Food Chemistry 172 (2015) 183–189

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Review

Effect of glycosylation patterns of Chinese eggplant anthocyanins andother derivatives on antioxidant effectiveness in human colon cell lines

http://dx.doi.org/10.1016/j.foodchem.2014.08.1000308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +86 2134207074 (X. Wang).E-mail addresses: pjing@sjtu.edu.cn (P. Jing), xingyaw@gmail.com (X. Wang).

Pu Jing a,⇑, Bingjun Qian a, Shujuan Zhao a, Xin Qi c, Ludan Ye a, M. Mónica Giusti d, Xingya Wang b,⇑a Research Center for Food Safety and Nutrition, Key Lab of Urban Agriculture (South), Bor S. Luh Food Safety Research Center, School of Agriculture & Biology,Shanghai Jiao Tong University, Shanghai 200240, Chinab College of Pharmaceutical Science, Zhejiang Chinese Medical University, Zhejiang 310053, Chinac Horticultural Engineering Institute, Tianjin Academy of Agricultural Sciences, Tianjin 300122, Chinad Department of Food Science and Technology, The Ohio State University, OH 43210, USA

a r t i c l e i n f o

Article history:Received 23 April 2014Received in revised form 21 August 2014Accepted 23 August 2014Available online 6 September 2014

Keywords:AntioxidantsDelphinidin derivativesHT-29HCT-116Glycosylation

a b s t r a c t

In this study, we compared the scavenging ROS of anthocyanins from Chinese eggplant var. Niu Jiao Qieand other delphinidin derivatives with different glycosylation patterns in HT-29 and HCT-116 cell lines.The eggplant anthocyanins were isolated and identified using LC–MSn and 1H/13C NMR as delphinidin-3-[(400-trans-p-coumaroyl)-rhamnosyl (1 ? 6)glucoside]-5-glucoside, also known as nasunin. Delphinidinderivatives with glycosylation only on C3 (delphinidin-3-glucoside, 3-sambubioside, or 3-rutinoside)exhibited greater effects on ROS reduction as compared to delphinidin derivatives that have glycosylationon C3 and C5 (delphinidin-3,5-diglucoside > delphinidin-3-rutinoside-5-glucoside). Nasunin has glyco-sylation on C3 and C5 and an acyl group (p-coumaric acid), demonstrated the least effect on ROS reduc-tion. Meanwhile, their ROS reduction activities were consistent with glutathione reductase proteinexpression levels in HT-29. Although not potent in ROS reduction, nasunin and its deacylated derivativesprotected cells from DNA damage in a dose-dependent manner. Taken together, our results suggest thatthe anthocyanins isolated from Chinese eggplant var. Niu Jiao Qie and other delphinidin have antioxidantactivities in colon cancer cells and also protect cells from DNA damage.

� 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1842. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

2.1. Chemicals, cells and media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1842.2. Preparation and analysis of nasunins from eggplants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1842.3. Identification of eggplant anthocyanins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1842.4. Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1852.5. Cellular ROS level (DCF assay) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1852.6. Western blot analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1852.7. DNA damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1862.8. Statistics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

3.1. Structure of anthocyanins in Chinese eggplant hybrid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1873.2. Modulation of cellular ROS level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1883.3. Expression levels of antioxidant proteins and DNA-damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

184 P. Jing et al. / Food Chemistry 172 (2015) 183–189

1. Introduction

Anthocyanins are water-soluble vacuolar pigments found inmost species in the plant kingdom, and are responsible for thered, purple, and blue colors of many fruits, vegetables, cerealgrains, and flowers (Giusti & Wrolstad, 2003). The common struc-tures of anthocyanins are cyanidin, pelargonidin, peonidin, del-phinidin, petunidin, and malvidin with various glycosylation andsometimes acylation patterns. Anthocyanins have been shown toexhibit many biological and pharmacological functions, includingantioxidant activities (Rahman, Ichiyanagi, Komiyama, Hatano, &Konishi, 2006) and can protect cells from DNA damage (Esselen,Fritz, Hutter, & Marko, 2009; Fritz, Roth, Holbach, Esselen, &Marko, 2008). Therefore, anthocyanins may play an important rolein the prevention of cancer development and other diseases (Buneaet al., 2013; Lim et al., 2013).

