Rapid Analysis and Guided Isolation of Astragalus Isoflavonoids by

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Rapid Analysis and Guided Isolation of Astragalus Isoflavonoids by UHPLC-DAD-MSn and Their Cellular Antioxidant Defense on High Glucose Induced Mesangial Cells Dysfunction Dan Tang, Ying-Bin Shen, Zhi-Hua Wang, Bao He, You-Hua Xu, Hong Nie, and Quan Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02949 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 2, 2017

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Journal of Agricultural and Food Chemistry

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Rapid Analysis and Guided Isolation of Astragalus Isoflavonoids by

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UHPLC-DAD-MSn and Their Cellular Antioxidant Defense on High

3

Glucose Induced Mesangial Cells Dysfunction

4

Dan Tang†1, Ying-Bin Shen‡1, Zhi-Hua Wang†, Bao He#, You-Hua Xu§, Hong Nie†*,

5

Quan Zhu§,#*

6

Affiliations

7

†

8

and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632,

9

P.R. China.

Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM

10

‡

11

P.R. China

12

§

13

Science and Technology, Macau, P.R. China.

14

#

15

Guangzhou 510530, P.R. China

Department of Food Science and Engineering, Jinan University, Guangzhou 510632,

State Key Laboratory of Quality Research in Chinese Medicine, Macau University of

Institute of Kidney Diseases, Guangdong Consun Pharmaceutical Group,

16 17

*Corresponding authors

18

Hong Nie

19

Tel: 0086-20-8522 2810; Fax: 0086-20-8522 4766; E-mail: [email protected]

20 21

Quan Zhu

22

Tel: 0086-20-8226 7589; Fax: 0086-20-8201 7468; E-mail: [email protected]

23

1

the authors contribute equally to this paper.

24

1

ACS Paragon Plus Environment


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ABSTRACT

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Isoflavonoids including isoflavones, isoflavans and pterocarpans, the principal

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components in Astragalus membranaceus, have a great deal of versatile

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health-promoting benefits. In this work, as a continuation of our searching for

29

bioactive constituents from A. membranaceus, a fast UHPLC-DAD-MSn method was

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firstly used to analyze the isoflavonoids profile of A. membranaceus roots extract.

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Twelve diverse isoflavonoids with subclass of isoflavones, isoflavans and

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pterocarpans

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characterized, of them eight major isoflavonoids were finally isolated guided and

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simultaneously quantified by the established fast UHPLC method. Furthermore,

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results confirmed for the first time that Astragalus isoflavonoid aglycones could

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attenuate mesangial cell proliferation and extracellular matrix (ECM) accumulation

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triggered by high glucose, and the primary mechanism might be via protecting

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intracellular antioxidant enzymes activities, and enhancing endogenous antioxidant

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function to lessen cellular oxidative damage induced by high glucose. Collectively,

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diverse Astragalus isoflavonoids antioxidants have the potential to ameliorate high

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glucose induced mesangial cells dysfunction through the regulation of the cellular

42

antioxidant defense.

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KEY WORDS: Astragalus isoflavonoids; mesangial cells dysfunction; antioxidant

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defense; UHPLC-DAD-ESI-MSn

occurred

in

glycoside/aglycone

pairs

45

2


forms

were

tentatively

the

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INTRODUCTION

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Phytoestrogens isoflavonoids, found abundantly in the Leguminosae family with

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isoflavonoid subclasses such as isoflavones, isoflavans and pterocarpans,1 have drawn

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a great deal of attentions due to their health benefits for numerous disorders and

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chronic diseases. For instance, clinical trials demonstrated that dietary intake of

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isoflavonoids

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membranaceus, an important functional food material of the Leguminosae family, is

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also rich in isoflavonoids. Its dried root, which can be boiled in tea and soup, is

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known as function foods all over the world, to improve body function against various

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diseases such as diabetes mellitus and its complications.4 Astragalus health food

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products could be obtained from

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membranaceus was also be classified as dietary supplement according to the Dietary

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Supplement Health and Education Act.5

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Isoflavonoids including isoflavones, isoflavans and pterocarpans, which commonly

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occur in glycoside/aglycone pairs forms, were reported to be primary metabolic

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components within A. membranaceus,6–8 and such isoflavonoids were associated with

