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A publication ofCHEMICAL ENGINEERING TRANSACTIONS
VOL. 106, 2023

The Italian Associationof Chemical Engineering

Online at www.cetjournal.it

Guest Editors: Jeng Shiun Lim, Nor Alafiza Yunus, Peck Loo Kiew, Hon Huin Chin
Copyright © 2023, AIDIC Servizi S.r.l.
ISBN 979-12-81206-05-2; ISSN 2283-9216

DOI: 10.3303/CET23106211

Bioactive Compound Potential of Brassicaceae Sprouts andthe Effect of UV-B Radiation on their Antioxidant Activity
Nur I. M. Rasida,b, Fazrena Nadia Md Akhira,*, Azila Abd Aziza, Iwane Suzukib,Shinya Takahashib
Department of Chemical and Environmental Engineering, Malaysia-Japan International Institute of Technology, Universiti
Teknologi Malaysia, Jalan Sultan Yahya Petra, 54100 Kuala Lumpur, Malaysiab
Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japanfazrena@utm.myaCurrent trend of climate change and depletion of ozone layer may cause the amount of ultraviolet (UV) radiationin the atmosphere to increase and thus, affecting biodiversity and ecosystem. Plants are one of the most affectedorganisms due their need of sunlight to carry important process of photosynthesis and the presence of UV
radiation may result in detrimental effects on them. However, with controlled amount of UV radiation exposure
as abiotic stress condition, plants will be able to develop a plant defense chemical and present an alternative
source of drug discovery as natural products. Plant secondary metabolites are important compounds largely
used in pharmaceutical industry and commonly found in Brassicaceae family plants such as broccoli sprouts,
red cabbage sprouts and kale sprouts. Previous studies suggested that Brassicaceae sprouts is a good sourceof phytochemicals and reported to exhibit bioactive properties including antioxidative and anti-inflammatory. In
this study, an overview of the effect of UV-B radiation exposure as abiotic stress on the amount of Brassicaceae
sprouts bioactive compound and antioxidant activities are presented and discussed. Brassicaceae sprouts aregrown and exposed to UV-B radiation under different sets of time and fresh samples of the sprouts are extractedwith ethanol before undergoing phytochemical analysis for total phenolic content and then proceed with DPPH
assays to study its antioxidant activity. Results suggest that UV-B radiation causes increase in double in theamount of total phenolic content for red cabbage sprout from 0.33 to 0.63 mg gallic acid/g-1 and increase in theantioxidant activity of kale sprout from 0.74 to 0.80 mg Trolox/g-1 from Brassicaceae species at 5th-min ofexposure. This study is important to understand the increment of UV radiation from ozone layer depletion forplants particularly from Brassicaceae family to adapt to these changes while applying them in different industries
including agriculture and health for their bioactive compounds.

1. Introduction
Depletion of protective ozone layer due to environmental issues caused by recent rapid industrialization andtechnology advancement resulted in research focus on its effect towards biodiversity and ecosystems. Thisphenomenon causes increment in the amount of harmful ultraviolet (UV) radiation reaching the earth’s surface
and its potential detrimental effects of the existing ecosystems and the progression of biodiversity (Dwivedi &Ahmad, 2023). Ozone layer depletion occurred mainly from chlorofluorocarbons (CFCs) andhydrochlorofluorocarbons (HCFCs) effluents by the industries and it is reported that the increase in theoccurrence of extreme climate change events also increases the depletion of ozone layer, and it will continue
to be prevalent in the future. The increase in the levels of UV radiation raise a concern of its detrimental effects
on organisms that require light to generate chemical energy. According to Soni et al. (2022), for photosynthesis,
plants utilize wavelengths in the range of 400 nm to 700 nm while UV radiation ranging from 100 nm to 400 nm
and categorized into three types which are UV-A, UV-B, and UV-C. While the radiation range does notcorrespond with photosynthesis process, UV-A is characterized as the least harmful radiation, and it does not
get absorbed in the ozone layer which ranging from 315 nm to 400 nm due to its low energy. Meanwhile, UV-C
is the most harmful range at 100 nm to 280 nm, but it is absorbed by the atmosphere and will not reach thePaper Received: 05 June 2023; Revised: 15 July 2023; Accepted: 22 September 2023
Please cite this article as: Rasid N.I.M., Akhir F.N.M., Aziz A.A., Suzuki I., Takahashi S., 2023, Bioactive Compound Potential of Brassicaceae
Sprouts and the Effect of UV-B Radiation on their Antioxidant Activity, Chemical Engineering Transactions, 106, 1261-1266
DOI:10.3303/CET23106211