Previous studies have shown that the type of glycosylation of theanthocyanidins may affect the total antioxidant capacity (Wang,Cao, & Prior, 1997; Yoshiki, Okubo, & Igarashi, 1995). Dependingon the anthocyanidin, different glycosylation patterns eitherenhanced or reduced the antioxidant power (Kähkönen &Heinonen, 2003; Wang et al., 1997). For example, glycosylation atC3 of anthocyanin skeleton significantly enhanced the ORAC valueof cyanidin that contains two hydroxyl groups at B ring, but notfor pelargonidin that has one hydroxyl group. Wang andco-workers (1997) also found that the glycosylation at C3 and C5 ofthe anthocyanin skeleton alleviated the antioxidant activity of bothcyanidin and pelargonidin. However, another study showed anopposite effect, namely that the intensity of the chemiluminescenceof the anthocyanins was in the order of delphinidin-3-p-cou-maroylrutinoside-5-glucoside (or nasunin) > cyanidin-3-digluco-side-5-glucoside > delphinidin > cyanidin > malvidin, suggestingthat glycosylation at C-3 and C-5 of the anthocyanin skeletonenhanced the chemiluminescence of both delphinidin and cyanidin(Yoshiki et al., 1995).

Eggplants (Solanum melongena L.) are rich in anthocyanins, withconcentrations as great as 85.7 mg/100 g (Wu et al., 2006). Egg-plant anthocyanins have been identified as delphinidin derivatives(Kuroda & Wada, 1935; Sakamura, Watanabe, & Obata, 1963; Wu &Prior, 2005). There are many varieties of anthocyanin-rich egg-plants worldwide. The source and variety of eggplants have a largeimpact on the glycosylation and acylation of delphinidin deriva-tives and thus could change their biological functions. The egg-plants, which were sampled from retail stores in United Stateswere identified as having delphinidin 3-rutinose-5-galactoside,delphinidin 3-rutinose-5-glucoside, delphinidin 3-glucoside, anddelphinidin 3-rutinoside as the major components (Wu & Prior,2005). However, the major anthocyanin in eggplants sampled fromJapan was identified as delphinidin-3-(p-coumaroylrutinoside)-5-glucoside, or nasunin (Kuroda & Wada, 1935; Sakamura et al.,1963), and was later found to consist of trans and cis isomers(Ichiyanagi et al., 2005).

Chinese varieties, also called Japanese eggplants, commonly areshaped like a narrow cucumber and are grown widely in China. Themajor anthocyanin in eggplants sampled from Shanghai localmarkets has been identified as delphinidin-3-[(400-p-coumaroyl)-rhamnosyl (1 ? 6)glucoside]-5-glucoside, namely nasunin, belong-ing to delphinidin-type anthocyanins (Jing et al., 2014). However,the antioxidant properties of nasunin as well as other delphinidinderivatives with various structures have not yet been studied com-paratively. Therefore, it is unclear if the glycosylation patterns ofdelphinidin derivatives have a key impact on their antioxidantactivity. In this study we studied anthocyanin structures in a hybridcultivar of Chinese eggplants, Niu Jiao Qie, and investigated theeffects of delphinidin derivatives with various glycosylation on

ROS levels and DNA oxidation damage in human colon cancer celllines HT-29 and HCT116. The mechanism involving the modulationof cellular antioxidant defense was explored.

2. Materials and methods

2.1. Chemicals, cells and media

Delphinidin, cyanidin, and cyanidin-3-glucoside werepurchased from Polyphenols (Sandnes, Norway). Delphinidin-3-glucoside, dephinidin-3-sambubioside, delphinidin-3-rutinoside,and delphinidin-3,5-diglucoside were purchased from Phytolab(Vestenbergsgreuth, Germany). Pelargonidin was purchased fromChromadex (Santa Ana, CA, USA). Monoclonal antibody quinoneoxidoreductase 1 (NQO1) and a-tubulin were purchased from CellSignaling (Beverly, MA). Monoclonal antibody c-glutamatecysteineligase catalytic subunit (c-GCLC) and glutathione reductase (GSR)were purchased from Abcam (Cambridge, MA). McCoy’s 5A med-ium (modified: without serum, with L-glutamine) and fetal bovineserum were purchased from HyClone (Logan, Utah). The bicinchon-inic acid (BCA) assay kit was purchased from Pierce (Rockford, IL).The Western Lightning TM Plus-ECL Enhanced ChemiluminescenceSubstrate assay kit was purchased from PerkinElmer (Waltham,MA). All other chemicals were purchased from Sigma–Aldrich(Shanghai, China).

