Food Chemistry 232 (2017) 621632

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

Evaluation of antioxidant potential of Artocarpus heterophyllus L. J33

variety fruit waste from different extraction methods and identification
of phenolic constituents by LCMS
Mohd Nazrul Hisham Daud a,b, Dian Nashiela Fatanah a, Noriham Abdullah a,c, Rohaya Ahmad a,d,
a
Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia
b
Food Safety and Forensic Department, Malaysian Agricultural Research and Development Institute, 43400 Serdang, Selangor, Malaysia
c
Malaysia Institute of Transport, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia
d
Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA, 42300 Bandar Puncak Alam, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Artocarpus heterophyllus J33 (AhJ33) fruit is a popular and valuable jackfruit variety in Malaysia. For
Received 24 June 2016 export, the pulp has to be separated from the skin which is usually discarded. Hence, the conversion
Received in revised form 1 April 2017 of the fruit waste to food products with economic value needs to be explored utilizing the waste to
Accepted 3 April 2017
wealth concept. This paper reports the evaluation of antioxidant potential of AhJ33 fruit waste (rind
Available online 5 April 2017
and rachis) extracts from three different extraction methods (maceration, percolation and Soxhlet).
The antioxidant potential was assessed by DPPH radical scavenging, FRAP and b-carotene bleaching
Keywords:
assays. The total phenolic and total flavonoid contents were estimated by TPC and the TFC assays. For
Artocarpus heterophyllus J33
Fruit waste
both rind and rachis, the maceration technique yielded extracts with the strongest antioxidant activities
Antioxidant which correlated with the highest TPC and TFC values. TOF LCMS analyses identified two phenolic acids
Total phenolic content as the major constituents responsible for the antioxidant activity of the active extracts.
Total flavonoid content 2017 Elsevier Ltd. All rights reserved.
DPPH
FRAP
LCMS

1. Introduction purpose, fruits usually undergo minimal processing to prolong the

Artocarpus heterophyllus J33 (AhJ33) variety is the most eco-
nomically important variety among Artocarpus species in Malay-
sia. In 2010 under the Economic Transformation Program, the
government has come out with Entry Point Project 7 (NKEA) in
which the agricultural sector focuses on improving the export
capacity of fruits and vegetables to the premium level in order to
increase local fruit exports. Until 2014, up to 5000 hectares of land
has been planted with this A. heterophyllus variety. In 2014, the
production of fruits was reported to be 33,788 tonnes with a pro-
ductivity of 11.2 (tonne/hectare) generating a total income of
about Ringgit Malaysia 107 million (Ministry of Agricultural and
Agro-Based Industry Malaysia, 2015). The fruits from this species
are exported widely throughout the world market mostly to Singa-
pore, China, Macau, Hong Kong, Middle East and Europe. For export
Corresponding author at: Faculty of Applied Sciences, Universiti Teknologi
MARA, 40450 Shah Alam, Selangor, Malaysia.
E-mail addresses: nazrul@mardi.gov.my (M.N.H. Daud), nashiela_beez@yahoo.
com.my (D.N. Fatanah), noriham985@salam.uitm.edu.my (N. Abdullah), rohayaah-
mad@salam.uitm.edu.my (R. Ahmad).
postharvest storage life and maintain freshness (Punan et al.,
2000). For the export of AhJ33 variety, only the pulps (with the
seed) are isolated while the rest of the fruit parts are usually dis-
carded as waste. Hence, the aim of our study was to convert
AhJ33 fruit waste to a food product with economic value based
on the reported nutritional properties of the fruit (Baliga,
Shivashankara, Haniadka, Dsouza, & Bhat, 2011).
Previous reports on A. heterophyllus found that extracts (includ-
ing the pulp, leaf and root bark) possess moderate to high antiox-
idant activities. In general, the methanolic, ethanolic and aqueous
crude extracts of A. heterophyllus showed more effective antioxi-
dant activity compared to the acetone extract (Baliga et al., 2011;
Biworo, Tanjung, Iskandar, & Suhartono, 2015). For the pulp, the
major chemical constituents which contributed to the antioxidant
activity in the methanol extract were reported to be b-carotenes
(Biworo et al., 2015). From the leaf, root and bark methanol
extracts, flavonoids have been reported to contribute significantly
to the antioxidant activities which were evaluated by the DPPH
assay (Ko, Cheng, Lin, & Teng, 1998). A preliminary phytochemical
study on A. heterophyllus has been reported for the skin of the fruit

