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Production of nigragillin and dihydrophaseic acidby biotransformation of litchi pericarp withAspergillus awamori and their antioxidant activities

http://dx.doi.org/10.1016/j.jff.2014.02.0011756-4646/� 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +86 20 37083042.E-mail address: yangbao@scbg.ac.cn (B. Yang).

Sen Lina,b, Jirui Heb, Yueming Jiangb, Fuwang Wub, Hui Wangb, Dan Wub, Jian Sunc,Dandan Zhangb, Hongxia Qub, Bao Yangb,*

aInstitute of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou

325027, ChinabKey Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences,

Guangzhou 510650, ChinacInstitute of Agro-Food Science & Technology, Guangxi Academy of Agricultural Sciences, Nanning 530007, China

A R T I C L E I N F O A B S T R A C T

Article history:

Received 18 November 2013

Received in revised form

2 February 2014

Accepted 4 February 2014

Available online 1 March 2014

Keywords:

Nigragillin

Dihydrophaseic acid

Aspergillus awamori

Litchi pericarp

Biotransformation

Biotransformation with Aspergillus awamori could degrade the bound bioactive compounds

of plant tissues into free form and induce the biosynthesis of novel chemicals. In this work,

two bioactive compounds with high levels were produced from litchi pericarp via

A. awamori fermentation. They were purified and identified to be nigragillin and dihydro-

phaseic acid by nuclear magnetic resonance spectroscopy and mass spectrometry. The

anticancer activity, DNA protection effect and antioxidant activity of these two compounds

were evaluated. Dihydrophaseic acid had significantly inhibitory effect against HepG2 and

HeLa cells with IC50 values of 41.92 ± 6.15 and 79.37 ± 9.78 lg/mL, respectively. Nigragillin

showed a better DPPH radical scavenging activity than dihydrophaseic acid. Dihydropha-

seic acid possessed good lipid peroxidation inhibition effect, hydroxyl radical scavenging

activity, and DNA protection effect even at a low dosage. The results indicated that

biotransformation with A. awamori was a good way to produce bioactive compounds.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Overproduction of reactive oxygen species (ROS) leads to

oxidative damage of biomacromolecules, such as DNA and

proteins, which increase the risk of some degenerative dis-

eases, like cancer and cardiovascular diseases (Adom & Liu,

2002). Dietary antioxidants from plants are supposed to assist

maintaining the balance of antioxidation and oxidation in vivo,

which is beneficial to human health. Thus, it is important to

obtain antioxidants from natural sources. Recently, increasing

attention has been paid to the production of bioactive com-

pounds from plant byproducts by microorganism conversion.

Grape pomace, pineapple waste, citrus peel, and banana peel

have been used to produce carotenoids and citric acid by

microorganism biotransformation (Buzzini & Martini, 2000;

Karthikeyan & Sivakumar, 2010). Aspergillus awamori, a black

filamentous fungi belong to Aspergillus genus, can produce di-

verse catalytic enzymes (Gottschalk, Oliveira, & Bon, 2010). It

has been used in food preparation since ancient time and

is generally recognised as safe (Schuster, Dunn-Coleman,

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 2 7 8 – 2 8 6 279

Frisvad, & van Dijck, 2002). Enhancement in antioxidant (Chen,

Huang, Lin, Hsu, & Chung, 2013) and antimutagenicity (Hung,

Wang, & Chou, 2009) were observed after fermentation with

A. awamori on several phenolics-enriched plant materials,

and increased total phenolics content is generally considered

as a key factor for bioactivity improvement. This viewpoint is

supported by our previous study when litchi pericarp was em-

ployed as the substrate (Lin et al., 2012). However, the produc-

tion of non-polyphenolic antioxidants from plant source with

microbial fermentation, which may also contribute to the in-

creased biological activity, is rarely studied.

Litchi (Litchi chinensis Sonn.) is an important tropical/sub-

tropical fruit with bright red pericarp tissue, which accounts

for approximate 20% by weight of the whole fresh fruit. Phen-

olics in litchi tissues were finely identified (Sarni-Manchado,

Roux, Guerneve, Lozano, & Cheynier, 2000; Wen et al., 2014).

However, little information regarding non-phenolics bioactive

components present in litchi pericarp is available, though

several non-phenolics antioxidants were reported in litchi

(Wang, Lou, Ma, & Liu, 2011), like stigmasterol (Jiang et al.,

2013). Compounds from litchi pericarp exhibit antioxidant,

anticancer and immunomodulatory activities (Zhao, Yang,

Wang, Li, & Jiang, 2006).