2.2. Preparation and analysis of nasunins from eggplants

Eggplants (S. melongena) var. Niu Jia Qie (No. V06B0003) wereprovided by Dr. Qi from the Horticultural Engineering Institute,Tianjin Academy of Agricultural Sciences, Tianjin, China. Theextraction and purification of eggplant anthocyanins were per-formed as described by Ichiyanagi et al. (2005). The peel of the egg-plant (5 kg) was immersed in 10 L of methanol containing 0.01%HCl and extracted for 2 h. Extracts were applied to a 600 � 50 cmAmberlite XAD-7HP column and washed with 0.01%-HCl water toremove water-soluble compounds. The anthocyanin fraction waseluted with 0.01%-HCl methanol. The anthocyanin fraction wasapplied onto a 100 � 2.5 cm open column packed with SephadexLH-20 and separated by 50% aqueous methanol containing 0.01%HCl. Anthocyanin fractions were further purified in an Agilent pre-parative HPLC system equipped with a semi-preparative ZORBAXEclipse XDB-C18 column. Anthocyanin peak fractions were col-lected and evaporated to dryness in a nitrogen-blow, and the puri-fied acylated delphinidin derivative (compound 1) from eggplantpeel was obtained.

The compound 2 was obtained by alkaline hydrolysis of egg-plant anthocyanins according to a previous method (Durst &Wrolstad, 2001). Anthocyanin-rich extracts from eggplant peelswere saponified in a screw-up test tube with 10 mL of 10% aqueousKOH for 8 min at room temperature in the dark. The solution wasthen neutralised and acidified by HCl (2 mol/L). The hydrolysatewas purified according to the same procedure as described above.

2.3. Identification of eggplant anthocyanins

The HPLC analysis was performed using a Finnigan SurveyorPlus system (Thermo Scientific, Waltham, MA, USA). Separationwas achieved by reverse phase elution on a 5 lm Shim-pack VP-ODS column (4.6 � 250 mm, Shimadzu, Kyoto, Japan) fitted witha 4.6 � 10 mm Shim-pack GVP-ODS guard column (Shimadzu,Kyoto, Japan). Solvents and sample were filtered through0.45 lm hydrophobic/hydrophilic membranes (Shanghai YaxingCorp., China) and 0.45 lm nylon membrane filters (Shanghai Mosu

P. Jing et al. / Food Chemistry 172 (2015) 183–189 185

Scientific Instruments and Materials, China), respectively. Thechromatographic conditions were: flow rate 1 mL/min, sampleinjection volume of 10 lL and mobile phase A (0.5 mol/L HCOOHin water) and mobile phase B (0.5 mol/L HCOOH in methanol). Agradient program was used as follows: 0–3 min, 40% B;3–10 min, 40–50% B; 10–12 min, 50–70% B; 12–15 min, 70% B.Spectral information over the wavelength range of 200–600 nmwas collected. The effluent from the column after the PDA detectorwas split into a 10:1 ratio and diverted to the Thermo LXQ LinearIon Trap system (Thermo Scientific, Waltham, MA, USA). Theapplied electrospray/ ion optics parameters were as follows: sprayvoltage, 4.5 kV; sheath gas (nitrogen), 50 arb; capillary tempera-ture, 300 �C; capillary voltage, 20 V; tube lens offset voltage,100 V. Spectra were collected using the full ion scan mode overthe mass-to-charge (m/z) range 200–2000. Collision-induced frag-mentation experiments were performed in the ion-trap usinghelium as collision gas.

1H/13C NMR spectra of anthocyanins were collected on a BrukerAVANCE 400 NMR spectrometer (Bruker Instruments, Billerica,MA) in TFA-d1/CD3OD (1:9) using tetramethylsilane as an internalreference.

2.4. Cell culture

Human colon cancer cell lines HT29 and HCT116 were pur-chased from the Chinese cell bank (Shanghai, China). Both of thecell lines were maintained in a humidified atmosphere with 5%CO2 at 37 �C in McCoy’s 5A medium supplemented with 10% fetalbovine serum (FBS) All results were based upon experiments withviabilities >85% and cell numbers not significantly decreased,compared to the solvent controls.