http://dx.doi.org/10.1016/j.foodchem.2017.04.018
0308-8146/ 2017 Elsevier Ltd. All rights reserved.
622 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632
(Sharma, Gupta, & Verma, 2015). However, to date, there has been
no specific reports on the antioxidant activities of the rind and
rachis extracts.
The extraction process is extremely important as the first step
in the treatment of plant materials for further phytomedicinal
screening and phytochemical investigations. The basic process for
extraction included steps such as pre-washing, drying of plant
materials, grinding and other steps to the kinetics of extraction
and increase the contact of sample surface with the solvents
(Sasidharan, Chen, Saravanan, Sundram, & Yoga Latha, 2011).
Solid-liquid extraction known as leaching or lixiviation has been
widely used for recovering secondary metabolite from plants using
solvents (Luque de Castro & Priego-Capote, 2010). The extraction
process usually involved thermal and non-thermal methods. For
thermal extraction, the Soxhlet method has been accepted as a
standard technique and remains as a primary reference in the
development of modern thermal extraction methods such as
microwave-assisted Soxhlet extraction and ultrasound-assisted
Soxhlet extraction. Traditionally, the non-thermal extraction pro-
cess has been carried out through maceration and percolation
especially to extract non-volatile phytochemical (Sarker, Latif, &
Gray, 2006). It has been reported that extraction methods may
affect the phytochemical constituents of a plant (Pothitirat,
Chomnawang, Supabphol, & Gritsanapan, 2010). Hence, for the first
phase of this study, this paper reports the evaluation of antioxidant
potential of AhJ33 fruit waste (rind and rachis) extracts from three
different extraction methods (maceration, percolation and Soxhlet)
evaluated by DPPH, FRAP and b-carotene assays. The phenolic and
flavonoid contents estimated by the TPC and TFC assays were then
correlated against these antioxidant activities. However, the inter-
pretation of the values took into account the limitation of these
assays, as reported by Pekal and Pyrzynska (2014). Finally, the
major antioxidant constituents which may be responsible for the
activities were identified using LCMS-TOF.
2. Materials and methods
2.1. General instrumentation
Solvents were evaporated using Buchi Rotavapor R210 at 45 C.
The UVVis was recorded on ELISA spectrophotometer Spectramax
Plus (Molecular Devices). Mass of compounds was obtained from
mass analyzer 6224 TOF LC/MS Agilent Technologies consisting
of an electrospray (ESI) source system run in negative mode.
2.2. Plant material
Non-seasoning type of Malaysian Artocarpus heterophyllus fruit
(AhJ33) for antioxidant and LCMS analysis was obtained from com-
mercial source at Taman Kekal Pengeluaran Makanan Lancang
Pahang, Pahang, Peninsular Malaysia. The rachis and rind of the
fruits were separated, cut into small pieces (2.0 mm) and oven-
dried at 40 C for 48 h.
2.3. Extraction process
The extraction approach was carried out using maceration, per-
colation and Soxhlet techniques. A mixture of ethanol and water
was used as the extracting solvent since numerous studies have
reported on its efficiency in extracting phenolic and polyphenolic
constituents (Laghari, Memon, Nelofar, & Laghari, 2011; Vongsak
et al., 2013). Ethanol-water mixtures are suited to penetrate the
hydrophobic areas of the vegetable matrix and help to precipitate
soluble proteins, facilitating further processing. The extraction
approach adopted in this study employs the method of Vongsak
et al. (2013) with some minor modifications.
2.3.1. Maceration
The sample (1.0 kg) was macerated with 2 L 70% ethanol for
72 h at room temperature (25 C) with occasional shaking. The
extract was filtered through a Whatman No. 1 filter paper and
the marc was re-extracted with the same amount of solvent. The
solvent was evaporated off to yield the crude rind maceration
extract (RDM) and crude rachis maceration extract (RCM).
2.3.2. Percolation
Solvent (70% ethanol) was added to the sample (1.0 kg) and the
mixture was allowed to stand for 1 h. The mixture was then trans-
ferred gradually (400 ml volume at a time until 4 L) into a fabri-
cated glass percolator (40 cm
6 cm) set at a flow rate of 1 ml/min
at room temperature (25 C) until the extraction completed. The
extract obtained was filtered and the solvent was evaporated off
to yield the crude rind percolation extract (RDP) and crude rachis
percolation extract (RCP).
2.3.3. Soxhlet
The sample (1.0 kg) was placed into a thimble and extracted
