolate extraction and analysis

Ten sprouts for radish varieties (0.15-0.22 g/sprout) and twenty sprouts for the species with smaller sprouts (0.02-0.04 g/sprout) were weighed and added to approximately 25ml boiling water for 6 minutes to inactivate myrosinase enzyme activity.  Sprouts were homogenised with an Ultra-Turrax (IKA Labortechnik) for two minutes and then centrifuged for 15 minutes at 14000rpm.  The supernatant was collected and filtered through a Whatmans No.1 filter paper.  The filtrate was made up to 20ml with distilled water and refiltered through a 0.2m syringe filter. Supernatants were analysed for glucosinolates by HPLC-UV and HPLC-MS as described by West et al. (2002). 

 

Quantification of individual glucosinolates was initially based on commercially available high-purity (99.3%) sinigrin (Fluka) and expressed as sinigrin-equivalents.  Because of the difficulty in obtaining standards for individual glucosinolates in sufficient amount for routine standard curves, the relative integrated absorbance areas for equimolar concentrations of glucoiberin, progoitrin, glucoraphanin, glucoraphenin and glucoerucin (Glucosinolate.com, Denmark) was calculated.  Conversion factors were then used to convert sinigrin-equivalents to actual glucosinolate concentrations.  In the case of glucodehydroerucin where no standard was available, the conversion factor for glucoerucin was used.

 

Results and Discussion

Of the five brassicaceous sprout samples tested (broccoli, kohl rabi, rocket, winter white radish and summer white radish), only rocket sprouts showed a significant change in glucosinolate levels over three weeks storage at 4°C (Fig. 1A).  In rocket, glucoerucin concentration declined after 1 week storage, while glucoraphanin declined after 2 weeks.  Changes in glucosinolate levels in broccoli, kohl rabi, winter white radish and summer white radish sprouts were not significant (Fig. 1B-1E), despite glucoerucin and glucoraphanin being also present in both broccoli and kohl rabi (Table 1).  Weight loss measurements generally were in the range of 0.2-2.1%, indicating loss of moisture was very small and therefore likely to have minimal impact on metabolism.  Similarly, measurement of oxygen and carbon dioxide within bags remained in the range 20.0-20.6% and 0.3-0.8%, respectively, indicating a non-modified atmosphere and aerobic conditions.

 

 

  

Figure 1. Glucosinolate content (glucoerucin , glucodehydroerucin , glucoraphanin , glucoraphenin , and glucoiberin ) in 7-day-old seed-sprouts of rocket (A), broccoli (B), kohl rabi (C), winter white radish (D) and summer white radish (E) during storage at 4C for 21 days.  Data are means of 3 replicates.  Least significant differences (p≤0.05) are indicated by vertical bars.

Glucoerucin and glucoraphanin have been reported to be the major glucosinolates present in rocket sprouts (Barillari et al. 2005).  The major glucosinolate, 4-mercaptobutyl glucosinolate, found in rocket leaves by Bennett et al. (2002) was not detected in sprouts in the present study or by Barillari et al (2005).  Although we did not detect 4-mercaptobutyl glucosinolate in the current study, we have found it to be a major component in sprouts of at least one rocket cultivar (Yates Australia), which would indicate that there is some variation in glucosinolate components between different rocket varieties.

 

Seven-day old broccoli sprouts used in this experiment contained glucoraphanin, glucoiberin and glucoerucin (Table 1), with trace amounts of sinigrin and progoitrin (data not shown).  This profile is similar to that reported by West et al (2002) whose HPLC protocol was used in our experiments.  The additional glucosinolates (4-hydroxyglucobassicin and glucoibervirin) present in 2-3 day old broccoli sprouts in the West study were not detected in the present 7 day old sprouts, although we have previously detected these in younger sprouts (2-3 days old) (data not shown).