In this study, litchi pericarp was fermented by A. awamori

and the production of two non-phenolics was observed. The

fermentation products were purified by column chromatogra-

phy and identified by electronic spray ionization-mass spec-

trometry (ESI-MS), as well as nuclear magnetic resonance

spectroscopy (NMR). The 2,2-diphenyl-1-picrylhydrazyl

(DPPH) radial scavenging activity, hydroxyl radical scavenging

activity, lipid peroxidation inhibition effect, DNA protection

activity, as well as anticancer activity of the purified com-

pounds were evaluated.

2. Materials and methods

2.1. Organism and plant material

Filamentous fungi A. awamori GIM 3.4 was obtained from

Guangdong Culture Collection Center (Guangzhou, China).

The strain was maintained on potato dextrose agar (PDA)

plate at 4 �C. For inoculum preparation, a loopful of spores

were transferred to a PDA plate and cultured at 30 �C for

3 days. The spore suspension was acquired by washing the

agar surface with sterile distilled water containing 0.1%

Tween 80, and successively adjusted to a concentration of

ca. 106 cfu/mL with sterile distilled water. The obtained spore

suspension was served as inoculums for further application.

Fresh fruit of litchi (L. chinensis Sonn.) cv. Huaizhi were col-

lected from a commercial orchard in Guangzhou (Guangdong,

China). The fruits were cleaned and peeled manually. The

pericarp tissues were collected, and then dried. After pulver-

ization, the pericarp was screened through a 40-mesh sieve.

2.2. A. awamori fermentation

Litchi pericarp powder (LPP) was weighted and then mixed

thoroughly with distilled water as the fermenting medium.

After sterilization (121 �C, 20 min), the medium was inoculated

with 1 mL of fresh or sterilized spore suspension. After being

thoroughly mixed, the inoculated medium was cultured for

5 days at 30 �C, and was stirred and mixed for every 24 h to

accelerate the release of fermentation heat.

2.3. Products preparation

The A. awamori-fermented LPP was extracted twice with 60%

ethanol. After filtrated through Whatman No. 1 filter paper,

the extract was concentrated using a rotary evaporator at

50 �C under vacuum to remove ethanol. The residual liquid

was further extracted twice with ethyl acetate. The water

fraction was applied onto a macroporous resin D-101 (Tianjin

Haiguang Chemical Co., Ltd., Tianjin, China) column and

eluted with ethanol/water in a stepwise manner. The column

was initially eluted using 10% ethanol–water solution and

then was successively eluted with 30%, 50%, 70%, and 90%

ethanol in a total volume of 1.5 L for each gradient. Fractions

1 (F1), firstly collected from the macroporous resin D-101 col-

umn eluted using 50% ethanol, was obtained by further puri-

fication on a Sephadex LH-20 column and a semi-preparative

HPLC (Shimadzu Corporation, Kyoto, Japan) equipped with a

PRC-ODS column (25 · 460 mm). The ethyl acetate extract

was dried and then was loaded onto a silica gel (200 mesh,

Qingdao Marine Chemical Co., Ltd., Qingdao, China) column

(50 · 1300 mm). The column was initially eluted with CHCl3,

and was sequentially eluted with a serial proportion of CHCl3/

CH3OH (9.8:0.2, 9.6:0.4, 9.2:0.8, and 9:1). Fraction 2 (F2), previ-

ously collected from the silica gel column eluted with CHCl3/

CH3OH (96:4), was obtained by further purification on a

Sephadex LH-20 (GE Healthcare, Buckinghamshire, United

Kingdom) column (10 · 1700 mm) eluted by CH3OH, followed

by a semi-preparative HPLC.

2.4. HPLC analysis

The chromatographic separation was performed on a Shima-

dzu LC-20 AT HPLC system (Shimadzu Corporation, Kyoto,

Japan). After filtration, 20 lL of samples were loaded on the

HPLC and separated by a Vydac C18 column (218 TP,

250 · 4.6 mm, Sigma–Aldrich, St. Louis, MO, USA) using 0.1%

trifluoroacetic acid in water (solvent A) and methanol (solvent

B) as the mobile phase, following the elution program (Yang &

Zhai, 2010): 0–5 min, 10% B; 5–35 min, 10–100% B; 35–40 min,

100% B; and 40–45 min, 10% B, with a flow rate of 1 mL/min.

The elution was carried out at room temperature and re-

corded at 280 nm.

