J Life Sci Res 2021; 18: 15-19© 2021 Osaka Prefecture University

Journal of Life Science Research
Online ISSN 2186-5809

Rapid paper

Irreversible degradation of vitamin C in broccoli during cryopreservation
Momoko YAMATO1, Midori SEIKE1, Akira WADANO2, Hiroshi INUI1, Toshio IWAKI † 1
1

Department of Clinical Nutrition, Faculty of Comprehensive Rehabilitation, Osaka Prefecture University, 3-7-30 Habikino, Osaka, Japan

2

Center for Research and Development of Bioresources, Osaka Prefecture University, 1-2, Gakuen-cho, Sakai, Osaka, Japan

Received 5 November 2021; accepted 15 November 2021Key words: ascorbic acid; dehydroascorbic acid; diketogulonic acid; food storage

enzymatically (Fig. 1). 5-8 The redox system is known as the1 Introduction
Ascorbic acid (ASC) or vitamin C is a water-soluble

glutathione/ascorbate cycle and is involved in the decomposition

vitamin with antioxidant properties, and its physiologicalof hydrogen peroxide and various hydroxylation reactions. Botheffects have long been recognized as antiscorbutic. 1, 2 ASC

ASC and DHA are known to act as vitamin C in vivo, and thepossesses many physiological effects, including its function as

total amount of ASC is described as vitamin C in the standard

a cofactor for enzymes that polymerize collagen and synthesize

tables of food composition in Japan. However, it has recently

catecholamines, and its clinical use based on pharmacologicalbeen shown that some DHA is metabolized to 2,3-diketoguloniceffects for cancer therapy.3, 4acid (DKG) via irreversible hydrolysis. DKG is not as active as

Vitamin C is typically present in the anionic state in thevitamin C and is further degraded to threonate and erythrulose

cell and produces monodehydroascorbic acid (MDA) during

under oxidative stress.9 The degradation of DHA in the metabolicoxidation. MDA is either reduced back to ASC by reductase orpathway of ASC has not been considered, and little is known

broken down to produce one molecule of ASC and one molecule

about the synthesis and degradation of DKG. However, thereof dehydroascorbic acid (DHA). DHA is then reduced to ASC

have been several reports on the toxicity of DKG due to its highby reductase or thiol compounds such as glutathione, non-reactivity with proteins.23-25

Fig. 1NADHNAD+GSSG

GSH

Dehydroascorbic acid reductase(DHAR)

Dehydroascorbic acid (DHA)

Ascorbic acid (ASC)Ascorbic acid peroxidase (APX)or Ascorbic acid oxidase (AAO)

H 2O

e-NADP+NADPH

2,3-Diketogulonic acid(DKG)

Monodehydroascorbate
reductase (MDAR)

Monodehydroascorbic acid (MDA)

L-Threonate, L-Erythrulose + oxalate

Fig. 1 Ascorbic acid metabolic pathway.
†Corresponding author, E-mail: iwaki@rehab.osakafu-u.ac.jp

16

In this study, we focused on the degradation pathway ofAfter incubation, 500 μL of ethyl acetate was added to each tube,

ascorbic acid in vegetables for clarifying the changes in theand the tubes were shaken at 200 rpm for 5 min and centrifugeddynamics of ascorbic acid under various storage conditions. Theat 12,000 × g for 2 min. The ethyl acetate layer and the aqueouspresent results will provide the important knowledge on foodlayer were separated, and 200 μL of the upper ethyl acetate layer

preservation and cooking.

was transferred to a 1.5 mL Eppendorf tube containing 100 μgof sodium sulfate. The tube was centrifuged at 12,000 × g for 22 Materials and Methods

min to remove water and impurities in the sodium sulfate layer,Chemicals

and 100 μL of the supernatant was collected in a vial for HPLCAll reagents were of the highest available quality.
Ascorbic acid, DHA, 2,6-dichlorophenolindophenol (DCIP),
tris [2-carboxyethyl] phosphine hydrochloride (TCEP), andanalysis.
To determine DHA and DKG, the addition of the oxidant
DCIP was omitted from the above procedure.

dinitrophenylhydrazine (DNPH) were purchased from Nacalai

For determining DKG, DHA was reduced to ASC by adding

Tesque (Kyoto, Japan). Meta-phosphate (MPA) was purchased

2 mM TCEP and incubating at room temperature for 15 min;from Santa Cruz Biotechnology (TX, USA).