with 70% ethanol (4 L) in a Soxhlet apparatus at boiling tempera-
ture for 5 h. The extract obtained was filtered through a Whatman
No. 1 filter paper. The solvent was evaporated off to yield the crude
rind Soxhlet extract (RDS) and crude rachis Soxhlet extracts (RCS).
2.4. Determination of scavenging activities
The chemical assay was based on method described by Ahmad
et al. (2005). The antioxidant potential of the crude extract of
AhJ33 fruit waste was assessed on the basis of their scavenging
activity on the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free
radical. All test samples were prepared by dissolving 1.0 mg of
samples into 1.0 ml 70% ethanol. A solution of DPPH was prepared
by dissolving 5.0 mg DPPH in 2.0 ml of methanol and the solution
kept in the dark at room temperature. Different concentrations of
the test samples were prepared in 96-well microtitre plates. Five
ll of methanolic DPPH solution was added to each well. The plate
was shaken to ensure thorough mixing before being placed in the
dark and wrapped with aluminum foil. After 30 min, the absor-
bance of the solution was analyzed using an ELISA reader at a
wavelength of 517 nm. Percentage inhibition by sample treatment
was determined by comparison with 70% ethanol treated control
group. All test analyses were run in triplicates and the readings
were averaged. Quercetin (Sigma, USA) was used as positive con-
trol. In the DPPH method, the antioxidant activity is described by
percent inhibition;
Percent inhibition Abs control
 Abs sample=Abs control100
2.5. Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was carried according to method of Dian,
Noriham, Nooraain, and Azizah (2015). The FRAP reagent was
freshly prepared by mixing 300 mM acetate and glacial acetic acid
buffer (pH 3.6), 20 mM ferric chloride and 10 mM 2-4-6-tripyridyl-
s-triazine (TPTZ) was made to 40 mM hydrochloride at a ratio of
10:1:1. All test samples were prepared by dissolving 1.0 mg of
sample into 1.0 ml 70% ethanol. Briefly, 0.1 ml sample/standard
was mixed with 3 ml FRAP reagent and 3 ml distilled water. The
mixture was incubated in the dark at 37 C for 8 min and the absor-
bance at 595 nm was then read using UVVis spectrophotometer.
 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632
The total antioxidant activity of AhJ33 variety fruit waste extracts
were determined against a standard of known FRAP values and was
expressed as lM of trolox equivalent (lM TE)/ml of extract.
2.6. b-Carotene bleaching assay
The b-carotene bleaching assay was based on method devel-
oped by Velioglu, Mazza, Gao, and Oomah (1998). The b-carotene
(0.2 mg in 1 ml chloroform), linoleic acid (0.02 ml) and Tween 20
(0.2 ml) were transferred into a round bottom flask. Chloroform
was removed at room temperature under vacuum at reduced pres-
sure using rotary evaporator. Following evaporation, 50 ml of dis-
tilled water was added and the mixture was then shaken
vigorously to form an emulsion. About 2 ml aliquots of the emul-
sion were pipetted into test tubes containing 0.2 ml of ethanol
(as negative control)/combination of BHA/BHT standard (as posi-
tive control)/AhJ33 variety fruit waste extracts and immediately
placed in a water bath at 50 C for 120 min. The initial Abs0 (at
time = 0 min) and final Abs120 (at time 120 min) was read at
470 nm. Antioxidant activity (AA) was expressed as percent of
inhibition relative to the control, using following formula:
Abssample  Abs120
Antioxidant activity %
100
Abs0  Abs120
2.7. Determination of total phenolic content
The total phenolic content for extracts and fractions were car-
ried out according to the Folin-Ciocalteu method as reported by
Harbourne, Marete, Jacquier, and ORiordan, (2009). All test sam-
ples were prepared by dissolving 1.0 mg of samples into 1.0 ml
70% ethanol. The reaction mixture contains 200 ll sample, 500 ll
Folin-Ciocalteu reagent (Merck, Germany), 1.5 ml of 20% sodium
carbonate and 7.8 ml of distilled water. The solution was mixed
and incubated for 2 h. The absorbance of the solution was then
analyzed using UVVis spectrophotometer at a wavelength of
760 nm. The total phenolic content was calculated as mg of gallic
acid equivalents per gram of dry extract (mg GAE/g DE).
2.8. Determination of total flavonoids content
The determination of total flavonoid content (TFC) was per-
formed based on aluminium chloride colorimetric method as
described by Kim, Jeong, and Lee (2003) using quercetin as a stan-
dard. All the test samples were prepared by dissolving 1.0 mg of
samples into 1.0 ml 70% ethanol. Total flavonoid content was