2.5. ESI-MS and NMR analysis

ESI-MS was run on a MDS SCIEX API 2000 MS/MS system in

both positive and negative ion modes in the range of m/z

50–800. The structural analyses of compounds F1 and F2 were

performed using 1H- and 13C-NMR on a Bruker AC 400 instru-

ment (Bruker, Rheinstetten, Germany). The spectra were re-

corded at 400 and 100 MHz, respectively. Furthermore, the

two dimensional NMR spectra including 1H-1H correlated

spectroscopy (COSY), heteronuclear single quantum coher-

ence (HSQC), and heteronuclear multiple bond correlation

(HMBC) were also recorded (Zhu et al., 2013). Samples were

280 J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 2 7 8 – 2 8 6

dissolved in CD3OD. The chemical shifts were expressed in

parts per million (ppm) relative to tetramethyl silane as an

internal standard.

2.6. In vitro antioxidant assays

2.6.1. Assay of DPPH radical scavenging activityCompounds F1 and F2 were dissolved in methanol at different

concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5 mg/mL).

The DPPH radical scavenging activities of F1 and F2 were

determined according to the method of Yang et al. (2012).

The absorbance was recorded by a spectrophotometer (Uico

Shangshai Instrument Co. Ltd., Shanghai, China) at 517 nm

against methanol as a blank. Methanol instead of DPPH solu-

tion was used as the control. The DPPH radical scavenging

activity (%) of the tested sample was calculated as [1 � (absor-

bance of sample � absorbance of control)/absorbance of

blank)] · 100.

2.6.2. Assay of hydroxyl radical scavenging activityThe hydroxyl radical scavenging activity of compounds F1

and F2 were determined using the method described by Jiang,

Jiang, Wang, and Hu (2005). Briefly, the hydroxyl radical was

generated by mixing 25 mM FeSO4, 2 mM sodium salicylate,

6 mM H2O2 and the samples at the range of concentration

0.1–2.5 mg/mL. Methanol instead of the sample was used as

the control. After incubating for 1 h at 37 �C, the colour

change of the mixture was recorded by a spectrophotometer

(Uico Shangshai Instrument Co. Ltd., Shanghai, China) at

520 nm. The hydroxyl radical scavenging activity of the inves-

tigated samples were calculated as follow: the scavenging rate

=[(absorbance of control � absorbance of sample)/absorbance

of control)] · 100.

2.6.3. Assay of lipid peroxidation inhibition capabilityThe lipid peroxidation inhibition was determined by the

method of Memarpoor-Yazdi, Mahaki, and Zare-Zardini

(2013). One mL of F1 (1 mg/mL) or F2 (1 mg/mL) were added

into a solution containing 0.13 mL of linoleic acid and 10 mL

of ethanol, then incubated in dark at 40 �C. The volume of

reaction solution was adjusted to 25 mL with phosphate buffer

(50 mM, 7.0). Linoleic acid oxidation was measured every 24 h

according to follow process: the resultant mixture (100 lL)

were mixed with ethanol (4.7 mL, 75%), ammonium thiocya-

nate (0.1 mL, 30%), and ferrous chloride (0.1 mL, 20 mM) in

3.5% hydrochloric acid. After 3 min, the colour change of the

mixture was recorded by a spectrophotometer (Uico Shangs-

hai Instrument Co. Ltd., Shanghai, China) at 500 nm, which

corresponded to the oxidation degree of linoleic acid.

2.7. Assay of DNA protective effect

Fenton-reaction mediated oxidation assay was conducted to

investigate the protective power of compounds F1 and F2 on

supercoiled DNA. Compounds were dissolved in methanol

and the concentration was adjusted to 50, 100, 200, 300 lg/

mL. The supercolied plasmid DNA was isolated by the follow-

ing process: firstly, the plasmid pUC19 was transformed to

Escherichia coli DH5a cells; then the cell was cultured in the

LB medium for 12 h at 37 �C on an orbital shaker (240 rpm).

Finally, plasmid DNA was obtained using the UNIQ-10 Plasmid

kit (Wuhan Sikete Science & Technology Development Co.,

Ltd., Wuhan, China) according the instruction supplied by

the manufacturer. DNA protective effect against Fenton-reac-

tion mediated oxidation damage was evaluated as described

by Lin et al. (2013). The resultant solution was then loaded

onto 1% agarose gel. After electrophoresis for 40 min under

120 V, the agarose gel was stained with 0.05% (w/v) ethidium

bromide and then analyzed with an image analyzer (Image

station 2000R, Kodak, New York, USA).

