Effects of C-Glycosides from Apios americana Leaves against

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Effects of C-glycosides from Apios americana leaves against oxidative stress during hyperglycemia through regulating MAPKs and Nrf2 Fujie Yan, yunyun yang, Lushuang Yu, and Xiaodong Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03163 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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Journal of Agricultural and Food Chemistry

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Effects of C-glycosides from Apios americana leaves against oxidative stress

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during hyperglycemia through regulating MAPKs and Nrf2

3

Fujie Yan, Yunyun Yang, Lushuang Yu, Xiaodong Zheng*

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Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, People’s

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Republic of China; Zhejiang Key Laboratory for Agro-food Processing, Zhejiang University,

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Hangzhou 310058, People’s Republic of China; Fuli Institute of Food Science, Zhejiang

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University, Hangzhou 310058, People’s Republic of China

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*Corresponding author,

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Xiaodong Zheng, Phone: +86 571 86098861, Fax: +86 571 86971139

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Email: [email protected]

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Postal address: Department of Food Science and Nutrition, Zhejiang University, Hangzhou

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310058, People’s Republic of China

14 15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment


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Abstract:

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Main components of Apios americana leaves extract (ALE) were flavonoid C-glycosides

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including vitexin (46.7%), schaftoside (18.9%) and orientin (4.32%). In vitro, ALE restored

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glucose consumption, glucose uptake and glycogen content in glucose-induced hepatic cells.

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Exposure of HepG2 cells to high glucose resulted in ROS and O2- accumulation, while ALE

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alleviated these increases by 47 ±0.68% and 68 ±0.74% respectively. Glucose increased JNK and

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decreased ERK1/2 and p38 phosphorylation, while ALE reduced p-JNK and p-p38 but not

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p-ERK1/2, accompanied by Nrf2, HO-1 and NQO1 down-regulation. In vivo, lifespan of

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Caenorhabditis elegans was more violently shortened by paraquat under hyperglycemia while

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ALE protected this damage in N2 worms (2.6 times extension) but not in daf-16 mutants.

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Furthermore, p38/PMK-1 and Nrf2/SKN-1 expressions in worms were suppressed by glucose,

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which were reversed by ALE treatment. These results suggest that ALE prevents glucose-induced

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damage via regulating specific MAPKs and Nrf2 pathways.

36

Keywords:

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Caenorhabditis elegans

Apios

americana

leaves;

C-glycosides;

oxidative

38 39 40 41 42 43 44 2


stress;

MAPKs;

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Introduction

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Type 2 diabetes is a chronic disease which is rapidly emerging as a global health challenge in 21st

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century.1 As living standards improvement, bad habits such as high-fat diets and sedentary

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lifestyles lead to a fast growth of diabetic patients numbers. Generation of excessive reactive

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oxygen species (ROS) leads to oxidative stress that disturbs intracellular redox status homeostasis

50

and induces cell apoptosis or death.2 Continuous oxidative stress impairs normal functions of cells,

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tissues and organs, potentially accelerating the process of some diseases such as cancer,

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hepatopathy, diabetes and obesity.3 Hyperglycemia induces formation of ROS through

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accumulating advanced glycation end products (AGEs) as well as activating some pathways like

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the protein kinase C (PKC) pathway.4 Therefore, strategies to decrease intracellular ROS levels

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under a hyperglycemic condition have a great significance in diabetes and its complication

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treatment.

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Apios americana Medik is originally cultivated in North America, which is regarded as a

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leguminous perennial vine that generates edible tubers. Nutritional values of A. americana tuber

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have drawn increasing attention for recent years because the tuber is abundant in mono- and

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oligosaccharides, protein, fatty acid and amino acid.5, 6 Like soybean, the tuber is a good source of

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isoflavones such as genistein and its derivative.7 Chemically, a novel isoflavone isolated from

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tubers was identified as genistein-7-O-gentiobioside, showing abilities of radical scavenging and

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antioxidative system activation.8 It has also been reported that diet with tubers could alleviate

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blood pressure elevation in spontaneously hypertensive rats.9 Although it is anticipated that

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flowers and leaves can also be used as new sources of functional food, their constituents and

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physiological effects have not been totally studied yet. Recently, in the flower extract, compound 3



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named caffeoyl β-d-glucopyranoside has been isolated and identified, which is considered as a

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kind of maltase inhibitor, exhibiting an anti-hyperglycemic effect in ICR and KK-Ay diabetic

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mice.10 Nevertheless, there is nearly no report about A. americana leaves—whatever their

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chemical constitution or bioactivity.

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In this study, we firstly isolated flavonoids from dried leaves of Apios americana Medik and

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identified main ingredients in the extract by LC-MS/MS analysis. Standards of flavonoids were

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used for further identification and quantification. And then, we aimed to investigate the underlying

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protective action of ALE against glucose-induced oxidative damage and possible signal pathways

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involved in vitro by using human hepatocyte as a model. Moreover, we assessed whether ALE

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exhibited positive effects against oxidative injury under a hyperglycemic condition in

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Caenorhabditis elegans.