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Earth’s surface. Next is UV-B, in the range of 280 nm to 315 nm, at high levels it will be detrimental towards
living things and ozone layer acts as a protection layer by absorbing it from reaching the Earth’s surface (Soniet al., 2022). As UV-B is the only type of UV radiation that can threaten the environment while reaches theEarth’s surface resulted from the depletion of the ozone layer, it is important to understand how this will affectplants biodiversity and its adaptation towards this abiotic stress. With the current trend of ozone layer depletion,
it is expected to continue rising in the future and some of the factors associating with this trend are climatechange, emission of greenhouse gases and volcanic eruptions. Brassicaceae family or formerly known asCruciferae has a wide range of species with rich contents of phytochemical for health promotion and isconsidered as one of the earliest groups of cultivated plant (Cámara-Martos et al., 2021). As one of the sourcesof important nutrition in food, species from Brassicaceae family is also ranked in top 10 of the most economicallyimportant plant families in the world due to its rich source of bioactive contents such as vitamin C, phenoliccompounds, and carotenoids (Cámara-Martos et al., 2021). Broccoli sprout, radish sprout, kale sprout and red
cabbage sprout are some of the examples categorized under Brassicaceae family are also known as superfood
for the health benefit they can offer which commonly found in traditional medicine and culinary (Šamec et al.,2018). With good environmental adaptations, Brassicaceae sprouts are grown worldwide for food consumption.
Bioactive compounds available in plants from Brassicaceae family has also been extensively analyzed byprevious research and known for their antioxidant activity, anti-inflammatory activity and gastro protective
associated with their presence of different types of phytochemicals (Šamec et al., 2018). Simple and costeffective approaches of cultivating Brassicaceae sprouts makes them commercially attractive in obtaining theirhigh-value phytochemical yields for health application. According to Soni et al. (2022), responses in plants froman increase in the level of UV-B is particularly important on its growth and development where the responses
vary depending on how UV-B light is perceived, how signals are transduced, and how gene expression isaltered. In addition to that, the application of UV-B radiation is also associated with simple manipulation, leaves
no residue, terminates instantly and most importantly, cost-effective for an ideal elicitation method which help
to increase the accumulation of bioactive secondary metabolites of plants via in vitro cultures (Gai et al., 2022).
The bioactivity potential of Brassicaceae sprouts under environmental stress (UV-B stress) with enhanced
accumulation of phytochemicals may benefit the consumer in providing alternative sources of nutrition otherthan synthetic ones. The main objective of this research is to evaluate the effect of UV-B radiation at different
exposure times as abiotic stress on total phenolic content and antioxidant activity of Brassicaceae sprouts.

