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Chiang Mai J. Sci. 2011; 38(3)

Chiang Mai J. Sci. 2011; 38(3) : 430-438
http://it.science.cmu.ac.th/ejournal/
Contributed Paper

Rapid Screening of Antioxidant Compounds in

Homemade Fruit Fermented Juice Using an On Line
LC-ESI-MS/MS and DPPH Assay
Nitra Nuengchamnong*, Saowanit Boonpathanasak, and Plubplueng Tepwitukkij
Regional Medical Sciences Center 9, Department of Medical Sciences, Ministry of Public Health,
Mueang, Phitsanulok, 65000, Thailand.
*Author for correspondence; e-mail: [email protected]
Received: 27 October 2010
Accepted: 29 November 2010

ABSTRACT
Fruit fermented juice used for health promotion was investigated for antioxidant
compound using an on line LC-ESI-MS/MS coupled to DPPH assay. The structural
elucidation of the active compounds was achieved by negative ionization LC-ESI-MS/MS.
Based on their mass spectra and fragmentation patterns related to antioxidant activity trace,
nine compounds showing strong DPPH scavenging were identied as; HHDP-glucoside,
Mucic acid, Monogalloyl glucoside, Monogalloyl diglucoside, Gallic acid, Galloyl quinic
acid, Digalloyl glucoside, Gallic derivative, Ellagic rhamnoside . In addition, the conrmation
of the active compounds was performed using LC-MS/MS in multiple reaction monitoring
(MRM) mode. Hydrolysable tannins were found as a major component responsible for
the antioxidant activity of this fruit juice.
Keywords: fruit fermented juice, hydrolysable tannin, LC-MS/MS, DPPH assay.
1. INTRODUCTION

Nowadays people concerned about their

health. They need food as functional foods
for health promotion and disease preventions.
Fruits and their products such as wine and
fermented fruit as juice are the popular form
of food supplement. The process of fermenting
is basically feeding sugars and nutrients in
solution to yeast, which return the favor by
producing carbon dioxide gas and alcohol.
Raw fruit juice naturally contains many yeasts,
moulds, and bacteria, derived from the surface
of the fruit. Substances in fermented
foods have been found to have a protective
effect against the development of cancer [1].

Epidemiological evidence has been provided

showing that constituents in fruits are
benecial to human health and contribute
to the prevention of degenerative processes
caused by oxidative stress [2,3]. Dietary intake
of plant phenolics are inversely related to
coronary heart disease [4] and act as anti-ulcer,
antispasmodic, antisecretory, or antidiarrhoeal
agents in the gastrointestinal tract [5]. Consumers need to be made aware of the numerous
benets of fermented foods, especially those
traditionally produced at the home scale.
The popularity of Maha Bambad (Biofermented tonic) which is promoted via Pa

Chiang Mai J. Sci. 2011; 38(3)

Chengs cable TV channel Super Cheng,

the juice, claimed as could cure many illnesses
ranging from skin diseases like psoriasis to
joint pains and pain caused by cervical cancer.
Meanwhile, Thai Public Health Minister
Jurin Laksanawisit told a press conference
on 28 January 2010 that Auntie Chengs
Nam Maha Bambad was highly acidic, had
no medical value and was contaminated with
Clostridium perfringens bacteria that can cause
nausea, vomiting, diarrhea as well as gangrene.
The food and drug administration (FDA) also
charges her for exaggerating the properties
of the liquid. The doubtful of healing power of
her fermented fruit was challenged the expert.
The word no medical value is used for the
adulterant of modern medicine. They did not
mention about the phytochemical compounds.
As we know that fruits and their products
contained many natural compounds that
benet for health.
That doubtful comes to the purpose of
the present study. The aim of this study is
to characterize the antioxidant compounds
from fruit fermented juice as formula from
the cable TV using an on-line LC-ESI-MS/
MS coupled to DPPH assay.
2. MATERIALS AND METHODS