DCIP treatment was omitted.12

Plant materials and thermal treatments

DKG values gave the DHA content and subtracting the DHA

Subtracting the value obtained for DKG from the DHA andFresh broccoli (Brassica oleracea L. var. Italica) waspurchased from a local market. Briefly, 100 mg of florets from aand DKG values from the total ascorbic acid value gave the ASCcontent.

broccoli head were washed with distilled water and then packedin a plastic bag. For thermal treatments, samples were stored at

HPLC analysis

4°C or -20°C for 0, 6, or 24 h. After the treatments, the samples

An HPLC system consisting of a Shimadzu LC10AD

were frozen in liquid N2 and homogenized with a mortar andpump and an SL-6B autosampler (Shimadzu, Kyoto, Japan)

pestle. The fine powders of the florets were thawed in 1 ml ofwas used. The DNPH derivatives were applied to a COSMOSIL

5% MPA, transferred into microtubes, and then centrifuged at

5SL-II column (4.6 mm i.d. x 150 mm, Nakarai tesque) and15,000 rpm for 10 min at 4°C. The supernatants were collected

eluted isocratically with ethyl acetate/hexane/2-propanol/and used as assay samples for ASC analysis, as described below.

acetic acid (50:47:2:1) at 1.0 ml/min. The absorption at 495
nm was recorded using an SPD-10A UV-VIS (Shimadzu)

Cooking

spectrophotometer.

For steaming, 50 g of florets were suspended in a domestic
steamer with 500 ml boiling water for 2 min. The surface
temperature of the broccoli was approximately 70°C with thisheat treatment.

Data analysis
Experimental points represent the mean ± SD of at least
three samples. For the statistical evaluation, student’s t test wasFor boiling, 250 ml of water in a 500 ml beaker was heatedused to test the significance of the differences in the mean content

to 100°C. Florets (50 g) were added to the boiling water for 2of ascorbic acid and its derivatives between control and samples.

min. The samples were drained and transferred into plastic bags.
For microwave cooking, 50 g of broccoli florets wereDifferences were considered significant at p ≤ 0.05. All statisticalanalysis were undertaken using Microsoft Excel ver. 16.

immersed in water and cooked in a microwave oven (Panasonic,Osaka, Japan) at a middle power level (500 W) for 2 min. The3 Results

surface temperature of the broccoli was approximately 65°C

3-1 Changes in ascorbic acid content in broccoli florets during

using this heat treatment. After cooking, the samples were frozen
in liquid nitrogen prior to extraction.

storage at low temperatures.
The total ascorbic acid content (ASA+DHA+DKG) in freshbroccoli was on average 95.7 mg/100 g fresh weight (FW) (Fig.

Determination of ASC, DHA, and DKG with DNPH2). The total ascorbic acid level in broccoli generally ranged

derivatization methodfrom 60.1 to 179.7 mg/100 g FW, with an average of 98.2

DNPH derivatization and ASC determination wereperformed according to the method of Omaye et al.

10and11Kishida et al. , respectively.

mg/100 g FW.14-20 The current value obtained here was slightly
lower; however, this variability can be related to cultivar,
maturity at harvest, and preharvest conditions. The amount ofFor the total ascorbic acid determination, the samples (500total ascorbic acid in the broccoli did not change significantlyµL) were added to 5 µL of 0.2% DCIP, mixed well, 2% thiourea

over a period of 24 h when stored at 4°C and -20°C. Several(500 µL) and 2% DNPH in 4.5 M H2SO4 (125 µL) was added,

studies have reported that the ascorbic levels in broccoli

and then incubated for 40 min at 60°C in closed glass test tubes.

decreased when it was stored for more than 2 days.18-21 However,17

it has been reported that the amount of total ascorbic acid hardly
decreased in a short period (approximately one day), which isconsistent with our result.18,21
80.0

70.0

(A) 4˚C

Total ascorbic acid (mg/100 g FW)

60.0

60.0

100
50.0

40.08030.06020.040

4. Boiling resulted in a significant loss of total ascorbic acid,(B) -20˚C

whereas steaming and microwaving resulted in less than a100
50.0

50% loss. In addition, boiling and steaming yielded more than

40.08050% DKG, and microwaving resulted in the highest amount of30.060

vitamin C remaining. The reason for this may be that the rapid

20.04010.020rise in the internal temperature of the food quickly deactivated

10.0200.0 000 hour

66 hour
2424hourTime (h)The change in total ascorbic acid levels following different
cooking treatments of the broccoli florets are shown in Fig.