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versatile health benefits such as antioxidant, anti-inflammatory, cardioprotective and

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anticancer properties.5,6,9–11 In addition, a series of our recently published researches

64

indicated

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dysfunction triggered by advanced glycation end products (AGEs) via ameliorating

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inflammation and apoptosis via estrogen receptor, which demonstrated that the two

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tested isoflavonoids could exert beneficial effects on diabetic complications including

could reduce the diabetes risk in populations.2,3 Astragalus

calycosin

and

health food market in USA because A.

calycosin-7-O-β-D-glucoside,

3


could

modulate

cell


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diabetic nephropathy (DN).12–16 However, the effects of other major isoflavonoids,

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especially such as isoflavans and pterocarpans, within A. membranaceus on DN and

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their underlying mechanisms were unclear yet.

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DN, a major public health concern in the past few decades, has become a primary

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cause of end-stage kidney disease with a high risk of morbidity and mortality

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worldwide.17,18 Hyperglycemia induced proliferation and excessive extracellular

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matrix (ECM) deposition of mesangial cell

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pathological features in the progression of DN. Therefore, the development of

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effective methods to ameliorate mesangial cell dysfunctions is pivotal for the

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treatment of DN. Oxidative stress induced by reactive oxygen species (ROS), i.e.

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superoxide anion radical (O2−•), singlet oxygen (O2), hydroxyl radical (•OH), and

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hydrogen peroxide (H2O2), is a leading cause of diabetes complications including

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DN.19–22 The experimental data demonstrated that the metabolic abnormalities of

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diabetes caused overproduction of mitochondrial O2−•, which is the central and major

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mediator of diabetes tissue damage with the activation of several downstream

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pathways in the development of the glomerular sclerosis in diabetes.23,24

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In this study, as a continuation of our discovering of bioactive agents from A.

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membranaceus for DN, fast UHPLC-DAD-MSn based on superficially porous

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particles (shell) was firstly adopted to analyze the isoflavonoids profiles in A.

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membranaceus roots. Meanwhile, eight main isoflavonoids were obtained from the

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extract of A. membranaceus roots guided by UHPLC. Considering that metabolism

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studies showed the isoflavones glycosides in A. membranaceus will be finally

has been identified the prominent

4


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converted into their corresponding aglycones in vivo, therefore, we investigated the

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renoprotective effects of four representative isoflavonoid aglycones, i.e. calycosin

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(CAL), formononetin (FOR), (6αR,11αR)-3-hydroxy-9,10-dimethoxypterocarpan

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(DPC), and (3R)-7,2’-dihydroxy-3’, 4’-dimethoxyisoflavone (DIF), on mesangial cell

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proliferation, ECM accumulation and cellular oxidative stress triggered by high

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glucose in vitro.

96

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MATERIALS AND METHODS

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General Apparatus and Chemicals

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UV spectra were determined using a Lambda 35 spectrophotometer (Perkin Elmer,

100

USA). IR spectra were recorded by a Paragon 500FTIR infrared spectrometer with

101

KBr pellets (Perkin Elmer, USA). NMR data were

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500 MHz spectrometer (Bruker, Switzerland). Chemical shifts were expressed as δ

103

values (ppm) with reference to the solvent signals. MSn data were determined using

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a Finnigan LCQ Fleet ion trap mass spectrometer (Thermo Finnigan, USA). Sephadex

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LH-20 (Pharmacia, Sweden) and Silica gel (200–300 meshes, Qingdao Marine

106

Chemical Inc., China) and were adapted for separation and purification. Thin-layer

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chromatography (TLC) was determined using pre-coated GF254 silica gel plates

108

(Qingdao, China). Chromatographic grade formic acid and acetonitrile were provided

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by Merck (Darmstadt, Germany). Deionized water was obtained from a Millipore

110

Synergy UV water purification system (Billerica, USA). All other reagents were

recorded by a Bruker Avance III

5



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derived from commercial sources.