 

Glucoraphanin was the major glucosinolate found in 7 day old broccoli sprouts, in agreement with previous reports by Fahey et al. (1997); Pereira et al. (2002) and West et al. (2002).  However, glucoraphanin, glucoiberin and glucoerucin concentrations of 1.70, 0.55 and 0.35 mol sinigrin equivalent·g-1 FW respectively (Table 1) were low when compared with other broccoli cultivars.  Although the differences may have been due to cultivar variation, the low values in our study may have been due to lower initial glucosinolate levels in the seed.  Measured glucoraphanin for the BroccoSprouts™ broccoli seed used in this study was 22.6 mol·g-1 which is approximately half the level quoted for BroccoSprouts™ broccoli seed in West et al. (2004). 

 

Table 1.  Average glucosinolate contents ( SE) of 7-day-old seed-sprouts stored at 4°C.  Values are also expressed as 'sinigrin-equivalents' for comparison with other studies (GR, glucoraphanin; GE, glucoerucin; GI, glucoiberin; GRe, glucoraphenin; GDE, glucodehydroerucin).

 

 

Glucosinolate concentration

 

mol·g-1 FW actual glucosinolate

 

mol·g-1 FW sinigrin equivalent

 

broccoli

kohl

rabi

winter

radish

summer

radish

 

broccoli

kohl

rabi

winter

radish

summer

radish

GR

5.55

6.36

 

 

 

1.70

1.95

 

 

(1.53)

(1.67)

 

 

 

(0.47)

(0.51)

 

 

 

 

 

 

 

 

 

 

 

 

GE

0.53

0.72

 

 

 

0.35

0.47

 

 

(0.38)

(0.41)

 

 

 

(0.25)

(0.27)

 

 

 

 

 

 

 

 

 

 

 

 

GI

1.19

3.98

 

 

 

0.55

1.83

 

 

(0.27)

(1.93)

 

 

 

(0.12)

(0.89)

 

 

 

 

 

 

 

 

 

 

 

 

GRe

 

 

11.44

3.34

 

 

 

3.55

1.04

 

 

(2.62)

(1.53)

 

 

 

(0.81)

(0.47)

 

 

 

 

 

 

 

 

 

 

GDE

 

 

6.89

16.56

 

 

 

4.53

10.88

 

 

(1.81)

(3.98)

 

 

 

(1.19)

(2.62)

 

 

In the present study, winter white radish and summer white radish sprouts were found to contain principally glucoraphenin and glucodehydroerucin, although in significantly different concentrations (Table 1).  Summer white radish sprouts contained twice the concentration of glucodehydroerucin and less than half the glucoraphenin content of winter white radish.  Apart from indicating there may be considerable variation in glucosinolate contents between radish cultivars, it also impacts on potential anti-cancer or anti-mutagenic activity, as the isothiocyanate derived from glucoraphenin would appear to have greater potency than that derived from glucodehydroerucin (Posner et al. 1994; Nakamura et al. 2001).

 

The present study indicates that storage of sprouts at 4°C, as recommended for domestic refrigerators by the USDA (2005), is unlikely to have significant affect on endogenous glucosinolate concentrations of broccoli, white radish or kohl rabi sprouts.  However, due to the limited number of replicates performed, these finding are tentative.  By contrast, rocket sprouts exhibited significant decline in glucoraphanin and glucoerucin, both of which have been shown to have derivatives with anti-cancer potential.  Consequently, if glucosinolate concentration is to be maximised, rocket sprouts should be consumed soon after purchase.

 

Acknowledgements

The work presented in this chapter is part of a paper that has been accepted to be published in the journal 'Postharvest Biology and Technology'.  Preliminary findings from this chapter were also presented as an oral presentation at the Australasian Postharvest Horticulture Conference held in Rotorua, New Zealand in 2005. 

 

Literature cited

Barillari, J., Canistro, D., Paolini, M., Ferroni, F., Pedulli, G. F., Iori, R., Valgimigli, L., 2005. Direct antioxidant activity of purified glucoerucin, the dietary secondary metabolite contained in Rocket (Eruca sativa Mill.) seeds and sprouts. J. Agric. Food Chem. 53, 2475-2482.

Bennett, R. N., Mellon, F. A., Botting, N. P., Eagles, J., Rosa, E. A. S., Williamson, G., 2002. Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L. (salad rocket). Phytochemistry 61, 25-30.