2.1 Materials
Gallic acid was obtained from Sigma
(St. Louis, MO, USA). Methanol (LC/MS
reagent) was purchased from JT Baker
(Mallinckrodt Baker, Inc. Phillipsburg, NJ,
USA) Formic acid (analytical grade) was
purchased for Merck (Darmstadt, Germany).
Water was puried using Elga USF system
(Bucks, England).
2.2 Fruit Fermented Juice Sample
Fruit fermented juice sample made from
the fruit, of Morinda citrfolia Linn, Phyllanthus
emblica Linn. Averrhoa carambola L. Sandoricum koetjape (Burm. f.) Merr., Rhizome of

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Zingiber cassumunar Roxb. Zingiber ofcinale

Roscoe and stem of Tinospora crispa (L.)
Miers ex Hook.f. & Thoms. The ratio of the
fermented formula was sugar: fruit: water
1:3:5 w/w/w and fermented for at least 4
month before use. The sample was collected
from the home of Mr. Suthep Srikreuw,
Wangpikul sub district,Wangthong, Phitsanulok, Thailand. The formula was from the
cable TV.
2.3 Sample Preparation
The fruit fermented sample was ltered
through a 0.2 m Nylon syringe lter (Chrom
Tech, Inc.MN, USA) and then direct injected
into the on-line antioxidant system and the
ltered samples were used for the determination of drug adulterant by LC-MS/MS. The
liquid samples also used directly for testing
the acidity and food poisoning bacteria.
2.4 Fruit acidity, Drug adulterant and Food
Poisonous Bacteria
The sample was tested for the acidity,
(pH meter, Mettler Toledo Multiseven,
Switzerland), drug adulterant as medical value
[6] and food poisoning bacteria; Bacillus
cereus, Staphylococcus aureus, Clostridium perfringens
and Salmonella [7].
2.5 LC-ESI-MS and Radical Scavenging
Detection Analysis
The HPLC was coupled on-line to MS and
a continuous ow DPPH assay as mentioned
before where the efuent of the analytical column was splitted to the MS/MS and the reactor cell for the DPPH reaction[8]. The delay
time between MS trace and visible trace was
ca 0.6 min. The Agilent 1,100 series HPLC
system (Agilent Technologies, Palo Alto, CA)
was coupled to a PE SCIEX API 4000 triple
quadrupole tandem mass spectrometer (Applied
Biosystem, Foster city, CA) equipped with
an electrospray ionization interface. The

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chromatographic separation was achieved

with a phenomenex Gemini column (5 Pm, 250
4.6 mm i.d.) (Phenomenex, Torrance, CA)
protected with an ODS C18 guard column,
o
operated at 25 C. The mobile phase consisted
of solvent A (1 ml formic acid in 1 L of
deionized water) and solvent B (methanol).
The elution program started from 90:10
solvent A:solvent B for 4 min, then changed
to 80:20 solvent A:solvent B in 6 min, linearly
increased to 10:90 solvent A:solvent B in 30
min. Then, the ratio of solvent A:solvent B
was constant at 10:90 for 5 min, then changed
to 90:10 solvent A:solvent B in 5 min and
kept constant at 90:10 solvent A:solvent B
for 5 min for reconditioning of the column.
Mass spectra were recorded within 55
min. The injection volume was 5 Pl. The ow
rate was set to 600 Pl/min. The Analyst 1.3.2
software was used for data acquisition and
processing. The full scan mass spectra from
m/z 100-1,000 amu were acquired in negative
ion modes. The optimum conditions of the
interface were as follows: ESI-negative; ion
spray voltage of - 4500 V, curtain gas (N2)
of 69 Kpa (10 psi), ion source gas 1 (air, for
nebulizing) of 450 Kpa (65 psi), ion source
gas 2 (air, for drying solvent) of 380 Kpa
(55 psi). The interface temperature was set
o
to 400 C. The entrance potential (EP) and
declustering potential (DP) were -10V and
-80V, respectively. The continuous ow
system for antioxidant activity detection,
consisted of an HPLC pump, LC20AD
prominence (Shimadzu, Kyoto, Japan),
home-made knitted reaction coil PEEK
tubing with an inner diameter of 180 Pm and
a total reaction coil volume of 100 PL. The
ow of 0.1mM DPPH was set to 200 Pl/min
and induced bleaching was detected as a
negative peak at 515 nm using the UV-VIS
detector (SPD 20AV, Shimadzu, Kyoto, Japan).
The LC solution software was used for data
acquisition and processing. The polarity