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Materials and Methods

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Chemicals. Vitexin (purity ≥ 98%), schaftoside (purity ≥ 98%), and orientin (purity ≥ 98%)

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were

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2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) was obtained from

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ApexBio (USA). Primary antibodies against Nrf2, HO-1, NQO1, p-ERK1/2, ERK1/2, FOXO1,

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PGC-1α and Lamin B were purchased from Abcam (Shanghai, China), primary antibodies against

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p-p38 MAPK, p38 MAPK, p-JNK, JNK and β-actin, fluorescent probes and WB/IP lysis buffer

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were purchased from Beyotime Biotechnology (Jiang Su, China). Other reagents were

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analytical-reagent grade from Aladdin (Shanghai, China).

purchased

from

Shfeng

Biological

Technology

(Shanghai,

China).

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Extraction of flavonoids from A. americana Medik leaves. A. americana Medik leaves were

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provided by Tianyu ecological agriculture limited liability company. The plant is originated from

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America and cultured in Fuyang, Dongzhou (Hangzhou, China). Dried leaves (100 g) were 4


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extracted by 70% ethanol (3 L) for 24 h at 4 °C. Filtered fluid was evaporated at 40 °C and then

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concentrates were loaded onto an equilibrated AB-8 macroporous resin column eluted with 80%

92

ethanol for further purification. ALE was obtained by lyophilization and stored at -80 °C before

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use.

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UPLC-TOF/MS analysis. An UPLC system (Waters, USA) coupled with a Triple-TOF Mass

95

Spectrometry System (AB SCIEX, TripleTOF 5600plus Framingham, MA, USA) was used to

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identify flavonoids compounds. The ingredients of mobile phase are 0.1% aqueous formic acid (A)

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and acetonitrile contains 0.1% formic acid (B). The linear gradient of phase B was: 0–2 min, 5%;

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2–25 min, 5–50%; 25– 35 min, 50–95%; 35–37 min, 95%; 37–40 min, 5%. The flow rate was 0.8

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mL/min and the injection amount was 5 µL. The mass spectrometry was operated in a negative ion

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mode at a temperature of 550 °C and the source voltage was 4.5 KV. Ions were recorded over the

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range from m/z 100 to 2000, and the wavelength for UV detector set at 280 nm. Further separation

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and quantification of flavonoids was conducted on Promosil C18 column (4.6 × 250 mm, 5 μm)

103

using an HPLC instrument (Thermo UltiMate 3000).

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Cell

culture

and

treatments.

HepG2

(hepatoma

cell

line)

and

LO2

(normal

105

human liver cell line) cells were cultured in DMEM supplemented with 10% fetal bovine serum,

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100 IU/mL penicillin and 100 μg/mL streptomycin at 37 °C, 5% CO2 atmosphere. ALE powder

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was always freshly dissolved in PBS buffer and used immediately. After reaching 70-80%

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confluence, the cells were washed with PBS twice and pretreated with or without ALE at different

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concentrations for 24 h and then incubated with normal glucose (5.5 mM) or high glucose (30 mM)

110

in the absence/presence of ALE at corresponding concentrations for another 36 h. Cells treated

111

with normal glucose (5.5 mM) were used as the negative control and cells treated with metformin

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(2 mM) were used as the positive control. 5



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MTT assays. Cells were seeded into a 96-well plate and MTT diluted with PBS at a final

114

concentration of 0.5 mg/mL was added to each well after treatments. After 4 h of incubation at

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37 °C, the formazan precipitate was dissolved in 150 μL DMSO, and the absorbance was

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measured at 570 nm with a spectrophotometer.

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Glucose consumption and uptake assays. The glucose consumption was estimated by the

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method of Yan et al. with modification.11 Cells were seeded into a 96-well plate at a density of

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3×103 cells/well with five wells left as blanks. After treatments, all media were removed and

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changed to the same. 24 h later, the glucose in the medium of each well was measured by a

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commercially available kit using the glucose oxidase method. Briefly, 2 µL medium was added

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into 200 µL reaction agents and incubated for 20 min at 37 °C, and then detected at 505 nm by a

123

microplate reader (Molecular Device, USA). Glucose consumption was calculated by the glucose

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concentrations of blank wells minus glucose concentrations in plated wells. The MTT assay was

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used to adjust the glucose consumption. There were five replicates for each treatment and the

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experiment was repeated three times.

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For the glucose uptake assay, cells were seeded into a 24-well plate at a density of 5×104

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cells/well and after the treatments cells were exposed to 0.1 mM 2-NBDG and 100 nM insulin for

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30 min at 37 °C. Images were obtained using identical acquisition settings on a fluorescence

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microscope.12

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Glycogen content assay. The cells were seeded into a 6-well plate and washed twice with PBS

132

and collected into centrifuge tubes after treatments. Samples were boiled in 30% KOH solution for

133

20 min, added 1.5 mL ethanol and then centrifuged for 15 min. Precipitates were dissolved in 0.5

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mL distilled water and 1 mL 0.2% anthrone (dissolved in 98% H2SO4) was added into each tube. 6


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After boiling for 20 min, OD values were detected at 620 nm and glycogen contents were

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employed glucose as a standard level and normalized to protein level by a BCA kit (Beyotime

137

Biotechnology, China).