2. Material and methods
Methods used in this study were divided into three parts, plants cultivation, UV-B treatment, and analysis ofbioactive activities.
2.1 Plant material and sprouting conditions
Three organic Brassicaceae sprouts seeds originated from Italy were purchased (broccoli sprout, red cabbage
sprout and kale sprout). Each type of the seeds was prepared in three containers with wet cotton. The seeds
were left to germinate in the dark for 3 days in a growth chamber at room temperature. After germination, thesprouts were exposed to light in growth chamber.
2.2 UV-B treatment
UV-B lamp was placed in a closed box and the distance between lamps and sprout cultures was set to obtain a
low radiation intensity (3 W/m2) monitored using a UV sensor. The sprouts cultures were exposed to the UV-B
radiation on the 7th day with different sets of exposure time (1 min, 2 min, 5 min and 10 min), where according
to, high radiation intensity and long UV-B exposure time may cause a reduce in sprouts quality due tooverexposure. The sprouts were left to react for three days in the growth chamber at room temperature.
No UV-B exposure was used as control in this study.
2.3 Preparation of Brassicaceae sprouts extracts
On 10th day, the crude extract of Brassicaceae sprouts was extracted using ethanol. 0.1 g of bagasse wasweighed, and 1 mL of ethanol was then added to the sample. The mixture was left to react for 48 h and prepared
for analysis on the 12th day.
2.4 Phytochemical analysis of Brassicaceae sprouts extracts
2.4.1 Total Phenolic Content (TPC)
Folin-Ciocalteu method was used in this analysis. Diluted extract of Brassicaceae sprouts (50 µl sample and 50µl extraction solution) of each sample (crude extracts) was added to a test tube and then 100 µl of Milli-Q wasadded to the sample. After that, 200 µl of phenol reagent (dilution 1:1) was then added to the extracted samples

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and prepared standard solutions and were left in the dark for 3 min before 200 µl of sodium carbonate (10%)
was added and left in the dark for 30 min. Then, the absorbance was measured at 750 nm using UV-V
spectrophotometer (Sanarat & Srihanam, 2019).
Gallic acid was used as a standard curve to calculate TPC and the results were expressed as mg gallic acid/gsample (Sanarat & Srihanam, 2019). Standard curve is a parameter that is used to compare the results of
unknown samples concentration with known concentration of standard this study.
2.5 Antioxidant activity by DPPH assay
DPPH assay was carried out to measure the free radical scavenging activity where a 10 μL of prepared
Brassicaceae sprouts extracts followed by mixing with 190 μL of DPPH working solutions. After incubation inthe dark at room temperature for 10 min, by using a spectrophotometer, the absorbance was read at 520 nm.
Trolox was used as standard solution.

3. Results and discussions
3.1 Red cabbage sprout
3.2.1 TPC
Red cabbage sprout is reportedly to have rich amount of vitamin c and this presence of interfering substance
may affect the measurement of TPC with Folin-Ciocalteu absorbance. Based on Figure 1, the results show thatthe amount of phenolic compound available in red cabbage sprout varies based on the amount of exposure time
towards UV-B. By comparing to sprouts condition without exposure to UV-B radiation, at minute 5 of UV-B
radiation, red cabbage sprouts show the highest increase in phenolic compound compared to 1st-minute, 2ndminute and 10th-minute.
Increases at the 5th-minute of exposure agrees with studies carried out by Martínez-Zamora et al., (2021) on
red cabbage sprout using different doses of UV-B (5, 10 and 15 kJ m-2) compared to untreated plant (control).
This shows a protective response of the plants toward the changes in environmental conditions as abioticstressor and helps to improve its resistance for future exposure.

Absorbance 750 nm

0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000

12510Exposure time (min)ControlUV-B

Figure 1: TPC of red cabbage sprout
3.2.2 Antioxidant activity
Antioxidant activity of red cabbage sprout was recorded as follows. At 1st-minute, 2nd-minute, and 5th-minute,there were slight increase in the amount of antioxidant activity of red cabbage spouts compared to the sprouts
at controlled condition (no UV-B exposure). While after 10th-minutes of UV-B exposure, the red cabbage sprout
shows a decline in antioxidant activity.

mg Trolox/ g fresh weight

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1.200
1.000
0.800
0.600
0.400
0.200
0.000

12510Exposure Time (min)Control

UV-B

Figure 2: DPPH of red cabbage sprout
As seen in Figure 1 and Figure 2, at higher amount of phenolic content, the antioxidant activity of the sprouts
was not increasing respectively. According to Nogueira (2018), different types of phytochemicals present indifferent type of sprouts may not contribute towards antioxidant activity and thus causes results with high amountof phenolic compound contain lower antioxidant activity. In addition to that, to detect antioxidant activity of every
different kind of phytochemical present in various Brassicaceae sprouts, DPPH method alone may not beenough for the analysis. Some other option of antioxidant activity analysis includes ABTS assay and FRAP withdifferent principles method which are not focused on this study.
3.2 Kale sprout
3.3.1 TPC