Chiang Mai J. Sci. 2011; 38(3)

of the signal output was reversed in order

to obtain positive signals. The system was
o
operated at 25 C. For the characterization of
antioxidant peaks, the fragment ions from
their corresponding parent ions in negative
mode were induced with collision gas (CAD)
of 41 Kpa (6 psi), collision energy (CE)
between-5 to-50V and collision cell exit
potential (CXP) of -6 V, DP in the range of
-20 to -110 V.
2.6 Peak Identication
Peak identication was performed by
comparison of the retention time and mass
spectrum with reference compounds. Tentative
identication of peaks of which standard
compounds were not available was obtained
by comparing their elution order and their
molecular ion with the data from the literature
and conrmation by multiple reaction monitoring
(MRM).
3. RESULTS AND DISCUSSION

The fruit fermented juice used as food

supplement for anti-diabetic anticancer and
health promotion of people in Wangthong
district, Phitsanulok. This sample showed high
acidity with pH 2.72. The food poisoning
bacteria; B.cereus/1ml, S.aureus/0.1ml, C.
perngens/1ml and Salmonella/25ml did
not found might be the high acidity of the
juice. There are no adulterant of drug as
medical value in the group of Tranquilizer
(alprazolam, diazepam, nitrazepam, triazolam),
Steroids (dexamethasone, prednisone, prednisolone), Antihypertensive (nifedepine), Antidiabetic (gliclazide, glimepiride, glipizide,
pioglitazone), and Antihistamines (epidine,
phenylpropanolamine, psuedoepedine).
An HPLC was coupled to a triple
quadrupole mass spectrometer and a DPPH
assay for the rapid identication of antioxidant
compound of the fruit fermented juice sample
as shown in the Figure 1. DPPH assay is

Chiang Mai J. Sci. 2011; 38(3)

based on the reduction of the stable radical,

DPPH, to the formation a non radical
form in the presence of hydrogen donating
antioxidant. The fruit fermented juice showed
an antioxidant activity by reducing DPPH to
the yellow coloured diphenylpicrylhydrazine
derivatives. The remaining of DPPH was
measured at wavelength 515 nm. In the

433

on-line DPPH assay monitors the activity of

the antioxidant compounds that separated
from the gradient reversed phase HPLC
system. The mass spectrometry was used to
elucidate the structures based on their
molecular weights and fragmentation patterns
of the active compounds.
There were tentative nine active anti-

Figure 1. HPLC separation of fruit fermented compounds with simultaneous antioxidant

activity assay and MS detection. a) The chromatogram from the antioxidant activity assay
detection at 515 nm. b) The total ion current (TIC) output from the ESI-MS in negative mode.
For peak assignments, see Table 1.

oxidant compounds in fruit fermented juice

(Figure 1a). The antioxidant compounds
were identied as; Peak 1 (tR= 6.9 min)
showed peak at m/z 481.4[M-H]-, and the
MS/MS analysis showed that fragmentation
of m/z 481.4 yield ellagic acid (m/z 300.9)
by comparison of the data with reported in
the literature [9], it is suggested that this
compound was hexahydroxyphenyl (HHDP)

glucoside. Peak 2 (tR= 10.3 min) with

[M-H]- at m/z 209.1, ([mucic acid-H]-) and
191.0 ([mucic acid H2O]-) that are proposed
as mucic acid. This compound was found in
the fruit juice of Emblica (Phyllanthus emblica
Linn) [10]. Peak 3 (tR= 11.7 min) with a
[M-H]- at m/z 493.0 and fragment ion at
m/z 330.0, and 313.0. It was proposed as
monogalloyl diglucoside. Peak 4 (tR= 12.9