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ROS, O2-, mitochondria numbers and mitochondrial membrane potential assays. Different

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fluorescent probes were used to measure intracellular ROS (CM-H2DCFDA, 10 µM), O2-

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(dihydroethidium, DHE, 20 µM) formations as well as mitochondria numbers (Mito-tracker, 20

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µM) and mitochondrial membrane potential (MMP) (RH123, 10 µM) alterations. Cells were

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washed twice in PBS buffer after treatments and incubated in free serum DMEM contain the

143

probe at 37 °C for 30 min. Then cells were washed three times with PBS buffer and detected on a

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fluorescence microscope (Nikon) at identical acquisition settings.

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Western blot. Cytoplasmic and nuclear protein extracts were prepared using a commercial kit

146

(Sangon, Shanghai, China) according to the manufacturer's instructions. Total protein extracts

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were prepared using the WB/IP lysis buffer. Equal amounts of protein were subjected to

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SDS-PAGE and transferred to PVDF membranes. Membranes were probed with primary

149

antibodies and then detected with horseradish peroxidase conjugated secondary antibodies using

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ECL detection system. β-actin was used as a loading control. Densitometry analysis was

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performed using the Image-pro plus 6.0 software (Media Cybernetic, Silver Springs, MD, USA).

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C. elegans maintenance. Strain N2 was provided by Dr. Aifang Du (Zhejiang University,

153

China) and the strains daf-16 (mu86), pmk-1 (ku25, AY102) and skn-1 (SPC 167) were provided

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by Dr. Dayong Wang (Southeast University, China), which were maintained at 20 °C on a standard

155

nematode growth medium (NGM) with E. coli OP50 as food resources. More detailed information

156

about mutants can be found in Wormbase (http://www.wormbase.org). 7



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Lifespan

analysis.

Synchronized

young

adult

worms

were

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grown

in

NGM

158

with 5-fluorodeoxyuridine (FudR, 50 μg/mL) and OP50 containing different agents during the

159

whole life span. A total of 30-40 young adults were cultured on each NGM and worms were

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counted every other day. Worms that did not move when gently touched with a platinum wire were

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considered as dead. The whole experiments were repeated three times.

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Quantitation of PMK-1 and SKN-1 expressions. Age-synchronized worms were treated with

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the MAE on the first day after hatching for 48 h and were then exposed to 1 mM paraquat

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with/without glucose in the medium for 24 h. After induction, the expression of PMK-1 and

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SKN-1 were measured directly through measuring the fluorescence intensity of the reporter

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protein GFP by fluorescence microscopy. Twenty randomly selected worms from each set of

167

experiments were mounted onto microscope slides coated with 2% agarose, anesthetized with 10

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μM levamisole, and capped with coverslips.

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Statistical analyses. Data are means ± SD. Statistical analyses of the data were performed with

170

SPSS for Windows Version 11.5. One-way ANOVA analyses with Duncan’s multiple range test or

171

t test were used to detect statistical significance, and differences were considered significant when

172

p < 0.05.

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Results and Discussion

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Identification and quantification of flavonoids. Firstly, we determined the possible

175

constituents of flavonoids in A. americana Medik leaves extract using UPLC-TOF/MS analysis.

176

Based on the results of LC-MS/MS, it was deduced that C-glycosyl flavones were in the majority

177

of ALE because a series of fragments [(M-H)-90]- and [(M-H)-120]- were measured. These two

178

fragment ions were the particular character of C-glycoside. Secondly, for 6-C-glycoside, the ion 8


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[(M-H)-90]- was less abundant than the ion [(M-H)-120]-, compared with 8-C-glycoside. Peak 2,

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peak 3 and peak 4 were respectively identified as schaftoside, orientin and vitexin instead of

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isoschaftoside, isoorientin and isovitexin (Fig. 1).13, 14 Comparison with retention time of authentic

182

standards, our deduction was verified and concentrations of these three compounds in the extract

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were also determined. Vitexin accounted for 467 ± 6.79 mg/g, schaftoside accounted for 189 ±

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3.83 mg/g and orientin accounted for 43.2 ±0.68 mg/g (Table 1). Besides, peak 1 (431[M-H]-) did

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not belong to C-glycosyl flavones but a kind of phenolic acid. Fragment ions 385[(M-H)-46]-, 223

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[(M-H)-46-162]- and 179 ([223-CO2]-), indicating a derivative of a sinapoyl hexoside.15 Recently,

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caffeoyl β-d-glucopyranoside has been identified in A. americana flowers methanolic extract,

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showing a strong maltase inhibitory effect.9 Considering similar flavonoids biosynthesis in the

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same plant, we presumed 179[M-H]- in peak 1 was more likely represent caffeic acid. Given its

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proportion in the whole peak area, more information needs to make sure the certain structure of

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peak 1.