2.8. Cytotoxicity assay

The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

bromide (MTT) method was employed for cytotoxicity assess-

ment using human lung cancer (A549), human cervical carci-

noma (HeLa), and human hepatoma (HepG2) cell lines. The

cells were grown in RPMI-1640 medium supplemented with

10% fetal bovine serum and cultured at 37 �C in a humidified

5% CO2 incubator. The cell viability assays was performed in

96-well microliter plates by the method described by Xu,

Xie, Hao, Jiang, and Wei (2010). Compounds F1 and F2 were

dissolved in dimethyl sulfoxide (DMSO) and diluted with

medium to a serial concentrations. The exponential growth

phase cells (10,000 cells/mL) were pre-cultured for 24 h. The

cells were then incubated with each concentration of test

compounds. The final concentrations of each compound in

wells were 100, 75, 50, 25, 12.5, and 6.25 lg/mL in tetraplicate.

The control was carried out with 200 lL of fresh medium con-

taining 0.5% DMSO instead of the test sample solution. After

incubation at 37 �C for 72 h, 20 lL of MTT (5 mg/mL) were

added and incubated for another 4 h. After removing the

supernatant, DMSO (150 lL) were added into each well and

the plate was vortexed for 15 min. The absorbance was re-

corded on a microplate reader (Bio-Rad model 550, Hercules,

California, US) at 570 nm. IC50 was defined as the concentra-

tion of tested compounds possessed 50% cell inhibitory. The

IC50 value was based on four individual experiments and ex-

pressed as means ± standard deviation.

3. Results

3.1. Production of F1 and F2

The HPLC chromatograms of A. awamori-fermented and non-

A. awamori-fermented LPP are present in Fig. 1(A). Many peaks

present in the non-fermented LPP were recognized as pheno-

lics, which were consistent with previous reports (Sarni-

Manchado et al., 2000). A large number of compounds were

eluted as a hump at the retention time of 15–35 min on the

chromatogram, which was further supported by the report

of Roux, Doco, Sarni-Manchado, Lozano, and Cheynier

(1998), who demonstrated the hump as procyanidins with dif-

ferent degrees of polymerization. After the fermentation with

A. awamori, the peak C1 disappeared and many new peaks (F1,

F2, and P1–P5) were emerged (Fig. 1). P1, P2, P3 and P5 have

been identified as isolariciresinol, quercetin 3-O-glucoside,

quercetin and kaempferol, respectively (Lin et al., 2014).

Interestingly, these new peaks also appeared when fermentation

15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0

uV(x100,000)

12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5uV(x100,000)

C1

F1

F2

P1:overlapped peak

P2

P3

P4

Phenolic products

P5

(A)

Non-fermented

Fermented

C1

F1

F2

P1P2

P3

P4

Phenolic products

P5

(B)

Non-fermented

Fermented

Fig. 1 – Chromatograms of A. awamori-fermented or non-A. awamori-fermented LPP. (A) LPP; (B), LPP residue.

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 2 7 8 – 2 8 6 281

on the residues of LPP was carried out, but were not observed

in the non-fermented residue Fig. 1(B). Furthermore, com-

pound F1 was also detected when fermented on Czapek agar

medium free of LPP (data not shown). Thus, it was hypothe-

sized that these compounds produced by A. awamori (Fig. 1)

were not originated from the extractable compounds, and

the bound compounds present in the extracted residues

might act as substrates for production of these new com-

pounds (except F1). While compound F1 whose production

was not affected by the absence of LPP was recognized as a

metabolite of A. awamori.

3.2. Identification of F1 and F2

After purification, the products were identified by ESI-MS, 1D-

and 2D-NMR. The chemical structures of F1 and F2 are shown

in Fig. 2A, and the identification was described as follows.

F1 was a yellowish powder, and its ESI-MS showed a quasi-

molecular ion at m/z 223.2 [M + H]+, m/z 245.2 [M + Na]+, m/z

261.2 [M + K]+, m/z 445.3 [2 M + H]+, m/z 467.4 [2 M + Na]+,

and m/z 255.2 [M + Cl]�, corresponding to the molecular

weight 222. The 1D- and 2D-NMR data of compound F1 in

CD3OD was present in Table 1. The hydrogen atom which

was linked to C-2 (H-2, d H = 6.39 ppm, d C-2 = 119.2 ppm)

showed H-H correlation with H-3 (d H = 7.19 ppm, d C-3 =

145.8 ppm) and had a coupling constant (J) of 14.8 Hz, which

indicated a trans-double bond. The H-H correlationship of

H-2/H-3, H-4/H-5 gave a conjugated double bond moiety in

F1. Furthermore, H-5 exhibited a double quadruple peak and

showed H-H correlation with H-6 means C-5 were directly

linked to C-6 (a methyl). This judgment was further supported

by the HMBC data. Totally, 13 carbon atoms were observed in13C NMR spectra. The distortion enhancement by polarization