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Chiang Mai J. Sci. 2011; 38(3)

min) with a [M-H]- at m/z 331.0 and fragmentation gave ion at m/z 270.7 and 211.0,
it was identied as monogalloyl glucoside
[11]. The ion m/z 270.7 and 211.0 formed
during the fragmentation of mono galloyl
glucose. Peak 5 (tR= 15.6 min) with a [M-H]at m/z 169.2 and a fragment ion at m/z 125.0
[M-H-CO2]-, this compound was identication as gallic acid which the retention time
and its mass data t with authenticated
compound. Peak 6 (tR= 17.6, 19.1 min) was
tentatively identied as galloylquinic acid
since it had a [MH] at m/z 343.1 and
fragmentation gave ions at m/z 190.8 and
168.9, corresponding to quinic acid and
gallic acid moieties [12,13]. Peak 7 (tR=18.6,
20.5, and 23.8 min) with [M-H]- at m/z 483.0
and fragmentation gave ion at m/z 331.3
(monogalloyl glucose), 312.9 (dehydrated
monogalloyl glucose), 271.0 digalloyl quinic
acid. Elimination of a galloyl ester group
(m/z 331), a subsequent loss of water (m/z

313) and the occurrence of gallic acid

(m/z 169) are also observed for the digalloylglucose anion (m/z 483) [14]. Peak 8
(tR= 26.3, 28.7 min) with [M-H]- at m/z 241.3
and fragmentation gave ion at m/z 169.0
[gallic acid-H]- 125.2 [gallic acid-H-CO2]-,
and 197.2 indicating that they were gallic
acid derivatives. These two compounds need
more data to identify. Peak 9 (tR= 35.0 min)
with [M-H]- at m/z 447.3 and fragmentation
gave ion at m/z 301.0 as ellagic rhamnoside.
The results were shown in Table 1 and conrmation of the identication using daughter
ion spectra in MRM mode were show in
Figure 2.
The component responsible for the
antioxidant activity of this fruit juice was
hydrolysable tannins. There are reported about the tannins in good and bad way.
The intake of a small quantity of some tannin promotes benecial health effects. They
are benecial to health due to their chemo-

Table 1. Identication of antioxidant compounds in fruit fermented juice using an LC-ESIMS-DPPH assay data in negative ionization, tR is the retention time of the peaks from the
antioxidant activity trace.
Peak no.

tR(min)

6.9

2
3

10.3

12.9

15.6

11.7

17.6
19.1
18.6

20.5
23.8

8
9
a

26.3
28.7
35.0

MS

ESI-MS (m/z)
MS/MS

481.4
209.0
493.0
331.0
169.0
343.0
343.0
483.0
483.0
483.0
241.3
241.3
447.3

300.9, 292.7, 190.8

191.0
331.0, 313.0
270.7, 211.0
125.0
191.0, 168.9
191.0, 168.9
331.3, 312.9, 271.0
331.3, 312.9, 271.0
331.3, 312.9, 271.0
168.9, 125.2, 197.3
168.9, 125.2, 197.3
301.0

compared with the standard compounds

Tentative Id

HHDP-glucoside
Mucic acid
Monogalloyl diglucoside
Monogalloyl glucoside
Gallic acida
Galloyl quinic acid
Galloyl quinic acid
Digalloyl glucoside
Digalloyl glucoside
Digalloyl glucoside
Gallic derivative
Gallic derivative
Ellagic rhamnoside

Chiang Mai J. Sci. 2011; 38(3)

Figure 2. Conrmation of the identication using daughter ion spectra in MRM mode.

435

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Chiang Mai J. Sci. 2011; 38(3)

Figure 2. Conrmation of the identication using daughter ion spectra in MRM mode.
(continued)
preventive activities against carcinogenesis
and mutagenesis [15] but on the other hand,
they may be involved in cancer formation,
hepatotoxicity or antinutritional activity [16].
It is not advisable to ingest large quantities of
tannins, since they may possess carcinogenic and anti-nutritional activities [17]. Thus, it
is important to determine the right dose of

the right kind of tannins to promote optimal

health.
4. CONCLUSIONS

A method for the analysis of antioxidant

compounds by RP-HPLC coupled to electrospray ionizationtandem mass spectrometry
and DPPH assay was applied to the fruit

Chiang Mai J. Sci. 2011; 38(3)

fermented juice. The results implied that this

on line technique was rapid and effective for
selectively analysis of the antioxidants. A
wide variety of hydrolysable tannin components were detected and their structure was
ascribed by daughter ion spectra obtained
after collision-induced dissociation. The nding
showed that these hydrolysable tannin could
be utilized as food supplement if moderate
intake.
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