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The flavone C-glycosides belong to important members of the flavonoid family, which are rich

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in many edible plants or herbs such as Lotus (Nelumbo nucifera) and Mung bean (Vigna radiata

194

L.). They possess a wide range of biological activities, including antioxidant, antidiabetic,

195

anti-inflammatory and antibacterial qualities.16 In the Apios americana leaves extract, we are very

196

interested in its biological activity since C-glycosides as the main constituents.

197

ALE prevented hepatic glucose metabolic disorder in high-glucose-induced cells. Fig. 2A

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showed that no toxicity of ALE from 10 to 400 μg/mL for both HepG2 and LO2 cells, suggesting

199

safety of ALE at these concentrations above. Cells with insulin resistance are a better in vitro

200

model to investigate type 2 diabetes since a variety of parameters related to glucose metabolism is 9



201

altered. Elevated glucose level plays a vital role in the development of insulin resistance, along

202

with excessive fat accumulation and ROS generation.2 In this study, two kinds of hepatic cells

203

were exposed to 30 mM glucose for 36 h and an obvious decrease of glucose consumption and

204

uptake was investigated in model cells, compared to the control, demonstrating that a model of

205

hepatic insulin resistance (IR) was successfully built. Compared with the IR model, ALE at

206

concentrations of 100 and 200 μg/mL was able to avoid the inhibited glucose uptake caused by

207

high-glucose (Fig. 2B). Additionally, ALE significantly increased the glucose consumption by

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21%/11%, 52%/3% 58%/24% and 79%/47% at 25, 50, 100 and 200 μg/mL of HepG2 and LO2

209

cells respectively, showing similar results with glucose uptake (Fig. 2C). Meanwhile, ALE also

210

increased glycogen levels in both cells (Fig. 2D). Metformin, as a positive control, showed the

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best protective effect on glucose consumption but less effective than ALE on glycogen synthesis.

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Leguminous plants like soybeans and mung beans are regarded as high nutritional value food

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contain a large amount of soluble dietary fiber, polyunsaturated fatty acids, vitamins and minerals

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as well as bioactive substances including flavonoids, phenolic acids, organic acids and so on.

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Interest of legumes as functional food ingredient in the diabetic diet is increasing. Genistein and

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daidzein, vitexin and isovitexin, γ-conglutin have been identified as bioactive compounds that may

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interfere with the metabolism of glucose no matter in vitro and in vivo research.17 Diet with mung

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bean sprout and seed coat those are abundant in vitexin and isovitexin could lower blood glucose,

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total cholesterol and triglyceride, and increase glucose tolerance in diabetic KK-A y mice. These

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two substrates also exhibited capacity of α-glucosidase inhibition and postprandial blood glucose

221

level reduction in mice.18 But there is few literatures published on antidiabetic quality of

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schaftoside, another main ingredient of our extract. 10


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Protective effects of ALE on high glucose-induced oxidative damage in HepG2 cells.

224

Considering similar above results between cancer and normal hepatic cells, we chose HepG2 cells

225

for further research since this cell line was more widely used for in vitro study related to insulin

226

resistance. Increase of antioxidant ability to inhibit ROS and superoxide anion (O2-) generation is

227

one of the strategies to prevent hepatotoxicity triggered by oxidative stress. As shown in Fig. 3,

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high glucose indeed obviously increased basal intracellular ROS and O2- levels, which was

229

consistent with previous reports.2, 19 Therefore, our initial goal was to determine whether ALE

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could reduce free radical generation under a hyperglycemic condition. Compared with high

231

glucose-treated HepG2 cells, pre-treatment with ALE decreased both ROS and O2- levels by

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approximately 47 ±0.68% and 68 ±0.74%, indicating that ALE is capable of reducing excess free

233

radical production. Additionally, glucose stimulation led to a MMP motivation while did not

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change mitochondria numbers, suggesting alterations in mitochondrial permeabilization that

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facilitates superoxide production in the mitochondria, amplifying oxidative stress and damage.

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ALE treatment not only prevented the increase of MMP but also partly enhanced mitochondria

237

numbers, showing a protective effect against mitochondria dysfunction from high glucose-induced

238

oxidative injury.

239

Flavonoids exhibit various beneficial properties on oxidative damage and chronic diseases no

240

matter in in vitro and in vivo trials, attributing to their powerful capacity of free radical scavenging.

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Vitexin occupies highest concentration in our extract, probably playing the most important role in

242

cellular protective effect. And this substrate has been report to have a significant anti-oxidative

243

activity.20 However, mechanisms of C-glycosyl flavones on oxidative stress alleviation at a

244

molecular level are still unclear, so we detected some key factors involved in oxidative damage 11



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resistance to find out possible targets of ALE.

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MAPKs and Nrf2 are involved in ALE-mediated oxidative stress resistance in HepG2 cells.