Fahey, J. W., Zalcmann, A. T.;,Talalay, P., 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56, 5-51.

Fahey, J. W., Zhang, Y., Talalay, P., 1997. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Natl. Acad. Sci. U.S.A. 94, 10367-10372.

Nakamura, Y., Iwahashi, T, Tanaka, A., Koutani, J., Matsuo, T., Okamoto, S., Sato, K., Ohtsuki, K., 2001. 4-(methylthio)-3-butenyl isothiocyanate, a principal antimutagen in daikon (Raphanus sativus; Japanese white radish). J. Agric. Food Chem. 49, 5755-5760.

O'Hare, T.J., Wong, L.S., Force, L.E., Irving, D.E., 2006 Glucosinolate composition and anti-cancer potential of seed-sprouts from horticultural members of the Brassicaceae.  International Symposium on Human Health - Effects of Fruits and Vegetables, 17th-20th August 2005, Quebec, Canada.  Acta Hort. (in press).

Pereira, F. M. V., Rosa, E., Fahey, J. W., Stephenson, K. K., Carvalho, R., Aires, A., 2002. Influence of temperature and ontogeny on the levels of glucosinolates in broccoli (Brassica oleracea Var. italica) sprouts and their effect on the induction of mammalian phase 2 enzymes. J. Agric. Food Chem. 50, 6239-6244.

Posner, G. H., Cho, C-G., Green, J. V., Zhang, Y., Talalay, P., 1994. Design and synthesis of bifunctional isothiocyanate analogs of sulforaphane: correlation between structure and potency as inducers of anticarcinogenic detoxication enzymes. J. Med. Chem. 37, 170-176.

Rose, P., Faulkner, K., Williamson, G., Mithen, R., 2000. 7-methylsulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes. Carcinogenesis 21, 1983-1988.

USDA, 2005. http://www.fsis.usda.gov/News_&_Events/NR_051905_01

West, L. G., Meyer, K. A., Balch, B. A., Rossi, F. J., Schultz, M. R., Haas, G. W., 2004. Glucoraphanin and 4-hydroxyglucobrassicin contents in seeds of 59 cultivars of broccoli, raab, kohlrabi, radish, cauliflower, brussels sprouts, kale, and cabbage. J. Agric. Food Chem. 52, 916-926.

West, L., Tsui, I., Haas, G., 2002. Single column approach for the liquid chromatographic separation of polar and non-polar glucosinolates from broccoli sprouts and seeds. J. Chromatogr. A  966, 227-232.

 

Chapter 7 

Industry assessment of sprouts

 

Sprouts with greatest potential to induce phase 2 detoxification enzymes and hence had greatest anti-cancer potential included (in decreasing order): radish (or daikon), broccoli, kohl rabi, rocket, wasabi, kale, garden cress and Chinese broccoli.   

 

Radish or daikon sprouts

Radish and daikon sprouts had by far the highest anti-cancer potential of the sprouts tested, potentially 4-5 times higher than broccoli sprouts if the action of epithiospecifier protein (ESP) inhibiting full conversion of glucoraphanin to sulphoraphane in broccoli is confirmed.  Radish and daikon do not contain ESP.  Radish sprouts are hottest (pungent) at a young stage.  Both glucoraphenin and glucodehydroerucin are pungent.

 

One of the most positive attributes of radish or daikon sprouts is that they are already in the marketplace (either as a pure 100% product, or in a mix with alphalfa and possibly other sprouts), indicating they are already accepted on a culinary basis to some degree.  Most, if not all, radish sprouts are actually daikon sprouts, as this tends to be the cheapest seed in large amounts available.  Seed commonly is un-named and may come as leftover seed from overseas breeding programs.

 

Radish sprouts can vary in appearance (some have red stems, similar in colour to red radish), so product differentiation from other sprouts would be readily achievable.  Currently, most radish sprouts are sold at a 3 day stage, at which the leaves may be yellow or beginning to turn green if exposed to light (as on supermarket shelves).   Cultivars apparently also vary in growth rate and postharvest shelf-life, which can be difficult to modulate if using an unnamed variety.