2.5. Cellular ROS level (DCF assay)

Oxidative stress in cells was quantified using the dichlorofluo-rescin (DCF) assay (Wang & Joseph, 1999). Cells were seeded into

0 10 20

(A) Compound 1 before saponification

Time (min)

mAU

0

200

400

600

Inten

sity (

mAu

)

Fig. 1. HPLC profiles of compound 1 (A) isolated from eggplant pe

black, clear-bottom 96-well plates at 6 � 104 cells/well with100 lL culture medium and allowed to grow for 24 h. After wash-ing with phosphate-buffered saline (PBS), six wells were incubatedat 37 �C for 1 h with 100 lL of 100 lmol/L compounds plus25 lmol/L 20,70-dichlorofluorescin diacetate (DCFH-DA, finalconcentration) dissolved in a treatment medium. The treatmentmedium was removed and 100 lL of PBS was then used to washeach well once. For the control wells, 100 lL culture medium con-taining no 2, 20-azobis (2-amidinopropane) dihydrochloride(600 lmol/L, AAPH) was added to the blank wells. Next, 100 lLof culture medium containing AAPH was added to other wells.The increase of fluorescence (FI), was measured in a microplatereader (Infinite F200, Tecan) every 5 min for 1 h (ex/em: 485/538 nm) at 37 �C. FI was calculated using the following equation:

CAA unit ¼ 100�R

SARCA

� �� 100 ð1Þ

SA: sample area; CA, control area.Quercetin, pelargonidin, and cyanidin were used as the positive

controls. Each plate included control and blank wells: control wellscontain cells were treated with DCFH-DA and AAPH; blank wellscontaining cells were treated with DCFH-DA and no AAPH. Thisexperiment was repeated three times.

2.6. Western blot analysis

Cells (1.5 � 106 HT-29; 1.5 � 106 HCT-116) were allowed togrow for 48 h and then incubated 1 h with the 100 lg/mL testedcompounds in McCoy’s 5A medium and supplemented with100 U/mL catalase to remove the hydrogen peroxide (H2O2) formedwithin cell culture medium (Bellion et al., 2009; Fritz et al., 2008).Pelargonidin and cyanidin were used as the positive controls. Afterappropriate treatment, cell lysate preparation and Western blotwere performed as previously described (Wang, Kingsley,Marnett, & Eling, 2011). All of the proteins were collected from cellsafter treatment. A total of 30 lg protein was electrophoresed on a

mAU

0

500

1000

1500

2000

2500

Time (min)

Inten

sity

(mAu

)

(B) Compound 2 after saponification

0 5 10

els and compound 2 (B) after saponification of compound 1.

186 P. Jing et al. / Food Chemistry 172 (2015) 183–189

10% SDS–polyacrylamide Tris–Hcl gel (Bio-Rad) and electrophore-sed at 170 V for 1 h. Separated proteins were transferred onto aPVDF membrane at 100 V for 30 min on ice. After the transfer,membranes were blocked with 5% non-fat dry milk in 1� TBST(50 mM Tris pH7.5, 150 mM NaCl, 0.1% Tween-20) at room temper-ature for 1 h. The membrane was probed with primary antibodies(NQO1, c-GCLC, GSR and a-tubulin) according to the manufac-turer’s instruction (Cell Signaling, MA). The signals were detectedusing the Western Lightning Plus-ECL enhanced chemilumines-cence substrate according to the manufacturer’s instruction.

2.7. DNA damage

HT-29 and HCT-116 cells (3 � 105 in 5 mL medium containing10% fetal calf serum v/v) were spread into petri dishes (5 cm)and allowed to grow for 48 h. In the experiments with single com-pounds, HT-29 cells were treated for an additional 1 h with solvent

Fig. 2. MSn spectra of eggplant anthocyanins: (A) delphinidin-3-[(400-trans-p-coumglucoside.