247

After a long high glucose exposure, phosphorylation of c-Jun N-terminal kinase (JNK) increased

248

to 2.7 times, and phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (ERK1/2)

249

decreased to 0.6 times and 0.05 times respectively, compared with the control value. ALE

250

pre-treatment prevented the phosphorylation of JNK, more suppressed the phosphorylation of p38,

251

even lower than the control, but did not restore ERK expression levels. Glucose also stimulated

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nuclear factor erythroid 2-related factor 2 (Nrf2) expression both in cytoplasm and nucleus,

253

accompanied with heme oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase 1 (NQO1)

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levels up-regulation. Besides, glucose induced forkhead box protein O1 (FOXO1) and PPARγ

255

coactivator 1α (PGC-1α) transcriptional activities and ALE repressed these alterations (Fig. 4).

256

In liver, FOXO1 can bind to glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate

257

carboxykinase (PEPCK) target DNA sequence directly to regulate their expression levels. Both

258

enzymes participate in critical processes of gluconeogenesis, playing vital roles in glucose

259

homeostasis.21 Moreover, PGC-1α and its target genes are implicated in hepatic gluconeogenesis,

260

oxidative phosphorylation and mitochondrial biogenesis.22 Enhancement of FOXO1 and PGC-1α

261

transcriptional activities by glucose might lead to an enhancement of gluconeogenesis so that

262

produced more glucose, which further stimulated insulin resistance formation. ALE inhibits these

263

increases, resulting in reduced glucose consumption and glycogen content recovery in liver cells

264

that is under a hyperglycemic environment.

265

Studies based on cells indicate the impairment of insulin signaling by phosphorylation of

266

insulin receptor substrate proteins (IRS) on serine and threonine residues, which might contribute 12


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to insulin resistance in metabolic disease.23 JNK can be facilitated by ROS and cause IRS1 serine

268

phosphorylation to impair insulin signal transduction. Besides, activated JNK promotes FOXO1

269

activity by promoting its movement to the nuclear.24 Considering JNK is vital for insulin

270

resistance development in hepatic cells,25 we infer that ALE firstly suppresses excessive free

271

radicals generation in cells under high glucose environment and then JNK phosphorylation is

272

inhibited due to decreased ROS levels so that insulin signaling normal transduction is recovered,

273

which explains improvement of glucose consumption, glucose uptake and glycogen synthesis.

274

Restriction of Nrf2 and two of its targets HO-1 and NQO1 are investigated in high glucose–

275

induced HepG2 cells when pre-treated with ALE. It is a stress response that Nrf2 is stimulated in

276

cells when they suffer from extra glucose to prevent themselves from more damage and Kim et al.,

277

also reported that Nrf2 expression in liver was significantly increased after CCl4 administration in

278

mice.26 But most studies exhibited that flavonoids showed protective effects against hepatic

279

oxidative stress via activating Nrf2, which were different from our results. We suppose ALE itself

280

has already directly scavenged excessive free radicals to lower elevated ROS levels so that

281

stimulation by ROS is also impaired. Accordingly, there is no need to activate Nrf2 pathway since

282

threaten of oxidative damage has already been lifted. Similarly, p38 was also decreased because

283

no enough stimulating factor to induce its expression. Vitexin has been reported to protect brain

284

against ischemia injury by down-regulating p-JNK and p-p38, 27 implying vitexin in the ALE may

285

also play an important role in MAPKs mediation in hyperglycemic hepatocytes. However, it is still

286

unclear why p38 levels were even lower than the control when treated with ALE. More research is

287

needed to determine the relationships between protective effects of ALE in cells and MAPKs/Nrf2

288

expression alterations. These data demonstrated that the involvement of specific mitogen-activated 13



289

protein kinases (MAPKs) (JNK and p38) in ALE mediation of Nrf2 pathway.

290

ALE prevents drastic lifespan shorten of C. elegans suffered from oxidative stress under

291

hyperglycemia

292

Caenorhabditis elegans is a good in vivo model to study various diseases associated with age

293

and oxidative stress because of its high similarity with human gene and short life span. Therefore,

294

we selected C. elegans for further experiments to investigate whether protective effects of ALE

295

were evolutionarily conserved.

296

Fig. 5A demonstrated that lifespans of worms were extended after grown in the medium added

297

into ALE at 100 μg/mL, compared to untreated ones. To test the protective ability against glucose

298

toxicity, we added 50 mM glucose into medium, which could lead to a shortened life span while

299

ALE could partly reverse this damage, showing the longevity-promoting effect. Moreover, we

300

uncovered that exposing worms to paraquat shortened lifespan and glucose could accelerate this

301

process of aging, resulting in shorter longevity. Again, ALE partly relieved this damage in

302

wild-type nematodes but failed to increase the survival rate in daf-16(-) mutants (Fig. 5B and

303

Table 2). DAF-16/FOXO is necessary for pathogenic bacteria resistance and longevity extension

304

in worms. Lee et al. demonstrated that glucose decreased life span by inhibiting DAF-16/FOXO

305

and dramatically shortened the long life span in daf-2 mutants.28 It is suggesting that

306

DAF-16/FOXO should be one of the targets of ALE to play an antioxidant activity.