 

Broccoli sprouts

Despite containing ESP, broccoli sprouts still had high anti-cancer potential relative to most other sprouts tested.  If a cultivar could be identified without ESP, broccoli sprouts would have higher anti-cancer potential than radish sprouts.  Currently, no broccoli cultivars have been identified.  Broccoli sprouts are described as having a peppery taste (attributable to glucoraphanin), and are not as pungent as radish sprouts.  At the same time, they have been described as not having a particularly nice flavour.  Broccoli is a 'Western' vegetable, but is very popular in Asian countries, and is a common stir-fry ingredient.

 

Broccoli sprouts were the first sprout to be identified as having potential to induce phase 2 enzymes, and have received considerable media attention in the United States.  In addition, a company 'BroccoSprouts' has been established marketing broccoli sprouts, in particular varieties said to be high in glucoraphanin.  Varietal names are not publicly available.  Broccoli and its principal glucosinolate, glucoraphanin, have had most research conducted in relation to anti-cancer activity.  In this regard, supporting evidence for high level health claims is more likely to be available in the short term for broccoli sprouts.

 

Broccoli sprouts are smaller in size than radish sprouts, and currently less readily available than radish sprouts.  Cost of seed has been cited as one restraint to growing broccoli sprouts in Australia, although imported seed (in bulk) can be cheaper.  Broccoli sprouts tend to be sold with green seed-leaves. 

 

Kohl rabi sprouts

Taxonomically, kohl rabi is related to broccoli (as with cauliflower, Brussels sprouts, kale, calabrese and cabbage) and also contains ESP which reduces its anticancer potential (about half that of broccoli sprouts).  It was included in the project, along with a number of other 'Western' vegetables for relative comparison. 

Kohl rabi is not a well known vegetable, and this is apparently a factor would could make marketing kohl rabi sprouts difficult.  It is probable that obtaining sufficient kohl rabi seed for commercial sprouting would also be currently difficult. 

 

Rocket sprouts & rocket leaves

Rocket is not strictly an 'Asian vegetable' as it originates from western Asia.  Rocket sprouts do not appear to contain ESP and their anti-cancer potential is moderate (about half that of broccoli sprouts).  Rocket is well-known by the Australian public, particularly as a salad leaf constituent. 

 

Interestingly, rocket leaves had a calculated anti-cancer potential similar or slightly higher than rocket sprouts.  This was the only situation where mature tissue was actually better than sprouts.  In a comparison of all mature vegetables tested, rocket leaves had highest anti-cancer potential (three times higher than daikon root, broccoli florets or wasabi stem).

 

The main negative with rocket in general is that the glucosinolates in rocket do not appear to store particularly well.  The reason for this is unknown, but it does not appear to be due to physiological breakdown of plant tissues.

 

Considering that rocket leaves have a similar health benefit to rocket sprouts, and they are already marketed as leaves, there would seem to be no added health value in developing rocket sprouts, apart from as a different means of delivery.  The name itself may serve as a selling point in juice bars - 'Rocket juice' to get you going in the morning.

 

Wasabi sprouts

Wasabi sprouts are an interesting alternative to wasabi (the pungent green substance often found in Japanese meals).  The difficulty with growing these however is likely to make their production minimal.  Firstly, the seed of wasabi, unlike most members of the brassica family, is 'wet' (as opposed to a hard seed), and probably linked to wasabi growing close to running water.  This means the seed has only a short life before it becomes unviable.  And secondly, the optimum growing temperature for wasabi is a low 14°C, above which it becomes more susceptible to disease. 

 

In Australia, mature wasabi stems are well sought after in the restaurant trade (much 'wasabi' found in restaurants is often not wasabi, but coloured horseradish).  Because of the lack of supply, mature wasabi commands a premium price, and so the economic incentive to grow wasabi sprouts is less apparent.  As seeds are not normally utilised, seed harvest is not currently practised, making a source of seed potentially difficult to find. 