80.0

Total ascorbic acid (mg/100 g FW)

70.0

3-3 Changes in DKG content in broccoli due to cooking

0.0 000 hour

6 24 hour
246 hourTime (h)Fig. 2 Changes in ascorbic acid content in broccoli florets
Fig.temperatures.
4during storage at lowA; stored at 4°C, B; stored at -20˚C for 0, 6, or 24 h. Data
are the mean of three determinations.

the ascorbate oxidase, which in turn suppressed the oxidationand degradation of vitamin C. Furthermore, Vallejo et al., 19
reported that the total amount of vitamin C decreased by 40%
when broccoli was heated in a microwave oven, which isconsistent with this result. Vallejo et al.,19 revealed that boilingfor 5 min or less suppressed the decrease in total vitamin C,
whereas Lisiewska et al.,15 concluded that the total vitamin C

Changes in DKG content in broccoli stored at 4°C and -20°C.
3-2 

decreased to 0% after 15 min of boiling.

The proportions of ASC, DHA, and DKG in the total; DKG

ascorbic acid in fresh broccoli were 75%, 7%, and 18%,; DHA; ASC

respectively (Fig. 3). Furthermore, the sample stored at 4°C
exhibited a 5% increase in DKG in 24 h, whereas DKG
increased to approximately 60% of the total ascorbic acid, whenstored at -20°C (p = 0.03). The ratio of DHA in the total ascorbic
acid did not change owing to storage and was approximately 7%.
These results suggested that the oxidation of ASC to DHA did
not stop; however, DHA was rapidly broken down to DKG. Asfor the slight increase in the total ascorbic acid content after 24
h of storage at -20°C, it is possible that the DNPH derivatization
method detected carbonyl compounds other than DHA, such as

freshboil

steam microwave

Fig. 4 Effect of different cooking methods on theDKG contents of broccoli.
The meanings of the abbreviations are as follows:
fresh broccoli without cooking; boil, boiling; steam,steaming; microwave, microwave oven.

ketone bodies.13 In addition, as there is almost no difference inthe amount of the total ascorbic acid levels between 6 h and 24
h at -20°C, it is possible that some by-products were synthesized
by the freezing process and detected as the total amount ofascorbic acid. The results showed that when fresh broccoli wasfrozen at -20°C, ASC was rapidly oxidized and degraded, andthe ratio of active vitamin C (ASC+DHA) to the total amount ofascorbic acid decreased by approximately half.
; ASC,; DHA,; DKG

4 DiscussionOwing to the diversity and importance of its physiologicaleffects, it is crucial to obtain the required amount of vitamin
C from the diet. It is known that ASC, together with its oxide
DHA, is always maintained at a constant ratio by the glutathione
cycle, and ASC continues to be supplied even under transientstress conditions.6 However, such homeostasis is not maintained; ASC,; DHA,

1008060; DKG

in foods, and the fluctuation of the intracellular level of ASC
has not been comprehensively investigated to date. In thestandard tables of food composition compiled in Japan, theamount of vitamin C is calculated using the DNPH method,implying that the measured values include DKG, which does40

not have vitamin C activity.26 It has recently been reported

20

that DHA has a poor absorption rate in the intestinal tract.13 In
addition, as DKG has a dicarbonyl structure, it is a substrate0Fig. 3 Changes in DKG content at different storage
temperatures.
A; stored at 4°C, B; stored at -20˚C for 0, 6, or 24 h.

for the Strecker reaction and is involved in the production ofmelanoidins, implying that it may be involved in the productionof carcinogens.22 In this context, it is important to elucidate
the detailed profile of ascorbic acid in foods and the metabolic
pathways involved. In this study, we analyzed the changes in the18

accumulation of ASC, DHA, and DKG resulting from variousof ascorbic acid with chemical derivatization and high-storage and processing treatments. The results showed that DKG

performance liquid chromatography. Anal Chem 1992;

accumulated in broccoli up to approximately 50% after storage64(13), 1505-7.

at -20°C. Although the reason for this is not clear at this time,

12Lykkesfeldt J. Determination of ascorbic acid anddehydroascorbic acid in biological samples by high-it may be that the degradation of DHA is accelerated at -20°C
27compared to 4°C, and that the accumulation of by-products

performance liquid chromatography using subtraction

detected as DNPH derivatives is increased at -20°C. The lattermethods: Reliable reduction with tris [2-carboxyethyl]

is currently being analyzed using gas chromatography or liquid

phosphine hydrochloride. Anal Biochem 2000; 282(1),chromatography-mass spectrometry. It was also suggested that
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on the storage of food materials and highlight the importance ofAgric Food Chem 2002; 50(1), 227-9.

comprehensive food material analysis by metabolome analysis.

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Albrecht JA, Schafer HW, Zottola EA. Relationship of
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