307

AY102 animals were developed by introducing the acEx102 extrachromosomal array into

308

KU25 animals, resulting in intestinal PMK-1::GFP expression.29 Meanwhile, a gst-4p::GFP

309

reporter was well established in SPC 167 strain to determine SKN-1 activity.30 In our study, both

310

PMK-1/p38 and SKN-1/Nrf were inhibited by glucose intervention even worms suffered from 14


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311

stimulation of paraquat. These intense inhibitions undermined ability of animals against oxidative

312

stress, leading to damage aggravations and ALE intervention partly recovered SKN-1 and PMK-1

313

expressions (Fig. 5C and D). The degree of SKN-1 in C. elegans and Nrf2 proteins in mammals

314

function is remarkably conservative. These similarities imply that SKN-1 provides a powerful

315

model for exploring how Nrf2 proteins are modulated, as well as how they affect the development

316

and functions of normal tissues in vivo. SKN-1 in C. elegans participates in many regulatory

317

pathways associated with different stresses and it is also modulated by the insulin-like/IGF-1

318

pathway for the promotion of longevity. In response to oxidative stress such as paraquat, heat and

319

hydrogen peroxide, PMK-1/p38 has been found to directly phosphorylate SKN-1 to activate its

320

activity.31 Lee et al. has elucidated that vitexin from V. angularis elevated the survival rates of C.

321

elegans against heat and oxidative stress, which was partly due to intracellular ROS reduction as

322

well as expressions of superoxide dismutase and heat shock protein activation.32 And these results

323

in C. elegans further confirmed potential protective effects of ALE on glucose-induced oxidative

324

stress.

325

In vivo studies, we observe that C. elegans is more sensitive to paraquat under hyperglycemic

326

conditions and PMK-1 and SKN-1 expressions restrain is involved in this sensitivity. ALE

327

intervention well prevents lifespan shorten in N2 strain accompanied with PMK-1 and SKN-1

328

expressions restoration. However, effects were not shown in daf-16 mutant, suggesting

329

DAF-16/FOXO is one target for ALE. These findings are in agreement with the reports that many

330

C-flavones reveal antioxidant and antidiabetic capacity. Given the homology of PMK-1 and

331

SKN-1 genes to their human counterparts, our studies further indicate that beneficial effects of

332

ALE on glucose-induced damage are associated with MAPK and Nrf2 regulation. Therefore, ALE 15



333

can be served as a potential new strategy to help improve human health, especially for some

334

chronic diseases like type 2 diabetes.

335

Abbreviations: ALE, Apios americana leaves extract; IR, insulin resistance; C, control; O2-,

336

superoxide; ERK, extracellular signal-regulated kinase; MMP, mitochondrial membrane potential;

337

NQO1, NAD(P)H quinine oxidoreductase 1; Nrf2, nuclear factor erythroid 2-related factor 2;

338

JNK, c-Jun N-terminal kinase; MAPKs, mitogen-activated protein kinases; FOXO1, forkhead

339

box protein O1; PGC-1α, PPARγ coactivator 1α; ROS, reactive oxygen species; HO-1, heme

340

oxygenase-1; PQ, paraquat; Glu, high glucose treatment.

341

Acknowledgements

342

This work was supported by grants from Zhejiang Provincial Natural Science Foundation of China

343

(No. LZ14C200001). Some strains were kindly provided by Dayong Wang (Southeast University,

344

China).

345

References

346

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K., Lifespan Extending and Stress Resistant Properties of Vitexin from Vigna angularis in

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431

Figure captions

432

Figure 1. (A) HPLC elution profiles of flavonoids in A. americana Medik leaves. The peak

433

numbers were underlined and corresponded to those in Table 1. (B) MS/MS information about

434

four compounds. (a) peak 1; (b)peak 2; (c)peak 3; (d)peak 4.

435

Figure 2. Preventive effect of ALE on glucose metabolic disorder in HepG2 and LO2 cells with

436

insulin resistance induced by high-glucose (HG). (A) Cell viability. Cells treated with ALE at

437

various concentrations for 24 h and cell viability was measured by the MTT method. (B) Uptake

438

of 2-NBDG into hepatic cells. (a) control; (b) high-glucose exposure; (c) Metformin-2 mM; (d)

439

ALE-100 µg/mL; (e) ALE-200 µg/mL; Densitometry analysis was performed using the Image-pro

440

plus 6.0 software. At least six images per treatment were quantified by the software. (C) Glucose

441

consumption from culture medium in hepatic cells. Vertical lines represent standard deviations of

442

five replicates. C, control; HG, cells incubated with glucose (30 mM) for 36 h; Met, cells 20


Page 20 of 33

Page 21 of 33


443

pretreated with metformin (2 mM) for 24 h and then incubated with glucose (30 mM) in the

444

presence of metformin for another 36 h. (D) Glycogen contents. Vertical lines represent standard

445

deviations of three replicates. *Significantly different (p < 0.05) from the control group;

446

#

447

different (p < 0.05) from the control group; ɛSignificantly different (p < 0.05) from the HG-treated

448

group (for LO2 cells).

