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Received: 30 March 2018 Revised: 17 October 2018 Accepted: 30 October 2018 DOI: 10.1111/jfbc.12744
FULL ARTICLE
Apple phlorizin attenuates oxidative stress in Drosophila melanogaster Hao Wang1,2
| Zhenou Sun1,3 | Dong Liu1 | Xiang Li1 | Rizwan‐ur Rehman4 |
Huali Wang5 | Zijian Wu6 1 State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin, China 2
Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science & Technology, Tianjin, China 3
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China 4
Center for Food Safety Standards, The University of Lahore‐Gujrat Campus, Pakistan 5
China National Center for Food Safety Risk Assessment, Beijing, China
6
College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin, China Correspondence Hao Wang, State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, China. Email:
[email protected] Zijian Wu, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China. Email:
[email protected] Funding information National Natural Science Foundation of China, Grant/Award Number: 31201322; National Science & Technology Pillar Program during the 12th Five‐year Plan Period, Grant/Award Number: 2012BAD33B05; Tianjin City High School Science & Technology Fund Planning Project, Grant/Award Number: 20100609
Abstract Apple phlorizin has a lot of applications owing to its antioxidant and hepatoprotective properties. This study explored the antioxidant effects and life span‐prolonging ac‐ tivity of apple phlorizin in Drosophila melanogaster. Treatment with apple phlorizin was found to significantly extend the life span and ameliorate the age‐related decline of locomotor function. This life span‐extending activity was associated with the in‐ creased activity of superoxide dismutase, catalase, mRNA expression of glutamate– cysteine ligase catalytic subunit, cap‐n‐collar (cnc, homologue of mammalian Nrf2 gene), Keap1, and deacetylase sir2, as well as the downregulation of methuselah. Computational analysis suggested phlorizin could work as a Nrf2 activator and exert its biological activities by interfering with the Keap1 and Nrf2 binding. Therefore, it was concluded that the antioxidant and anti‐aging effects of phlorizin might, at least in part, be mediated through the cooperation with the endogenous stress defense system.
Practical applications Phlorizin, from apple peel, has been used as a nutrient for over 100 years. To date, despite extensive research on phlorizin, a report on its effect on the antioxidant sys‐ tem in fruit flies is yet lacking. This report demonstrates that phlorizin can exert a protective effect on antioxidant issues and prolong life in fruit flies, which is valuable in the rational utilization of phlorizin in functional foods. KEYWORDS
anti‐aging, antioxidation, Drosophila melanogaster, molecular docking, phlorizin
1 | I NTRO D U C TI O N
damage macromolecules such as mitochondrial and nuclear DNA, alter or activate cellular signaling pathways, or cause apoptotic or
The occurrence of many diseases such as cancer, diabetes type II,
necrotic cell death (Finkel & Holbrook, 2000). This may be due to
neurodegenerative diseases, and cardiovascular diseases increases
inefficient sensors that detect increases in cellular ROS concentra‐
exponentially with chronological age. Most of these diseases are
tions, or to an increased rate of ROS production that exceeds the
linked to an increase in reactive oxygen species (ROS), which can
scavenging capacity of cells.
J Food Biochem. 2018;e12744. https://doi.org/10.1111/jfbc.12744
wileyonlinelibrary.com/journal/jfbc
© 2018 Wiley Periodicals, Inc.
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Phlorizin, one of the polyphenol compounds in apples, has been used as a subject for physiology research for more than 100 years
the remaining alive flies were transferred to a new vial containing the same diet until all the flies died.
and possesses a variety of potent properties including blood glu‐ cose reduction and antitumor activities (Masumoto, Akimoto, Oike, & Kobori, 2009). Deng et al showed that phlorizin had a significant
2.4 | Climbing assay
protective effect against acute hepatotoxicity induced by CCl4 in
Locomotor function of fruit flies was assessed using a climbing assay
rats, which might be due to its free radical‐scavenging effect, inhibi‐
as described previously by Peng with slight modifications (Peng et
tion of lipid peroxidation, and ability to increase antioxidant activity
al., 2012). Briefly, male flies (n = 200, 20 flies per vial) were placed
(Deng et al., 2012). Rezk et al showed that phlorizin had a potent
in a plastic vial and given 10 s to climb up. At the end of each trial,
antioxidant activity in peroxynitrite scavenging and inhibition of lipid
the number of flies that climbed up to a vertical distance of 8 cm
peroxidation (Rezk, Haenen, & Vijgh, 2002).
or above was recorded. Each trial was performed three times. Flies
Drosophila melanogaster, one of the most commonly used
were tested at selected time points during the survival assay.
models, has emerged as an excellent genetic model to study the complexity of the aging process (Bolukbasi et al., 2017). Humans actually share a huge number of conserved biological pathways and disease‐causing genes with this tiny insect (Fan et al., 2015). Despite extensive research on phlorizin, little is known about
2.5 | Stress resistance assay 2.5.1 | Paraquat challenge assay
its interaction with the genes involved in delaying aging and
Paraquat (PQ) can generate superoxide anion radicals (Michaelis &
prolonging the life span of a whole organism in vivo. Therefore,
Hill, 2002). The 2‐day‐old male Oregon K wild‐type flies (n = 800)
the present study evaluated the antioxidant and anti‐aging
from the same generation were divided into four groups, with 200
activities of phlorizin in D. melanogaster and the underlying
flies in each group (20 flies per vial). Flies were maintained on their
molecular mechanisms.