expressed as milligram of quercetin equivalents per gram of dry
extract or mg QE/g DE.
2.9. LCMS profiling and identification
Profiling of the crude extracts was carried-out using a combina-
tion of chromatographic separation Agilent HPLC 1200 series and
mass analyzer 6224 TOF LC/MS Agilent Technologies consisting
electrospray (ESI) source system. The chromatographic instrument
was Agilent HPLC 1200 series system equipped with an auto sampler
(G1367D), binary pump 1260 (G1312B), column compartment
(G1316B) and Diode Array Detector 1260 DAD (G1315C) for spectro-
scopic scanning. The chromatographic separation was performed
using GL Sciences - Inertsustain Column C-18 (250 mm
4.6 mm,
i.d., 5 mm) and temperature set at 40 C for both left and right side.
The liquid chromatography (LC) parameters such as injection vol-
ume was set for 5ul at auto sampler (G1367D), binary pump was
set till 45.01 min with post time 4.99 min, flow rate 0.6 ml/min,
minimum pressure 1 bar and maximum pressure 400 bar, max flow
623
gradient 100 ml/min. Two set of solvents used known as solvent A
and B. Solvent A and solvent B consist of H2O: MeOH (8:2) with
0.1% formic acid and ACN with 0.1% formic acid. Combination of both
solvent in LC system was set at a ratio of solvent A: solvent B, 95:5
with gradient elution: from 5% solvent B at 0 min, 55% solvent B at
30 min to 100% solvent B at 40 min. The eluent was monitored with
a diode array detector at k 254, 280 and 310 nm.
The acquisition method for mass spectrometer was set for dual
ESI for ion source with minimum range MS 100 and maximum at
1000. The gas temperature was set at 350 C, drying gas flow at
8 L/min, nebulizer gas at 30 psi, capillary voltage at 4000 V, frag-
ment voltage at 175 V, skimmer voltage at 65 V, and OCT 1 RF at
750 V. Scan segment was carried out for both positive (+) and neg-
ative () mode. Data processing was performed using Agilent
MassHunter Workstation software. The reference masses involved
were TFA anion (112.985587) and TFA adduct (1033.988109) in
order to verify the high resolution of mass spectrometry, which
confirmed that parent ion and source induced dissociation frag-
ment could provide the accurate mass information.
Identification of constituents were carried-out using TOF LCMS
data processer (Agilent MassHunter Workstation) and online data-
base. Mass value m/z obtained from ESI chromatogram for sub-
fraction was analyzed using LCMS tool system known as Formula
calculator. Data such as molecular formula and mass (m/z) for the
selected peak on the chromatogram was generated by the system.
Analysis for mass to charge ratio (m/z) on selected peaks was
carried-out using ESI Scan experiment. The major target for mass
to charge ratio (m/z) in this experiment was the major values or
known as base peak and other related value the shows high possi-
bility and accuracy according to LCMS Formula calculator analy-
sis. All generated data was based on calculated mass (m/z) of the
exact compound and the range of accuracy was less 7.0 ppm. The
molecular formula and mass value obtained from Formula calcula-
tor was further subjected to online database for identification of
constituent structures. Two major online databases known as
Chemspider and Drugbank were used in order to facilitate identifi-
cation for structure of the constituents.
2.10. Statististical analysis
All experiments were run in triplicates. Statistical analyses were
conducted with the Statistical Analysis System (SAS) 9.1.3 software
package. Analyses of variance were performed by ANOVA proce-
dures. Significant differences (P < 0.05) were determined by least
square means comparison.
3. Results and discussion
The investigation on the effect of three extraction methods on
the percent yield, antioxidant activities, total phenolic content
and total flavonoid content of AhJ33 variety fruit waste yielded
the following results:
3.1. Percent yield
Table 1 shows the percent yield of crude extracts obtained from
the three extraction methods for the rind and rachis of AhJ33 variety
along with other antioxidant values. The results showed that the
extraction method played an important role in extracting phyto-
chemicals. For the rind, the percolation method yielded the highest
percent yield of crude extract with 33.8%, followed by the macera-
tion method with percent yield of 28.0% and Soxhlet with the lowest
percent yield of 12.1% (RDP > RDM > RDS). Similarly, for the rachis,
the same trend was observed where the yield of crude extract was
in the order of RCP > RCM > RCS, as shown in Table 1.
 624 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632