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Extraction and Isolation

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The A. membranaceus dried roots were provided by Bozhou Huikang Inc. (Anhui,

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China). The dried roots (10 Kg) were minced finely and extracted with 80% ethanol

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under reflux (3×80L each) for 1 h each time. Then, the mixed extracts were filtered

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and concentrated in vacuum to form the syrup (~2.8 Kg), which was re-suspended in

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water (80 ◦C), then kept in the freezer (2-8 ◦C) overnight. The supernatant liquor was

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fractionated by D101 macroporous resin chromatography (100×14 cm, I.D.) using

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water and 80% ethanol. The 80% ethanol fraction solvent was removed in a vacuum

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and then re-suspended in water, which was partitioned with ethyl acetate (5 × 1000

121

mL each). The yield ethyl acetate-soluble residue (38 g)

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column

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chloroform-methanol system (50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, v/v), and finally

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methanol to furnish 21 portions (Fr.1~21) based on TLC and UHPLC-DAD analysis.

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Fr.19 was submitted to Sephadex LH-20 column (120×3 cm, I.D.) with methanol to

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yield compound 1 (324 mg). Compound 4 (98 mg) was obtained from Fr. 17 by direct

127

crystallization from methanol. Compounds 5 (97 mg) was obtained from Fr.14 by

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repeated silica gel column chromatography (300 mesh, 40 × 3 cm, I.D.) eluted with

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chloroform-methanol (15:1, v/v) and followed by Sephadex LH-20 column

130

chromatography (120 × 2.5 cm, I.D.) using methanol as eluent. Fr.10 was applied to

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Sephadex LH-20 column (120 × 3 cm, I.D.) with methanol to produce compound 6

(200~300

mesh,

50

×

6

cm,

I.D.)

was submitted to silica gel consecutively

6


eluted

with

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(260 mg). Fr.15 was submitted to silica gel column (300 mesh, 30 × 4 cm, I.D.) with

133

chloroform-methanol (10:1, v/v), and followed by Sephadex LH-20 column

134

chromatography using methanol to yield compound 7 (82 mg). Compound 9 (173 mg)

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was obtained from Fr.2 by crystallization from methanol. Fr.10 was separated by

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repeated silica gel column (300 mesh, 40 × 4 cm, I.D.) eluted with petrol-acetone

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system (5:1→1:1, v/v), and recrystallized with methanol to afford compound 10 (63

138

mg) and compound 11 (55 mg). The structures of compounds 1, 4-7, 9-11 all were

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elucidated by UV, IR, MS and NMR in comparison with the data in the

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references.25–27 The purities of all isolated isoflavonoids were >98% determined by

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analytical UHPLC-DAD-MS.

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UHPLC-DAD-ESI-MSn analysis of isoflavonoids

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Rapid analysis of isoflavonoids was performed on an Agilent 1200 HPLC

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ChemStation

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Chromatographic separation was achieved with an Agilent Poroshell Zorbax EC C18

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column (2.1 × 100 mm, 2.7 µm). A gradient elution program was employed using

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0.2% formic acid aqueous solution (A) and acetonitrile (B) at flow rate of 0.4 mL/min:

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0–8 min, 15-35% B; 8–12 min, 35-55% B; 12-17 min, 55-80% B; 17-20 min, 80% B;

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followed by 5 min of re-equilibration. The column temperature was 30 ◦C, and the

150

analytes were monitored at 280 nm. The chromatographic peaks of the analytes were

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assigned according to the retention times and UV spectra of the reference compounds.

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Quantification of targeted isoflavonoids was performed with external standards, using

controlling

software

(Agilent

Technologies,

7


with

Germany).


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linear curves generated between 0.20 and 40 µg/mL.

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ESI/MSn analysis were performed on a Finnigan LCQ Fleet ion trap mass

155

spectrometer with Xcalibur version 2.0 software (Thermo Finnigan, San Jose, CA,

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USA) in a positive ion mode. The MS analysis conditions were set as follows: ion

157

spray voltage, 3.5 kV; capillary temperature, 300 ◦C; tube lens offset voltage, 30 V;

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capillary voltage, 15 V; sheath gas (N2) flow rate, 40 arbitrary units; auxiliary gas (N2)

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flow rate, 5 arbitrary units; nebulizing gas, nitrogen (99.999%); collision gas, helium

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(99.999%). The full-scan MS data were monitored within the range of m/z 100 to 650.

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A data-dependent program was set in order to the most abundant ions in each scan

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could be captured and submitted to further MSn analysis. The collision energy for

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MSn was adjusted to 30-40% of the maximum.