corresponding control diet or phlorizin diet at 25°C for 25 days to examine the resistance of flies to superoxide‐induced stress. Then,
2 | M ATE R I A L S A N D M E TH O D 2.1 | Materials and reagents
25‐day‐old male flies were collected after starving for 2 hr and then transferred to new vials containing a filter paper saturated with 1 ml of 20 mM paraquat diluted in a 6% glucose solution. The number of dead flies was counted every 2 hr until all the flies died.
Phlorizin (>98%) was obtained from Jianfeng Natural Product Co., Ltd., Tianjin, PRC. Superoxide dismutase (SOD) assay kit using the hydroxylamine method, catalase (CAT) assay kit for visible light,
2.5.2 | H2O2 challenge assay
and malondialdehyde (MDA) assay kit were purchased from Nanjing
H2O2 is also used to examine the resistance of flies against OH−‐in‐
Jiancheng Biotech Group Co., Ltd, Nanjing, PRC; TRIzol Reagent
duced oxidative stress (Fan et al., 2017). Flies were maintained on
and cDNA Synthesis SYBR Green Kit were obtained from Takara
their corresponding control diet or phlorizin diet at 25°C for 25 days.
Biological Engineering Co., Ltd., Japan.
Similarly, on day 25, the Oregon K wild‐type flies were starved in empty vials for 2 hr at 25°C and transferred to new vials with a filter
2.2 | Animal model and culture conditions
paper impregnated with 30% H2O2 diluted in a 6% glucose solution. The number of dead flies was counted every 2 hr until all the flies died.
In this study, 2‐day‐old male Oregon K wild‐type flies from the same generation were kept in a humidified, temperature‐con‐ trolled incubator at 25°C and 50% humidity.
2.6 | Enzyme activity assay
The standard formulation was adopted to prepare a basal diet
The 2‐day‐old Oregon K wild‐type male flies (800 flies in total)
as reported previously (Zhang et al., 2014). In brief, 750 ml of diet
were grouped into four categories, each group containing 200 flies
contained 72 g of glucose, 72 g of cornmeal, 10 g of yeast, and 6 g
(n = 200 each). They were maintained on their corresponding control
of agar; 40 ml of preservative (75% ethanol containing 1% ethyl p‐
diet or phlorizin diet at 25°C for 45 days. The male flies were col‐
hydroxybenzoate) was added into the diet to prevent mold growth.
lected under CO2 anesthesia after starving for 2 hr and recording
The experimental diets were prepared by adding 0.5, 1.0, or 2.0 mg
the average body weight in each group and stored at –80°C. SOD
phlorizin to the control diet per milliliter.
and CAT activities and MDA content were calculated as per the in‐ structions of the corresponding kits.
2.3 | Life span assay The 2‐day‐old male Oregon K wild‐type flies (n = 800) from the same
2.7 | mRNA expression assay
generation were divided into four groups, with 200 flies in each
Flies (45‐day‐old) were collected, and total RNA was extracted
group (20 flies per vial). Dead flies were counted every 3 days, and
using the commercial extraction agent TRIzol (Takara, Japan).
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Subsequently, RNA was subjected to reverse transcription to syn‐
the most conformations was considered as the most suitable con‐
thesize cDNA in a thermocycler (MyCycler, Bio‐Rad, CA, USA),
formation. The resulting conformations of 2D were visualized in the
with the program set as initiation for 15 min at 37°C, followed by
Discover studio 3.5 Client.
incubation at 85°C for 15 s. The synthesized cDNA was stored at –20°C. The target genes were copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase
2.9 | Statistical analyses
(CAT), glutamate–cysteine ligase catalytic subunit (GclC), cap‐n‐col‐
Data were expressed as means ± SD. The significance of differences
lar (cnc), methuselah (MTH), Keap1, and deacetylase sir2 (dSir2). The
between the samples was assessed using Student t tests and one‐
rp49 transcript was used as the reference for quantifying the relative
way analysis of variance. Differences were considered significant
transcript level of each selected gene (Yu‐zhi, Li‐ying, Li, Xue‐mei, &
when p 0.05). The mean and maximum life spans of phlorizin‐treated groups were higher than those of the control group. The con‐
2.8 | In silico assay
trol group had a mean life span of 46.33 days. The three phlori‐
Docking studies on the interaction between two compounds (phlo‐
zin‐treated groups had 46.25, 47.88, and 50.58 days of mean life
rizin and curcumin, a Nrf2 activator had been reported) with Crystal
span, respectively. Both 1.0 and 2.0 mg/ml phlorizin treatment
Structure of the Kelch‐Neh2 Complex from Homo sapiens obtained
extend the mean life span and the maximum life span significantly
from the Protein Data Bank, with the accession ID: 2FLU (1.5 Å)
(p