Absorbance 750 nm

As discussed above for TPC of red cabbage sprouts, kale sprout shows a slight increase in the amount ofphenolic compound at 5th-minute of UV-B exposure. While at 1st-minute, 2nd-minute, and 10th-minute, the amountof phenolic compound recorded significant amount of declination compared to the controlled condition. Theresult that shows increase in the TPC at the 5th-minute of UV-B exposure compared to untreated plant iscorrelated to results that showed an increase of 22% of TPC at all UV-B doses of 5, 10 and 15 kJ m-2 of kalesprouts after 10 days of sprouting (Castillejo et al., 2021).

0.600
0.400
0.200
0.000

12510Exposure time (min)ControlUV-B

Figure 3: TPC of red kale sprout
3.3.2 Antioxidant activity
As for antioxidant activity of kale sprout, there was slight increase on the 1st-minute, 2nd-minute and 5th-minuteof UV-B radiation. Meanwhile at 10th-minute, the antioxidant activity of kale sprout decreased after exposed toUV-B radiation. According to research by Castillejo et al. (2021) at different UV-B doses, the antioxidant activityof kale sprouts changes accordingly where an increase was observed only at 5 kJ m-2 compared to untreatedplants by 28%.

mg Trolox/ g fresh
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1.000
0.500
0.000

12510Exposure Time (min)ControlUV-B

Figure 4: DPPH of red kale sprout
3.3 Broccoli sprout
3.4.1 TPC

Absorbance 750 nm

Broccoli sprouts show a decrease in phenolic compound for 1st-minute and 2nd-minute compared to controlled
condition and increase in phenolic compound on 5th-minute and 10th-minutes of UV-B exposure. The same
sprout shows an increase of 160% of untreated sprout after UV-B exposure (Martínez-Zamora et al., 2021).
0.400
0.300
0.200
0.100
0.000

12510Exposure time (min)ControlUV-B

Figure 5: TPC of broccoli sprout
3.4.2 Antioxidant activity

mg Trolox/ g fresh
weightBased on Figure 6, broccoli sprouts show a decrease in every different exposure time of UV-B radiation (1stminute, 2nd-minute, 5th-minute, and 10th-minute). The decrement in antioxidant activity is more significant as theexposure time increases, while a report on the effect of UV-B, UV-C and UV-B + C showed 55% increase afterUV-B exposure (Martínez-Zamora et al., 2021). This showed a varied responses according to UV-B doses.
0.600
0.400
0.200
0.000

12510Exposure Time (min)ControlUV-B

Figure 6: DPPH of broccoli sprout
According to Figure 6, at 10th-minute of UV-B exposure, a significant decrease in the antioxidant activity wasobserved from 0.42 to 0.24 mg Trolox/g-1 due to overexposure to UV-B radiation as seen in Figure 7.

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Overexposure to UV-B radiation can occur when Brassicaceae sprouts are exposed to the radiation for too longand the sprouts become reduces in size, productivity, and quality (Lemmons, 2023).

Figure 7: Broccoli sprouts 3 days after 10 min UV-B exposure (in red box)

4. Conclusions
In conclusion, UV-B radiation as abiotic stress affects the sprouts growth both chemically and physically. At
certain minute of exposure, the Brassicaceae. sprouts reacts positively observed through the increases inphenolic content and antioxidant activity as seen in kale sprout at 5th-minute exposure. There were also somedecrements observed in antioxidant activity of broccoli sprout after exposure of UV-B radiation and negativechanges on the plant growth. A good understanding on the plants’ behaviors towards environmental changes
may contribute to agricultural and health industries with their rich source of bioactive compounds.
Acknowledgments
The authors are thankful for the opportunity to do the research with the support from Malaysia-Japan
International Institute of Technology (MJIIT), Universiti Teknologi Malaysia and University of Tsukuba, Japan
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