control (1% v/v DMSO), 20 lmol/L menadione, and tested com-pounds in a serum-free medium in the presence of 100 U/mL cata-lase (Bellion et al., 2009; Fritz et al., 2008). About 1� 106 cells/mlwas prepared for analysis with the comet assay kit according tothe manufacturer’s protocol (Research Bio-lab, Beijing, China).Briefly, 10 lL of cell suspension and 90 lL of low melting agarosewere mixed and spread on a slide coated with normal meltingagrose. Cover slips were placed on the slides and stored at 4 �Cfor 4 min to allow the agarose to solidify. The slides with coverswere placed in lytic solution for 1.5 h. After cell lysis, slides werewashed with distilled water and then placed in electrophoresisbuffer (pH P 13) for 30 min to allow the DNA unwind completely,followed by a gel electrophoresis at 4 �C for 30 min (25 v, 300 mA).The slides were washed with distilled water and dipped in ethanolfor 10-min hydration. Finally the slides were stained with ethi-dium bromide for 4 min and examined with an LSM 510 Metamicroscope (Carl Zeiss, Jena, Germany).

aroyl)-rhamnosyl (1 ? 6)glucoside]-5-glucoside; (B) delphinidin-3-rutinoside-5-

0

5

10

15

20

25

30

35

40

45

Fluo

resc

ence

redu

ctio

n %

of c

ontro

l

Compounds (100 µmol/L)

HT-29 HCT-116

Fig. 3. Effects of delphinidin derivatives on AAPH-induced cellular ROS levels in HT-29 and HCT-116 cells after 1 h incubation. Results are presented as mean and standarddeviation. Cy-3-glu, cyanidin-3-glucoside; Dp-3-glu, delphinidin-3-glucoside; Dp-3-rut, delphinidin-3-rutinoside; Dp-3-samb, delphinidin-3-sambubioside; Dp-3,5-diglu,delphinidin-3,5-diglucoside; Dp-3-rut-5-glu, delphinidin-3-rutinoside-5-glucoside; nasunin, delphinidin-3-[(400-trans-p-coumaroyl)-rhamnosyl (1 ? 6)glucoside]-5-glucoside.

29KDa

73KDa

56KDa

52KDa

29KDa

73KDa

56KDa

52KDa

NQO1

-Tubulin

-GCLC

GSR

NQO1

-Tubulin

-GCLC

GSR

Fig. 4. Effects of delphinidin derivatives on NAD(P)H: quinone oxioreductase 1 (NQO1), c-glutamate cysteine ligase catalytic subunit (c-GCLC) and glutathione reductase(GSR) expression in HT-29 and HCT-116 cells. Dp-3-glu, delphinidin-3-glucoside; Dp-3-rut, delphinidin-3-rutinoside; Dp-3-samb, delphinidin-3-sambubioside; Dp-3,5-diglu,delphinidin-3,5-diglucoside; Dp-3-rut-5-glu, delphinidin-3-rutinoside-5-glucoside; nasunin, delphinidin-3-[(400-trans-p-coumaroyl)-rhamnosyl (1 ? 6)glucoside]-5-glucoside.

P. Jing et al. / Food Chemistry 172 (2015) 183–189 187

2.8. Statistics

Statistical analysis of data was performed by one-way analysisof variance (ANOVA) using SPSS (version14.0, SPSS Inc., Chicago,IL, USA). Differences among means were compared using leastsignificant difference (LSD) at p = 0.05.

3. Results and discussion

3.1. Structure of anthocyanins in Chinese eggplant hybrid

The HPLC chromatograms of eggplant peel extracts before andafter saponification are shown in Fig. 1. Two peaks have been

detected around at 8 and 4.8 min in Fig. 1A and B respectively, witha maximum visible wavelength of absorbance at 520 nm. The com-pound 1 in Fig. 2A gave a m/z value of 919 [M]+ as well as fragmentsat values of 757 [M�hexoside]+, 465 [delphinidin+ hexoside]+ and303 [delphinidin]+, and a week signal of fragment at m/z 611[delphinidin+ rutinoside]+. Compound 2 was obtained after alkalinehydrolysis of compound 1 gave a molecular ion of m/z 773 in Fig. 2B,where a mass deduction of m/z 146 from compound 1 molecular ion(m/z, 919) is responsible for an acyl group of m/z value [p-coumaricacid-H2O]. Compounds 1 and 2 had the same fragments of m/z equalto 611, 465 and 303, indicating that both compounds containedrutinose, hexose, and delphinidin as a partial structure whereascompound 1 had p-coumaric acid as an acyl group.

(A)

(B)

Fig. 5. Effects of anthocyanins from eggplant peels on Md-induced DNA damage in HT-29 cells. (A) delphinidin-3-rutinoside-5-glucoside; (B) delphinidin-3-[(400-trans-p-coumaroyl)-rhamnosyl (1 ? 6)glucoside]-5-glucoside.