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Cell culture

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The rat glomerular mesangial cell HBZY-1 (China Center for Type Culture Collection,

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China) were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM,

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GIBCO) containing 10% fetal calf serum (FCS; Sijiqing Biological Engineering Inc.,

168

China), 100 µg/mL streptomycin and 100 U/mL penicillin, at 37 ◦C in a 5% CO2/95%

169

air incubator. Cells were passaged twice a week at 1:2 split.

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Assay for mesangial cells proliferation

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The inhibition of high glucose triggered rat mesangial cells proliferation was assessed

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by methyl thiazolyl tetrazolium (MTT).12 Briefly, cells in exponential growth were

8


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seeded in 96-well flat-bottomed plates (Corning Costar Inc., USA) as a density of

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2×105 per well in DMEM (5.6 mM glucose) containing 5% FCS. After cell confluence

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reached, the medium was replaced with FCS free DMEM for 24h to make sure

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growth arrest in a cell incubator. Then, cells were treated by either 5.6 mM (control

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group, Ctr) or 30 mM (high glucose group, HG) D-glucose for 48 h with or without

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tested isoflavonoids at 12.5, 25 and 50 µM, respectively. α-tocopherol (purity>96%,

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Sigma Aldrich, Germany) as a positive control. Then the cells were further incubated

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for 4 h to form formazan crystals after addition of MTT (Sigma, USA). Absorbance

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values were recorded at 490 nm by a microplate reader (Molecular Devices

182

Corporation, Sunnyvale, CA), and the proliferation rate was obtained by

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normalization.

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Biochemical measurement of mesangial collagen

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To assess collagen accumulation, quantification of hydroxyproline in the cells culture

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supernatant was carried out according to the commercial kit protocols (Nanjing

187

Jiancheng Bioengineering Institute, China). Briefly, the cells culture supernatant

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sample was hydrolyzed in 6 M hydrochloric acid for 16 h, and hydroxyproline

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contents were determined with a color-based reaction using a standard curve.

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Collagen accumulation was finally measured by multiplying the hydroxyproline

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contents by a factor of 8.228.

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Measurement of intracellular ROS

193

The intracellular ROS level was evaluated by loading cultured cells with the 9


cell


194

fluorescence probe dihydroethidium (DHE).29 In brief, cells were incubated with 5

195

µM DHE at 37 °C for 30 min in PBS according to manufacturer's protocols. Then

196

cells were washed for three times with PBS to remove the free DHE and to be

197

recorded by an Olympus BX51 Fluorescence microscope (Olympus Corporation,

198

Japan). The florescence intensity was analyzed by Image-Pro Plus software version

199

6.0 (Media Cybernetics Inc., USA). The fluorescence intensity was evaluated by the

200

mean intensity of six random squares with fixed size in the fluorescence pictures.

201

202

Determination of intracellular antioxidant enzyme activity and malondialdehyde

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The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione

204

peroxidase (GSH-Px) as well as the content of malondialdehyde (MDA) were

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measured by commercial colorimetric assay kits (Nanjing Jiancheng Bioengineering

206

Institute, China) according to the protocols. Briefly, cell growth in a 24-well plate was

207

arrested by treating with FCS free DMEM for 24 h in a cell incubator. Then, cells

208

were exposed to either 5.6 mM (control group, Ctr) or 30 mM (high glucose group,

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HG) D-glucose for up to 48 h with or without tested isoflavonoids at 50 µM,

210

respectively. α-tocopherol was set as the positive control. At the end of this period, the

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cell culture supernatant was assayed for the content of MDA. The cells were washed

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twice with PBS, collected and sonicated in sample buffer, then the sonicated mixture

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was centrifuged with 10,000 × g for 15 min at 4 °C to collect the supernatant. The

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protein content of cell lysates was measured by an enhanced BCA Protein Assay Kit 10


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(Beyotime Biotechnology, Shanghai, China) with bovine serum albumin as standard.

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The activity of each enzyme was calculated in unit per mg of protein (U/mg protein).

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Statistical analysis

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Data were present as mean ± SD of three independent experiments unless otherwise

219

indicated. One-way ANOVA was used for statistical analysis followed by Dunnett

220

post test with SPSS version 20.0 (SPSS Inc., USA). A value p

Rapid Analysis and Guided Isolation of Astragalus Isoflavonoids by

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