transfer (DEPT) experiment revealed that there were four CH3

groups, two CH2 groups, and six CH groups present in molec-

ular structure of F1. A carbonyl group (d 169.1 ppm) directly

linked to C-2 (HMBC data) was found. The moieties C-2 0, C-

3 0, C-7 0, and C-5 0, C-6 0, C-9 0 (Fig. 2A) can be assembled in the

same way. The molecular weight of those above mentioned

moieties was 194 in total. Take the fact that molecular weight

of F1 was 222 into consideration, additional 2 nitrogen atoms

(molecular weight = 28) were included in F1. Moreover, the

downfield chemical shift of H-2 0, H-6 0, and H-8 0 indicated that

C-2 0, C-6 0, and C-8 0 were directly connected to nitrogen atoms.

Concentration (mg/mL)0.0 .2 .4 .6 .8 1.0 1.5 2.0 2.5H

ydro

xyl r

adic

al s

cave

ngin

g ac

tivity

(%)

30

40

50

60

70

80

90

100F1 (Nigragillin)F2 (Dihydrophaseic acid)

(C)

Time (d)

Abso

rban

ce a

t 500

nm

(Lip

id p

erox

idat

ion

degr

ee)

0.0

.5

1.0

1.5

2.0

2.5

3.0(D)Control

1 mg/mL F1 (Nigragillin)1 mg/mL F2 (Dihydrophaseic acid)

Concentration (mg/mL)0.0

0 1 2 3 4 5 6

.2 .4 .6 .8 1.0 1.5 2.0 2.5 3.0

DPP

H ra

dica

l sca

veng

ing

activ

ity (%

)

10

30

50

70

0

20

40

60NigragillinDihydrophaseic acid

(B)(A)

Fig. 2 – Chemical structures of nigragillin and dihydrophaseic acid (A) and their antioxidant activities. DPPH radical

scavenging activities (B); Hydroxyl radical scavenging activity (C); Lipid peroxidation inhibition activity (D). Value are

mean ± standard deviation of three determinations.

Table 1 – 1H, 13C-NMR data (d in ppm, J in Hz) of nigragillin in CD3OD.

Position dH, J H-H COSY dC DEPT HMBC

1 169.1 C H-2, H-3

2 6.39 (1H, d, J = 14.8) 7.19 119.2 CH H-3*, H-6*

3 7.19 (1H, dd, J = 10.8, 14.7) 6.39, 6.31 145.8 CH H-5, H-6*

4 6.31 (1H, m) 1.85, 6.14, 7.19 131.4 CH H-2, H-5*, H-6

5 6.14 (1H, dq, J = 6.5, 6.5, 6.4, 13.2) 1.85, 6.31 140.0 CH H-3*, H-4, H-6

6 1.85 (3H, dd, J = 6.5, 12.51) 6.14, 6.31 18.9 CH3 H-5

2 0 4.55 (1H, m) 2.58*, 2.87*, 1.27 48.6 CH H-7 0

3 0 2.87 (1H, dd, J = 5.1, 12.3) 4.55* 53.4 CH2 H-7 0, H-8 0

2.58 (1H, dd, J = 5.1, 12.3) 4.55*

5 0 3.07 (1H, m) 1.04, 3.41*, 4.05* 57.2 CH H-3 0*, H-8 0, H-9 0

6 0 4.05 (1H, d, J = 12.9) 3.07* 44.2 CH2 H-9 0

3.41 (1H, d, J = 12.6) 3.07*

7 0 1.27 (3H, d, J = 6.7) 4.55 16.9 CH3 H-3 0

8 0 2.41 (3H, s) 42.8 CH3 H-3 0*

9 0 1.04 (3H, d, J = 6.5) 3.07 9.5 CH3 H-5 0*, H-6 0

* Indicated as weak correlation ship.

282 J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 2 7 8 – 2 8 6

Based on the data acquired and previous report (Isogai et al.,

1975), compound F1 was identified as nigragillin. Nigragillin

general recognized as a metabolite of black Aspergillius was

first isolated from Aspergillius niger-fermented broth (Caesar,

Jansson, & Mutschle, 1969). It was also detected in the extract

of Zephyranthes candida, but it was possibly originated from

the entophytes of Z. candida (Luo et al., 2012).