188 P. Jing et al. / Food Chemistry 172 (2015) 183–189

Further structural information on the positions of glycosylationand acylation on the delphinidin was obtained with 1H, 13C NMRanalyses and it confirmed that compounds 1 and 2 were delphini-din-3-[(400-p-coumaroyl)-rhamnosyl (1 ? 6)glucoside]-5-glucosideand delphinidin-3-rutinoside-5-glucoside respectively, whichwere previously characterised in regular Chinese eggplant (Jinget al., 2014). The chemical shift (d 6.27 and 7.55) and largeJ-coupling constants (15.88 Hz) of H-7 and H-8 for p-coumaric acidindicated that p-coumaroyl group in compound 1 has a trans con-figuration (Giusti, Ghanadan, & Wrolstad, 1998). Taken together,we conclude that compound 1 isolated from the Chinese eggplantvar. Niu Jia Qie is delphinidin-3-[(400-trans-p-coumaroyl)-rhamno-syl (1 ? 6)glucoside]-5-glucoside, and that the compound 2 isdelphinidin-3-rutinoside-5-glucoside.

3.2. Modulation of cellular ROS level

The influence of delphinidin derivatives on ROS level is shownin Fig. 3. Generally, delphinidin derivatives more effectivelyreduced AAPH-induced ROS levels in HT-29 than in HCT-116 cellsafter a one-hour treatment. Cyanidin-3-glucoside and quercetin asreference compounds diminished fluorescence by 35–38% of thecontrol, exhibiting the highest ROS-reducing levels among testedcompounds in HT-29 and HCT-116 cells (p < 0.05), whereas thepelargonidin only reduced 2–10% ROS of the control (p < 0.05).The results were consistent with the previous reports (Wanget al., 1997) that cyanidin-3-glucoside had the highest ORACactivity, among anthocyanins including the delphinidin, cyanidin,pelargonidin, malvidin, peonidin, and their derivatives with differ-ent sugar linkages as determined by the chemical-based oxygenradical absorbance capacity (ORAC) method.

Delphinidin-3-glucoside, 3-sambubioside, or 3-rutinosideexhibited ROS reduction by 29.4% and 29.5%, 34.0% and 27.7%,and 32.3% and 25.9% in HT-29 and HCT-116 cells, respectively,almost as high as quercetin and cyanidin-3-glucoside. Other antho-cyanins showed lower effectiveness in the following order: del-phinidin-3,5-diglucoside > delphinidin-3-rutinoside-5-glucoside >delphinidin > nasunin in both cells. Delphinidin-derivatives glycos-ylated at position C3 exclusively, including glucose, sambubiose orrutinose, exhibited greater ROS-reducing effects than delphinidin-

3,5-diglucoside and delphinidin-3-rutinoside-5-glucoside in HT-29cells and HCT-116 cells. Those results suggest that the glycosyla-tion at C3 and C5 of the anthocyanin skeleton reduced the antiox-idant activity using ORAC assay in human colon cells, consistentwith Wang et al. (1997)’s study using the chemical ORAC assay,but inconsistent with Yoshiki et al. (1995) results using a chemilu-minescence assay. It has been shown that regardless of whetherthe glycosylation of delphinidin contributed to an enhanced anti-oxidant activity was sometime dependent on the evaluation assay,such as the ORAC assay (Wang et al., 1997) and the chemilumines-cence assay (Yoshiki et al., 1995). Additionally, delphinidin-3-[(400-p-coumaroyl)-rhamnosyl (1 ? 6)glucoside]-5-glucoside or nas-unin, showed �9% ROS reduction in both cell lines, whereas the dea-cyl nasunin (delphinidin-3-rutinoside-5-glucoside), increased theROS reduction to �17% and �12% in HT-29 (p = 0.012) andHCT-116 cells, respectively, indicating that the acylation to delphin-idin glycosylation appeared to reduce their antioxidant activity.