 

Kale sprouts

Kale is grown in western countries and Asia as a vegetable, but is not generally recognised as an 'Asian vegetable'.  Although kale sprouts rated moderately for anti-cancer potential (it is actually related to broccoli), kale is one of the few vegetables which contains a goitrogenic (goitre-inducing) glucosinolate.  This is one of the reasons why kale as a mature vegetable has lost popularity in today's society.

 

Considering the perceived and potentially real health risks associated with kale, we would not recommend it as a sprout (sprouts with high levels of progoitrin included sensposai, choy sum and Japanese turnip).  Additionally, the many types of kale available and the large number of glucosinolate profiles present in these, would make it difficult to develop a consistent product.

 

Garden cress sprouts

Garden cress is a 'Western' vegetable sold as short stems.  As sprouts have a similar anti-cancer potential to stems there is no health benefit to growing sprouts.  Garden cress seeds also produce very small sprouts which may be difficult to market. 

 

Chinese broccoli sprouts

Chinese broccoli sprouts have similar anti-cancer potential to garden cress sprouts, and like garden cress, the sprout has a similar anti-cancer potential to the mature vegetable, which would preclude growing them for any greater health benefit.

 

Other brassicaceous sprouts

All other 'Asian' brassicaceous vegetables (pak choy, tatsoi, mizuna, komatsuna, senposai, choy sum, Chinese cabbage, Chinese mustard, Japanese turnip) and 'Western' brassicaceous vegetables (broccoli raab, cabbage, black mustard, white mustard) had lower anti-cancer potential to the above species.  Consequently, there is little justification to introduce these as a sprout based on health benefit.

 

Chapter 8

Addressing Regulatory Issues Relating to Anti-Cancer claims

 

All food claims in Australia and New Zealand are regulated by FSANZ (Food Standards Australia New Zealand) under Standard 1.2.7.  The standard is currently under review. 

 

In regard to health, claims can be broken down into: 

1.    General level claims (permitted where substantiated)

2.     High level health claims (prohibited unless approved by FSANZ)

3.     Therapeutic claims (prohibited)

 

In the case of glucosinolates and their chemoprevention properties (ability to induce detoxification enzymes to protect against carcinogens), only the first 2 types of claim are relevant.

 

General level claims include:

1.     nutrition content claims (eg. product contains glucosinolate; product contains glucoraphanin)

2.    general level health claims (eg. product contains calcium…good for strong bones; product contains glucoraphanin…with long-lasting antioxidant activity)

Nutrition content claims include both 'absolute' claims and 'comparative' claims (eg. radish sprouts contain 100 times more glucoraphenin than mature radishes).  For biologically active substances (such as glucosinolates, lycopene etc.), you can state on a product: "source of glucosinolate" or "contains glucosinolate" or similar relating to an individual glucosinolate.  This can be placed anywhere on the product, such as across the front label.  

 

A nutrient content claim cannot include a descriptor.  For example, you cannot say "Radish sprouts are a great source of glucosinolates".  Rather, you can say: "Radish sprouts are a natural source of glucosinolates".  Substantiation for claims is required to be held by the manufacturer of the food.  For nutrition content claims, the food must contain on average the level referred to in the claim, and to be preferably established using laboratory analysis with recognised and validated methods. 

 

Current advice from FSANZ for biologically active substances is that comparative claims (such as radish sprouts contain 100 times more glucoraphenin than mature radishes) cannot be made, as it is currently uncertain what level of increase constitutes a bioeffective dosage.  You can however, list the absolute level of the compound (eg. total glucosinolate, or individual glucosinolates of interest) on the nutrition label of the product.

 

General level health claims include both function claims and risk reduction claims that reference a non-serious disease/condition.  These are currently not possible for glucosinolates, as they must be accompanied by data for recommended daily intake, and what % a serving size contains.  For general level health claims, either authoritative generally accepted evidence should be held, pre-approved nutrient-function statements should be used, or FSANZ can make an assessment of all available evidence.  

 

High level health claims include:

1.  relationships between the food and a health effect or reference to a serious disease or biomarker (eg. product contains calcium…reduces risk of osteoporosis; product contains glucoraphanin…reduces risk of colorectal cancer).