449

Figure 3. ROS, O2-, mitochondrial numbers and MMP levels alterations of HepG2 cells in the

450

presence of glucose and ALE. Cells with different treatments were incubated with CM-H2DCFDA,

451

DHE, Mito-tracker or RH123 for (A) ROS, (B) O2-, (C) mitochondrial numbers and (D) MMP

452

analysis. (a) Control; (b) Cells with high glucose treatment (model cells); (c) Model cells treated

453

with ALE at 50 µg/mL; (d) Model cells treated with ALE at 100 µg/mL. Images were captured on

454

a fluorescence microscope at identical acquisition settings. (E) The quantitative analysis of

455

fluorescence from six images with same treatments. Data represent means ± SD. *Significantly

456

different (p < 0.05) from the control group; #Significantly different (p < 0.05) from the HG-treated

457

group.

458

Figure 4. ALE treatment altered expressions of proteins associated with oxidative stress. (A)

459

Protein levels of HO-1, NQO1, ERK, JNK and p38 MAPK in total cell extracts. (B) The intensity

460

of bands corresponding to HO-1 and NQO1 were corrected by β-actin. The intensity of p-ERK,

461

p-JNK and p-p38 were corrected by respective total ERK, JNK and p38 MAPK protein levels to

462

obtain relative measures of ERK, JNK and p38 MAPK phosphorylation among samples. (C)

463

Effect of ALE on intracellular distribution of Nrf2, PGC-1α and FOXO1 in HepG2 cells. (D) The

464

intensity of bands corresponding to cytoplasm and nuclear was corrected by β-actin and Lamin B

Significantly different (p < 0.05) from the HG-treated group (for HepG2 cells). ōSignificantly

21



465

respectively. Foxo1 of control in nuclear was too low to be identified by the software. Vertical

466

lines represent standard deviations of three replicates. *Significantly different (p < 0.05) from the

467

control group; #Significantly different (p < 0.05) from the HG-treated group.

468

Figure 5. Protective effect of ALE on C. elegans challenged by paraquat under hyperglycemia. (A)

469

Effect of ALE on longevity in N2 under normal and high glucose conditions. (B) Lifespan of N2

470

and daf-16 under different conditions. Mean lifespans of each treatment were summarized in Table

471

2. Con, control; ALE, worms cultured in medium contained ALE (100 µg/mL); Glu, worms

472

cultured in medium supplied with glucose (50 mM); Glu+ALE, worms cultured in medium

473

supplied with glucose (50 mM) and ALE (100 µg/mL); PQ, worms cultured in medium contained

474

parquat (1 mM); PQ+Glu, worms cultured in medium contained parquat and glucose (50 mM);

475

PQ+Glu+ALE, worms cultured in medium contained parquat and glucose with ALE (100 µg/mL)

476

protection; (C) PMK-1 expression in mutant AY102 worms. (D) SKN-1 activity was imaged for

477

GFP expression indicative of SKN-1 activation of the gst-4p::gfp reporter in mutant SPC167

478

worms. (a) control; (b) paraquat (1 mM) (c) paraquat under a high glucose (50 mM) condition (d)

479

paraquat under a high glucose condition in the presence of ALE (100 µg/ml) (E) The quantitative

480

analysis of fluorescence. Densitometry analysis from at least 10 worms per treatment was

481

performed using the Image-pro plus 6.0 software. Values with different letters above are

482

significantly different, p < 0.05, one-way ANOVA test.

22


Page 22 of 33

Page 23 of 33


Table 1. Major compositions identified from Apios americana leaves extract Tentative identification

Concentration (1gram)

Peak No.

Rt (min)

1

12.98

Sinapic acid derivative

-

2

14.06

Schaftoside

189 ±3.83 mg

3

14.83

Orientin

43.2 ±0.68 mg

4

15.98

Vitexin

467 ±6.79 mg

Table 2. Effects of Apios americana leaves extract on the lifespan of Caenorhabditis elegans Strain

Treatment

N

Mean ±SD (days)

Control

90

16.9 ±2.35

Wild type (N2)

ALE

90

19.8 ±1.86

(Fig. 5A)

Glu

95

11.1 ±1.28

Glu+ALE

90

14.4 ±2.03

Control

90

21.3 ±1.54

PQ

90

11.7 ±0.98

Glu+PQ

90

7.20 ±0.67

Glu+PQ+ALE

90

18.9 ±1.08

Control

100

13.1 ±1.21

daf-16 (mu86)

PQ

100

10.8 ±0.87

(Fig. 5B)

Glu+PQ

100

7.65 ±0.65

Glu+PQ+ALE

100

9.38 ±0.53

Wild type (N2) (Fig. 5B)

23



(A)

(B) (a)

