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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
© 2005 Rural Industries Research and Development Corporation. All rights reserved.
ISBN 0 642 (…RIRDC
to assign)
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 Tim.O’Hare@dpi.qld.gov.au
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
PO Box 4776
Phone: 02
6272 4819
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.
Most of our publications are available for viewing, downloading or purchasing online through our website:
downloads at www.rirdc.gov.au/fullreports/index.html purchases at www.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 ivAcknowledgements 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.
MethodologyApproach 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 glucosinolatesA 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).
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