High level health claims include both biomarker claims and risk reduction claims that reference a serious disease/condition.  Currently, there is no high level health claim approved for glucosinolates and reduction in risk of cancer in Australia, the UK, or the USA.  Based on the current research thrust, it is likely that the first high level health claims will be made for broccoli in regard to glucoraphanin/sulphoraphane and reduced risk of gastrointestinal cancers.

 

General media & Point of sale educational material

The general media such as television reports, radio interviews, magazine and newspaper articles are all valid methods of informing the general public of the links between certain foods and their corresponding health benefits.  Often, this has more impact than product labelling alone, and is not as constrictive as food labelling.  Product labelling is helpful however to support general health information released to the public.

 

Point of sale 'educational' material released in conjunction with a product by the company producing the product in likely to be seen as an extension of the 'label', and therefore health claims in such material may infringe current food standard regulations.   General health information by other sources however (eg. a free booklet on the "Benefits of sprouts to Health" by an independent nutritionist) would not infringe any regulation.

 

Conclusions

For products sold within Australia and New Zealand, current health claims in relation to glucosinolates are limited to absolute nutrient claims, such as "Radish sprouts are a natural source of glucoraphenin", together with levels of individual and/or total glucosinolates listed on the nutrition panel. Different regulations apply for other countries.

 

As increasing medical evidence becomes available, data can be assessed by FSANZ for both general level health claims and higher level health claims.  It is expected that the first claims will be in regard to broccoli and glucoraphanin.

 

Recommendations

Asian vegetables with anti-cancer potential

 

Based on the glucosinolate profiles of the Asian vegetable species tested in this project, daikon/red radish, rocket, and wasabi were estimated to have greatest anti-cancer potential in regard to inducing phase 2 detoxification enzymes.  Of these, daikon and red radish sprouts showed most potential, possibly outperforming broccoli sprouts by up to five times.

 

From an anti-cancer perspective, sprouts were by far the most viable potent source of glucosinolates.  Seeds, although higher in glucosinolates, contain erucic acid which is thought to have detrimental effects related to cardiovascular health, while mature vegetables tended to be 20-100 times lower in glucosinolates than their sprout equivalents. 

 

The exception to this was rocket, in which the leaves had similar levels to sprouts.  On the other hand, the glucosinolates in rocket did not have good postharvest stability, which is important as both salad leaves and sprouts are usually placed in refrigerators by consumers after purchase, rather than eaten immediately.  Other sprouts tested (daikon, broccoli, kohl rabi) showed good glucosinolate stability.

 

Daikon and red radish sprouts are also considered to be highly marketable, as sprout companies are already familiar with them, the public is familiar with radish (radish sprouts are generally grown from daikon seed), and apart from daikon radish sprouts potentially out-performing broccoli sprouts, they are considered to have a nicer flavour by many who have tasted both.

 

In regard to sprouts, growth stage had a major impact on glucosinolate concentration, with younger sprouts having higher glucosinolate concentration than older sprouts.  This, together with consumer preference should be kept in mind when developing a marketable product.  In regard to daikon sprouts, growing temperature did not impact significantly on anti-cancer potential, although cultivars did show different responses. 

 

It is likely that cultivar selection will influence anti-cancer potential, with some cultivars having inherently higher levels of glucosinolate, or a more potent profile of glucosinolates than others.  It is therefore recommended that cultivar performance for glucosinolate levels should be tested as it would for other characteristics.  Other factors such as cost and availability of seed, growth characteristics, appearance, flavour and postharvest shelf-life should also be taken into account.

 

Current Australian food regulations allow nutrient content claims in regard to glucosinolates, but not health claims.  Consequently, a claim on a label such as 'source of glucosinolate' or 'natural source of glucoraphenin' is permitted, together with the amount on the nutrient content table.  However, qualifiers such as 'rich source of glucosinolate' or actual health claims 'reduces risk of colorectal cancer' are not currently allowed.  General awareness of potential health benefits from glucosinolates can be made through the media, and often it is this, and not food labelling, that can influence purchase.