“Let Food be Thy Medicine”
Investigating Nutriceutical Properties of Cruciferous Vegetables
Jillian KonyndykA, Jenna TenneyA, Herb FyneweverB, and David KoetjeA
Departments of BiologyA and Chemistry & BiochemistryB, Calvin College, Grand Rapids, MI 49546
Objectives
• Develop and optimize methodologies to assess the effects
of different types of cooking on the nutriceutical
properties of cruciferous vegetables. These include:
1. A methodology for assessing myrosinase activity in
cruciferous sprouts before and after cooking.
2. A methodology for measuring antimicrobial activities
of isothiocyanates against gram-positive and gramnegative bacteria.
Introduction
• The Greek philosopher Hippocrates once said, “Let food
be thy medicine.” In recent years, studies have shown
that plant-based diets have significant health advantages.
In fact, the term “nutriceuticals” has been coined to
describe natural compounds that have both nutritional
and pharmaceutical benefits.
• Cruciferous vegetables, such as broccoli, radish, daikon,
watercress, and cauliflower, have significant nutriceutical
properties. Increased consumption of these vegetables
has been associated with a decreased risk of cancer
(Navarro et al. 2011), cardiovascular disease (Yuan et al.
2009), and infectious diseases (Sofrata et al. 2011).
• Cruciferous vegetables contain glucosinolates, secondary
metabolites that are hydrolyzed by myrosinase enzyme
to release bioactive isothiocyanates (ITCs, Figure 1).
Curiously, glucosinolates and myrosinases are located in
different cells and only come together to produce ITCs
when plant tissues are damaged, such as during chewing.
Assessing Myrosinase Activity
Method Development
•
Broccoli sprouts are reputed to have high gluosinolate
levels. Thus, we adopted this as our model system.
•
Production of glucose (Figure 1) in broccoli extracts
incubated with sinigrin (a glucosinolate) provides a
convenient means for measuring myrosinase activity.
•
Key components of our method include:
1. Removal of endogenous glucosinolates from broccoli
extracts using Amicon
Ultra 10kD spin columns.
Antibacterial Properties of Isothiocyanates
•
Model gram-positive (Staphylococcus epidermidis) and
gram-negative (Escherichia coli) bacteria were selected
for assessment of antibacterial activities of different
classes of ITCs: allyl-ITC (AITC) from broccoli, benzyl-ITC
(BITC) from garden cress, and phenethyl-ITC (PEITC)
from radish.
•
Bacteria were grown to A600 of 0.1, centrifuged, and resuspended in different ITCs. Growth curves (Figures 3
and 4) demonstrate that all these ITCs have at least
some antimicrobial activity and that both bacteria
species are more sensitive to BITC than to AITC or PEITC.
2. Use of 150 μM sinigrin
substrate in each
reaction.
3. Assessment of glucose
production via Amplex
Red (Invitrogen, Figure
2) spectroscopy.
Figure 2. Amplex Red glucose
assay is based on assessment of
resorufin absorbance. (invitorgen.com)
Effect of Cooking on Myrosinase Activity
• Alfalfa (negative control), broccoli, and radish sprouts
were prepared using the following common cooking
methods: raw, microwaving, boiling and steaming.
Table 1. Myrosinase activity after cooking of sprouts.
Figure 3. Effects of ITCs on
growth of E. coli.
Figure 4. Effects of ITCs on
growth of S. epidermidis.
Conclusions
Figure 1. Myrosinase-catalyzed production of isothiocyanates from
glucosinolates in cruciferous vegetables. (wikipedia.org)
• Our research focused on optimizing methodologies so
that undergraduate students in an introductory cellular
biology and genetics laboratory would be able to assess
the effects of different food preparation methods on the
production of ITCs in cruciferous vegetables.
Acknowledgment
This research was funded by grant #DUE-1140767 from
the National Science Foundation’s TUES program.
Interpretation of Results
• Broccoli sprouts, which are more tender than radish
sprouts, appear to be more sensitive to cooking
methods. This may reflect differences in cell damage.
• Since myrosinase activity declines after cooking, fewer
ITCs would be produced. This negatively affects the
nutriceutical quality of cruciferous vegetables.
References
•
•
•
• We have developed and optimized a new method for
measuring myrosinase activity in cruciferous vegetables.
• Cooking methods have different effects on myrosinase
activity of broccoli and radish sprouts. From our results,
we can infer that raw cruciferous vegetables are the best
sources of nutriceutical ITCs, and boiling has the most
serious detrimental effects on ITC availability.
• ITCs have antimicrobial activity. E. coli and S. epidermidis
are more sensitive to BITC than to AITC or PEITC.
Navarro SL, F Li, JW Lampe (2011) Mechanisms of action of isothiocyanates in cancer chemoprevention: an update. Food Funct 2: 579-587
Sofrata A, EM Santangelo, M Azeem, A-K Borg-Karlson, A Gustafsson, K Pütsep (2011) Benzyl isothiocyanate, a major component from the roots of Salvadora persica is highly active against gram-negative bacteria. PLoS One 6(8): e23045.
Yuan G, B Sun, J Yuan, Q Wang (2009) Effects of different cooking methods on health-promoting compounds of broccoli. J Zhejiang Univ Sci B 10(8):580-588.