F2 was isolated as a colorless powder. The molecular

weight of F2 was determined to be 282 base on the quasi-

molecular ion peak at m/z 305.4 [M + Na]+ and 281.5 [M–H]�

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 2 7 8 – 2 8 6 283

in the ESI–MS. The 1D- and 2D-NMR data of F2 in CD3OD was

present in Table 2. Three olefinic protons at dH 5.76 (1H, s, H-

2), 7.97 (1H, d, J = 16.0 Hz, H-4), and 6.52 (1H, d, J = 16.0 Hz, H-5)

were observed in 1H-NMR spectra. The large coupling content

(J) of H-4 and H-5 indicated a trans-conjugated double bond

moiety in F2. These results were further confirmed by the

HMBC and H-H COSY data (Table 2). Fifteen carbon atoms

were observed in 13C NMR spectra, and the DEPT experiment

further revealed that there were three CH3 groups, four CH2

groups, three CH groups, and five quaternary carbon atoms.

The quaternary carbon atom C-3 exhibited a weak correla-

tionship with H-2 and H-4 as well as a strong correlationship

with H-5 in HMBC spectra. This suggested that C-3 was di-

rectly linked to C-2 and C-3. The correlation (H-H COSY and

HMBC) profiles of C-6 and H-6 revealed that the methyl group

(C-6) was located at C-3. Moreover, a carbonyl group (d13C = 169.4 ppm) directly connected to C-2 (HMBC data) was

found. Based on the data acquired and previous report (Ngan

et al., 2012), compound F2 was identified as dihydrophaseic

acid. Dihydrophaseic acid and its glycosyl-conjugated prod-

ucts were detected in the aqueous-organic extract of leave

(Raschke & Zeevaart, 1976), stem (Ngan et al., 2012), seed

(God-evac et al., 2012), and fruit (Hirai & Koshimizu, 1983) of

various plants. However, it has not been reported in litchi

pericarp before. In this study, dihydrophaseic acid could be

produced from litchi pericarp by A. awamori fermentation

(Fig. 1). Additionally, dihydrophaseic acid was also obtained

from the fermented residues of LPP (Fig. 1B). As the glyco-

syl-conjugated dihydrophaseic acid was reported in many

plants (God-evac et al., 2012; Ngan et al., 2012), we hypothe-

sized that dihydrophaseic acid in A. awamori-fermented LPP

was initially bounded to cell wall polysaccharide via esters

or glycosidic bond (un-extractable), and then released via A.

awamori fermentation.

3.3. Antioxidant activity of F1 and F2

In the present study, the antioxidant of compounds F1 and F2

were determined on the basis of their abilities to scavenge

Table 2 – 1H, 13C-NMR data (d in ppm, J in Hz) of dihydrophase

Position dH, J H-H COSY

1

2 5.76 (1H, s) 2.09*

3

4 7.97 (1H, d, J = 16.0) 6.52

5 6.52 (1H, d, J = 15.9) 7.97

6 2.09 (3H, s) 5.76*

1 0

2 0 2.03 (m, overlap) 4.12

1.76 (m, overlap) 4.12

3 0 4.12 (2H, m) 1.66, 1.76, 1.8

4 0 1.85 (m, overlap) 4.12

1.66 (m, overlap) 4.12, 3.79*

5 0

6 0 3.79 (1H, dd, J = 1.8, 7.3); 3.7 (1H, d, J = 7.3) 3.79*

8 0

9 0 1.14 (3H, s)

10 0 0.92 (3H, s)

* Indicated as weak correlation ship.

DPPH radicals, hydroxyl radicals, and inhibit lipid peroxida-

tion, and a dose-dependent maner was observed. As shown

in Fig. 2B, Nigragillin possessed much higher DPPH radical

than dihydrophaseic acid. When 1 mg/mL nigragillin was em-

ployed, nearly 40% scavenging rate was observed, by contrast

less than 10% was observed with dihydrophaseic acid. While

dihydrophaseic acid exhibits much stronger hydroxyl radical

scavenging activity and lipid peroxidation inhibition potential

than nigragillin (Fig. 2C and D). The insecticidal effect of nigr-

agillin was reported (Isogai et al., 1975), while the DPPH radi-

cal scavenging activity, hydroxyl radical scavenging activity

as well as lipid peroxidation inhibition potential of nigragillin

was not documented before.