3.3. Expression levels of antioxidant proteins and DNA-damage

Regulation of c-GCLC, GSR and NQO1 protein expression in HT-29 and HCT-116 cells by delphinidin derivatives at 100 lmol/L wasstudied to clarify the potential influence on the observed variety ofROS levels in Fig. 4. For c-GCLC, there were no changes in proteinexpression that were observed in both cell lines. A distinct increaseof GSR protein expression was observed in HT-29 cell, not in HCT-116 (Fig. 4). For NQO1, delphinidin-3-rutioside 5-glucoside as thedeacyl nasunin and delphinidin-3-sambubioside up-regulated theprotein expression in HCT-116 cells, whereas other delphinidinderivatives either had no effect or even down-regulated theexpression level compared with the control in HT-29 and HCT-116 cells. The pelargonidin and cyanidin as positive controls didnot exhibit induction of these enzyme proteins in both cell lines.

Cyanidin did not show any induction of the enzyme proteins inspite of its reduction effect on AAPH-induced cellular ROS, suggest-ing that cyanidin might exhibit the antioxidant activity via differ-ent mechanism (s). One of possible mechanism(s) is thatanthocyanins including cyanidin could be bound to the HT-29and HCT-116 cell membrane and/or enter the cells, and then couldreact directly with peroxyl radicals produced by AAPH according to

P. Jing et al. / Food Chemistry 172 (2015) 183–189 189

the principle of cellular antioxidant activity proposed by Wolfe andLiu (2007), not via inducing expression of cellular antioxidantenzymes. The reduction of the cellular ROS of delphinidin deriva-tives appeared to be consistent with the expression levels of anti-oxidant enzymes in HT-29 cells, suggesting that the simulation ofantioxidant enzyme expression might be the main mechanismfor the cellular ROS reduction of delphinidin derivatives. Delphin-idin-3-glycosides including delphinidin-3-glucoside, delphinidin-3-rutinoside and delphinidin-3-sambubioside showed a betterROS-reduction via a greater GSR protein expression than dodelphinidin-3, 5-glycoside.

Additionally, it showed that delphinidin acted as an effectiveantioxidant within intact cells and maintain DNA integrity inhuman colon carcinoma cells if the formation of hydrogen peroxidewas prevented (Esselen et al., 2009). The nasunin and deacyl nas-unin in this study were found to reduce menadione-induced DNAdamage in HT-29 cells (Fig. 5). Deacyl nasunin seemed to protectslightly the DNA from damage at 50 lmol/L, whereas both com-pounds showed a distinct reduction of DNA-damage at 100 and200 lmol/L. However, for unknown reasons, nasunin and deacylnasunin were ineffective in HCT-116 at all concentrations tested(data not shown). Our data suggest that both nasunin and deacylnasunin may exhibit anti-oxidant activity and anti-DNA damageactivity in a cell type dependent manner.

4. Conclusions

Delphinidin derivatives with either monosaccharide or disac-charide at the C3 position, may contribute to improved antioxidantactivity via promoting GSR expression in HT-29. Additional sugarmoieties at the C5 position caused a decrease in the ROS-reducingeffectiveness. The acylation to delphinidin-derivatives appeared toreduce their antioxidant activity. The evaluation assays of antioxi-dant activities do have an impact on antioxidant evaluation com-pared to other previous results. Although not potent in ROSreduction in human colon cells (HT-29 and HCT-116), nasuninand deacyl nasunin reduced menadione-induced DNA damage inboth cell lines at 50–200 lmol/L in a dose dependent manner.

Acknowledgement

This study was founded by the National Nature ScienceFoundation of China (Grant No. 31371756).

References

Bellion, P., Olk, M., Will, F., Dietrich, H., Baum, M., Eisenbrand, G., et al. (2009).Formation of hydrogen peroxide in cell culture media by apple polyphenols andits effect on antioxidant biomarkers in the colon cell line HT-29. MolecularNutrition & Food Research, 53(10), 1226–1236.

Bunea, A., Rugina, D., Scont�a, Z., Pop, R. M., Pintea, A., Socaciu, C., et al. (2013).Anthocyanin determination in blueberry extracts from various cultivars andtheir antiproliferative and apoptotic properties in B16-F10 metastatic murinemelanoma cells. Phytochemistry, 95, 436–444.

Durst, R. W., & Wrolstad, R. E. (2001). Separation and characterization ofanthocyanins by HPLC. In R. E. Wrolstad, et al. (Eds.), Current Protocols in FoodAnalytical Chemistry (1st ed.) (Vol. 1, pp. F1.3.1–F1.3.13). NY: John Wiley andSons Inc.

Esselen, M., Fritz, J., Hutter, M., & Marko, D. (2009). Delphinidin modulates the DNA-damaging properties of topoisomerase II poisons. Chemical Research inToxicology, 22(3), 554–564.