(b)

24


Page 24 of 33

Page 25 of 33


(c)

(d)

Figure 1

25



(A)

Page 26 of 33

(B) HepG2

1.8

LO2

HepG2

*#

1.6

120

1.4 2-NBDG uptake (Fold of control)

100 Inhibition (%)

LO2

80 60 40

*# ɛ

*#

1.2

ɛ

ɛ

1 0.8

ō *

0.6 0.4

20

0.2 0 C

10

25

50

100

200

0

400

C

ALE (µg/mL)

HG

Met

100

200

ALE (µg/mL)

HepG2

LO2

(a)

(b)

(c)

26


(d)

(e)

Page 27 of 33


(C)

(D) 2.5 HepG2

*#

ōɛ *#

1.6 1.4

2

#

#

ɛ

1.2

ɛ

1 *

0.8

LO2

LO2

ō

ō

ō

0.6

Glycogen contents (Fold of control)

Glucose consumption (Fold of control)

1.8

HepG2

*

ōɛ ōɛ 1.5

1

ō

0.5

0.4 0.2

0

0 C

HG

Met

25

50

100

200

ALE (µg/mL)

Figure 2

27


C

HG

Met

100

200

ALE (µg/mL)


Page 28 of 33

(A)

(B)

(C)

(D)

(a)

(b)

(c)

(E) Con

HG

ALE-50

ALE-100

4 3.5

*

*

Fold of control

3 *

2.5 2 1.5

# b

*#

*#

* #

#

c

#

1 0.5 0 H2DCFDA

DHE

Mito-tracker

RH123

Figure 3 28


(d)

Page 29 of 33


(A) p-p38

p38

p-JNK

JNK

p-ERK

ERK

HO-1

NQO1

β-actin

G

β-actin C

HG

50 100 200 ALE (µg/mL)

C

HG 50 100 200 ALE (µg/mL)

(B) 1.2

p-JNK/JNK

1

2

*# *#

1.5 1 *# 0.5

0.8 0.6

*

0.4 0.2

0.8 0.6 0.4

*# *#

ALE (µg/mL)

2

HO-1

1.8

3.5

1.6

3 2.5

2 1.5

0.2

0 C HG 50 100 200

*

4

1 *#

0

4.5

p-ERK/ERK

1 Fold of control

2.5

Fold of control

Fold of control

3

1.2

p-p38/p38

* 0

C HG 50 100 200

*

*

0.5 *

C HG 50 100 200

ALE (µg/mL)

ALE (µg/mL)

29


Fold of control

*

Fold of control

3.5

*# *# *#

0

NQO1

*

1.4

1.2 1

#

0.8 0.6 0.4

*#

*#

0.2 0

C HG 50 100 200 ALE (µg/mL)

C HG 50 100 200 ALE (µg/mL)


Page 30 of 33

(C) Cytoplasm

Nuclear

Nrf2 Foxo1 PGC-1α β-actin

Lamin B C

HG 50 100 200 ALE (µg/mL)

C

HG

50 100 200 ALE (µg/mL)

(D) 1.6

Nrf2 cytoplasm

1.4

2.5

7

Nrf2 nucleus

*

1

1 0.8

# *# *#

0.6

ALE (µg/mL)

*#

3

1.5

1

# *#

FOXO1 cytoplasm

4

*#

3

2.5 2

*# *#

1.5 1 0.5

0 C HG 50 100 200

*

3.5

0.5

0 C HG 50 100 200

4

4.5

PGC-1α nucleus

1

0.2

0

*

2

0.4 0.5

*#

5

*

2

*# Fold of control

1.5

*#

Fold of control

Fold of control

*# *#

2.5

PGC-1α cytoplasm

6

1.2 2

*

Fold of control

*

Fold of control

3

0 C HG 50 100 200

ALE (µg/mL)

ALE (µg/mL)

Figure 4

30


0 C HG 50 100 200 ALE (µg/mL)

C HG 50 100 200 ALE (µg/mL)

Page 31 of 33


(A)

(B) Con

ALE

Glu

Con

Glu+ALE

PQ+Glu

PQ+Glu+ALE

100

100

Con

% Survival

40

60 40 20

20

0

5

10

15 20 Time (day)

25

30

daf-16

60 40

0 0

5

10

15 20 Time (day)

25

30

0

5

(C)

(a)

PQ+Glu+ALE

20

0

0

PQ+Glu

80 % Survival

80

60

PQ

100

N2

N2

80 % Survival

PQ

(b)

(c) 31


(d)

10 15 Time (day)

20

25


Page 32 of 33

(D)

(a)

(b)

(c)

(d)

(E) 3

SKN-1 a

a

2.5

a

2

2

Fold of control

Fold of control

2.5

PMK-1

1.5 b 1

bc

1.5 b 1 b

c

0.5

0.5 0

0 Con PQ Glu ALE

Con PQ Glu ALE

Figure 5 32


Page 33 of 33


TOC

33


Effects of C-Glycosides from Apios americana Leaves against

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