3.4. DNA protection effect

DNA is a well known biomacromolecule which plays an

important role in vivo. Oxidative damage to DNA can lead to

many diseases. In order to further understand the health ben-

efits of A. awamori-fermented LPP, the products nigragillin and

dihydrophaseic acid were subjected to the assessment of

plasmid DNA protection effect against Fenton-reaction medi-

ated hydroxyl radicals. Plasmid DNA exhibits three forms on

agarose gel electrophoresis, namely supercoiled circular

DNA (S form), open circular form (O form) and linear form

(L form). The hydroxyl radicals were able to cleave DNA

strand, resulting in the cleavage of supercoiled circular DNA

to open circular and linear forms. As shown in Fig. 3, S form

DNA was the dominant component and only slightly L form

and O form DNA can be detected in the incubated DNA with-

out Fenton-reaction liquid (Lanes a). Dramatically breakage of

S form DNA was observed when the plasmid DNA incubated

with Fenton-reaction liquid (Lanes b). While 50 lg/mL of nigr-

agillin significantly prevented the degradation of the S form of

DNA. The prevention effect was increased when 100 lg/mL of

nigragillin was employed. However, it was decreased when

treated with 200 lg/mL of nigragillin. Moreover, 300 lg/mL of

nigragillin could promote the degradation of S form DNA.

With regard to this fact, monitoring the production of

ic acid in CD3OD.

dC DEPT HMBC

169.7 C H-2*

119.4 CH H-4, H-6

151.6 C H-2*, H-4*, H-5, H-6

131.7 CH H-2, H-5, H-6

135.2 CH H-4*

21.4 CH3 H-2, H-4

87.9 C H-2 0, H-6 0*, H-9 0

46.2 CH2 H-4 0, H-9 0

5, 2.03 66.2 CH2 H-2 0, H-4 0,9 0*

44.7 CH2 H-2 0, H-6 0, H-10 0

49.4 C H-4 0*, H-6 0*, H-10’

77.4 CH2 H-4 0*, H-10 0

83.4 C H-4, H-5, H-2 0, H-4 0, H-6 0, H-9 0, H-10 0

19.7 CH3 H-2 0

16.5 CH3 H-4 0, H-6 0

284 J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 2 7 8 – 2 8 6

nigragillin during the fermentation process is required for the

food safety concern as A. awamori was popularly used in brew

food industry. Compound dihydrophaseic acid also exhibits

strong DNA protective effect, and such effect was increased

in relation to the increased concentration of dihydrophaseic

acid. The DNA protective effect of current seed extract which

contain a significant amount of dihydrophaseic acid and its

glycosyl derivatives were reported by God-evac et al. (2012).

Our previous study suggested that the DNA protective effect

of litchi pericarp extract was enhanced after fermentation

with A. awamori (Lin et al., 2012). In the present study, both

nigragillin and dihydrophaseic acid produced by A. awamori

fermentation can contribute to such enhancement.

3.5. Anticancer effects

The cytotoxicities of nigragillin and dihydrophaseic acid

against human lung cancer (A549), human cervical carcinoma

(HeLa), and human hepatoma (HepG2) cell line were evalu-

ated. Results showed that dihydrophaseic acid was active

against HepG2 and HeLa with IC50 value of 41.92 ± 6.15 and

79.37 ± 9.78 lg/mL, respectively (Table 3), while no significant

inhibitory activities were observed for A549 cell. The dihydro-

phaseic acid is recognized as a metabolite of abscisic acid in

plant. It is involved in maintaining the balance of abscisic

acid content in vivo. However, its in vitro pharmaceutical prop-

erties were rarely studied. Leu et al. (2012) reported the signif-

icant anti-inflammatory activity of dihydrophaseic acid.

Inhibitory activity of dihydrophaseic acid 3 0-O-b-D-glucopy-

ranoside against HepG2 was good (Ngan et al., 2012). This

study suggested that dihydrophaseic acid had a positive influ-

ence on tumor cell viability. In addition, nigragillin was not

significantly cytotoxic to A549, HeLa, and HepG2 cells in the

Fig. 3 – Electrophoretic patterns of plasma DNA with damage

solution in presence of compounds nigragillin (A) or

dihydrophaseic acid (B) at different concentrations (50–

300 lg/mL). Lanes a, electrophoretic patterns of intact DNA

(control); Lanes b, electrophoretic patterns of DNA with

damage solution (blank); The damage solution containing

50 mM •OH generated by mixing 2 lL of 50 mM hydrogen

peroxide and 2 lL of 5 mM ferrous sulfate. Super circular

DNA (S form); Linear DNA (L form); Open circular DNA

(O form).

tested concentration range. This result was in agreement

with the report of Luo et al. (2012).