Fritz, J., Roth, M., Holbach, P., Esselen, M., & Marko, D. (2008). Impact of delphinidinon the maintenance of DNA integrity in human colon carcinoma cells. Journal ofAgriculture and Food Chemistry, 56(19), 8891–8896.

Giusti, M. M., Ghanadan, H., & Wrolstad, R. E. (1998). Elucidation of the structureand conformation of red radish (Raphanus sativus) anthocyanins using one- andtwo-dimensional nuclear magnetic resonance techniques. Journal of Agricultureand Food Chemistry, 46(12), 4858–4863.

Giusti, M. M., & Wrolstad, R. E. (2003). Acylated anthocyanins from edible sourcesand their applications in food systems. Biochemical Engineering Journal, 14(3),217–225.

Ichiyanagi, T., Kashiwada, Y., Shida, Y., Ikeshiro, Y., Kaneyuki, T., & Konishi, T. (2005).Nasunin from eggplant consists of cis–trans isomers of delphinidin 3-[4-(p-coumaroyl)-L-rhamnosyl (1 ? 6)glucopyranoside]-5-glucopyranoside. Journalof Agriculture and Food Chemistry, 53(24), 9472–9477.

Jing, P., Zhao, S. J., Ruan, S. Y., Sui, Z. Q., Chen, L. H., Jiang, L. L., et al. (2014).Quantitative studies on structure-ORAC relationships of anthocyanins fromeggplant and radish using 3D-QSAR. Food Chemistry, 145, 365–371.

Kähkönen, M. P., & Heinonen, M. (2003). Antioxidant activity of anthocyanins andtheir aglycons. Journal of Agriculture and Food Chemistry, 51(3), 628–633.

Kuroda, C., & Wada, M. (1935). The coloring matter of eggplant (Nasu). Part II.Proceedings of the Imperial Academy (Tokyo), 11, 235–237.

Lim, S., Xu, J., Kim, J., Chen, T.-Y., Su, X., Standard, J., et al. (2013). Role ofanthocyanin-enriched purple-fleshed sweet potato p40 in colorectal cancerprevention. Molecular Nutrition & Food Research, 57(11), 1908–1917.

Rahman, M. M., Ichiyanagi, T., Komiyama, T., Hatano, Y., & Konishi, T. (2006).Superoxide radical- and peroxynitrite-scavenging activity of anthocyanins;structure-activity relationship and their synergism. Free Radical Research, 40(9),993–1002.

Sakamura, S., Watanabe, S., & Obata, Y. (1963). The structure of the majoranthocyanin in eggplant. Agricultural and Biological Chemistry, 27, 663–665.

Wang, H., Cao, G., & Prior, R. L. (1997). Oxygen radical absorbing capacity ofanthocyanins. Journal of Agriculture and Food Chemistry, 45(2), 304–309.

Wang, H., & Joseph, J. A. (1999). Quantifying cellular oxidative stress bydichlorofluorescein assay using microplate reader. Free Radical Biology andMedicine, 27(5–6), 612–616.

Wang, X., Kingsley, P. J., Marnett, L. J., & Eling, T. E. (2011). The role of NAG-1/GDF15in the inhibition of intestinal polyps in APC/Min mice by sulindac. CancerPrevention Research (Philadelphia), 4(1), 150–160.

Wolfe, K. L., & Liu, R. H. (2007). Cellular antioxidant activity (CAA) assay forassessing antioxidants, foods, and dietary supplements. Journal of Agricultureand Food Chemistry, 55(22), 8896–8907.

Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L.(2006). Concentrations of anthocyanins in common foods in the United Statesand estimation of normal consumption. Journal of Agriculture and FoodChemistry, 54(11), 4069–4075.

Wu, X., & Prior, R. L. (2005). Identification and characterization of anthocyanins byhigh-performance liquid chromatography–electrospray ionization–tandemmass spectrometry in common foods in the united states: Vegetables, nuts,and grains. Journal of Agriculture and Food Chemistry, 53(8), 3101–3113.

Yoshiki, Y., Okubo, K., & Igarashi, K. (1995). Chemiluminescence of anthocyanins inthe presence of acetaldehyde and tert-butyl hydroperoxide. Journal ofBioluminescence and Chemiluminescence, 10(6), 335–338.