The relatively high percent yield for RDP and RCP may be justi-

RDM (rind-maceration), RDP (rind-percolation), RDS (rind-soxhlet), RCM (rachis-maceration), RCP (rachis-percolation) and RCS (rachis-soxhlet). Values are expressed as mean standard deviation. Means with different letters
0.9256 0.0471

0.9577 0.0339
fied by the fact that, in the percolation technique, the saturated
solvent (70% ethanol) which is capable of extracting polar com-
TFC & Beta-

TFC & Beta-

carotene

carotene
pounds is permanently replaced by fresh or unsaturated solvent
that works its way down through the marc (Busia, 2016). However,
the Soxhlet technique which involves thermal reaction could cause
degradation of polar constituents (easily extractable by 70% EtOH)

0.9376 0.0469
0.8091 0.0103

leading to low yields. This could justify why the Soxhlet technique
TFC & FRAP

TFC & FRAP

was found to be more effective in the extraction of non-polar com-

pounds such as terpenes rather than polar compounds such as
phenolics and polyphenolics (Hossain, Al-Toubi, Weli, Al-Riyami,
& Al-Sabahi, 2013).
0.9911 0.0011

0.9931 0.0006
TFC & DPPH

TFC & DPPH

3.2. Antioxidant activity

The antioxidant activity for all crude extracts prepared from the
Percent yield, total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity as measured by the DPPH radical-scavenging, FRAP and beta-carotene assays.

three different extraction techniques, as measured by the DPPH,

0.8459 0.0621

0.8656 0.0735

FRAP and b-carotene bleaching methods along with their correla-

TPC & Beta

TPC & Beta

tion coefficients are shown in Table 1. In the DPPH assay, RDM

carotene

carotene

showed the highest scavenging activity with 94.4% inhibition fol-

lowed by RDP with 50% inhibition and RDS with 43.0% inhibition.
The rachis crude extracts from the three extraction methods
0.6884 0.0392