4. Discussion

The experiment results showed that nigragillin and dihydro-

phaseic acid could be produced from LPP with A. awamori fer-

mentation. Nigragillin was proved to be the metabolite of A.

awamori, while dihydrophaseic acid was demonstrated as

the bound compound to the cell wall of LPP which was re-

leased by A. awamori. The present study also evaluated the

DPPH radical scavenging activity, hydroxyl radical scavenging

activity, lipid peroxidation inhibition effect, DNA protection

effect as well as anticancer effect of nigragillin and dihydro-

phaseic acid. Results suggested that nigragillin possessed

DPPH radical scavenging activity, hydroxyl radical scavenging

activity, and DNA protection effect, while dihydrophaseic acid

exhibited good hydroxyl radical scavenging activity, lipid per-

oxidation inhibition effect, and DNA protection effect as well

as anticancer effect. DPPH radical is stable organic free radical

that accepts an electron or a free radical species and led to a

noticeable colour change from purple to yellow. Those com-

pounds with hydroxyl group and ethylenic bond are generally

recognized as having DPPH radical scavenging activity

(Bondet, Brand-Williams, & Berset, 1997). In this study, nigr-

agillin exhibited more potent effect on scavenging DPPH

radicals when compared with dihydrophaseic acid, in spite

of more hydroxyl groups were found in dihydrophaseic acid.

This indicated that nigragillin is a more potent electronic

donor than dihydrophaseic acid. Both nigragillin and

hydroxyphaseic acid exhibit high DNA protection effect

against Fenton-reaction mediated oxidative damage. How-

ever, high concentration of nigragillin seems to promote the

DNA damage. Our pervious study revealed that enhanced

DPPH radical scavenging activity and DNA protection effects

were concomitant with the increase of polyphenol levels

when fermentation on LPP with A. awamori (Lin et al., 2012).

In the past few years, a serial studies were conducted on

the bioconversion of plant materials using A. awamori, and

enhanced antioxidant activity was achieved after fermenta-

tion (Bhanja, Kumari, & Banerjee, 2009; Martins et al., 2011).

Among these studies, the authors usually ascribed the im-

proved antioxidant activity to the increased phenolics con-

tent via A. awamori fermentation. The present study

demonstrated these two non-polyphenol compounds (nigr-

agillin and dihydrophaseic acid) also contributed to the

enhancements of DPPH radical scavenging activity and DNA

protection effect. The anticancer activity of the fermented

product (dihydrophaseic acid) was also observed in this study.

A. awamori is an important filamentous fungi widely em-

ployed in traditional fermented food industry in East Asia.

With regard to the health benefits of the fermented food

(Han, Rombouts, & Nout, 2001; Kaneki et al., 2001), more work

on the compositional changes during fermentation which

may contribute to human health are needed. Moreover, as

demonstrated in this study that high concentration of nigr-

agillin (a metabolite of A. awamori) can promote DNA damage,

more efforts are required to monitor the production of nigr-

agillin during the fermentation process.

Table 3 – Cytotoxicities of nigragillin and dihydrophaseic acid against HepG2, HeLa, and A549 cell lines. Each valuerepresents mean ± standard deviation of four determinations.

Compounds IC50 (lg/mL)

HepG2 HeLa A549

Nigragillin >100 >100 >100

Dihydrophaseic acid 41.92 ± 6.15 79.37 ± 9.78 >100

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 2 7 8 – 2 8 6 285

5. Conclusions

In summary, two non-polyphenol compounds (nigragillin and

dihydrophaseic acid) were produced form litchi pericarp with

A. awamori fermentation. Nigragillin exhibits DPPH radical

scavenging activity and hydroxyl radical scavenging activity.

Dihydrophaseic acid possessed strong hydroxyl radical scav-

enging activity, lipid peroxidation inhibition effect, DNA pro-

tection effect and had an inhibitory effect on hepatoma

(HepG2) and cervical cells (HeLa). This work provided an

effective way of utilizing fruit byproduct as a readily accessi-

ble source of the natural antioxidants.

Acknowledgements

We are grateful for the financial support from Guangdong

Natural Science Funds for Distinguished Young Scholar (No.

S2013050014131), Youth Innovation Promotion Association of

Chinese Academy of Sciences, Pearl River Science and

Technology New Star Fund of Guangzhou, International

Foundation for Science (No. F/4451-2), Guangdong Natural Sci-

ence Foundation (No. S2011020001156), Guangdong Province

Group Team for Equipment Technology of High Efficiency

Drying and Cold Chain Transport of Agricultural Products,

Special Fund for Agro-Scientific Research in the Public Inter-

est (No. 201303073), and the PhD Start-up Fund of Wenzhou

Medical University (No. KYQD131109).

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