0.9920 0.0100

showed the same trend in the order of RCM > RCP > RCS with per-
Pearsons correlation coefficient

Pearsons correlation coefficient

cent inhibition of 62.0, 44.0 and 38.0%, respectively. Similarly, in

TPC & FRAP

TPC & FRAP

terms of the reductive ability of antioxidants, as evaluated by

the FRAP assay, RDM exhibited the highest value followed by
RDP and RDS with 26.4, 24.6 and 15.8 mM TE/ml of extract, respec-
Pearsons correlation coefficient strength value: Weak = 0.20.39; moderate = 0.40.59; Strong = 0.60.79; Very strong = 0.81.0.

tively. The rachis crude extracts from the three extraction methods
0.9999 0.0014

0.9931 0.0052

also showed the same trend in the order of RCM > RCP  RCS with
TPC & DPPH

TPC & DPPH

18.7, 15.7 and 15.6 mM TE/ml of dry extract, respectively. In the

b-carotene bleaching assay, the order of antioxidant activity was
found to be the same as in the DPPH and FRAP assays. The percent
**

**

bleaching of b-carotene for rind extracts was RDM > RDP > RDS
54.1 0.3b

25.2 0.8b

with 59.0, 54.1 and 43.0%, respectively. Similarly, for the rachis
28.9 0.9a
59.0 1.0a

25.3 0.4c

20.0 0.5c
bleaching

bleaching
carotene

carotene

extracts, the order of activity was RCM > RCP > RCS with values
Beta-

Beta-

of 28.9, 25.2 and 20%, respectively.

(%)

(%)

It is interesting to note that despite the moderate percent yield

TE/ml of dry

TE/ml of dry

for RDM and RCM, both exhibited the highest antioxidant activities
24.6 0.9b

15.7 0.3b
15.6 0.2b
26.4 0.7a

18.7 0.2a
15.8 0.5c
FRAP mM

FRAP mM

which indicated that the maceration process is effective in extract-

extract

extract

ing phytochemicals. This is justifiable because maceration softens

and breaks the plant cell wall in order to release the soluble phy-
tochemicals and hence preserving the active constituents (Luque
44.0 0.1b
50.0 0.1b
94.4 0.1a

62.0 0.1a
43.0 0.1c

38.0 0.1c
Inhibition

Inhibition

de Castro & Priego-Capote, 2010). In contrast, the percolation tech-

DPPH,

DPPH,

nique may not be an effective method. Although it could increase

(%)

(%)

the percent yield of extractive substances (as seen for RDP and
RCP), the solvent may not reach all areas causing interference
content, mg QE/g

content, mg QE/g

and finally lowering the efficiency of extraction of active compo-

Total flavonoid

Total flavonoid

b
a

nents. This is in line with a previous study as reported by Sarker

511.6 1.0b
871.4 1.2a

381.4 1.1c
dry extract

dry extract

429.4 1.1
660.9 1.1

300.6 1.1

et al. (2006). Furthermore, since 70% ethanol was used as the sol-
vent in this study, primary metabolite constituents such as sugars
could be easily extracted (Balto et al., 2016) contributing to the
overall percent yield but not the antioxidant activity. Between
content, mg GAE/g

content, mg GAE/g

the rind and rachis, the rind extract showed stronger antioxidant
Total phenolic

Total phenolic

activity indicating a significant difference in the composition of

dry extract

dry extract
79.5 0.1b

74.0 0.1b
84.9 0.1a

77.2 0.1a

73.5 0.1c
79.0 0.1c

their chemical constituents which can be estimated by the TPC

are significantly different (p < 0.05).

and the TFC assays.

3.3. Total phenolic content (TPC)

substances

substances
28.0 0.1b

29.0 0.1b
33.8 0.1a

43.9 0.1a
12.1 0.1c

16.3 0.1c
extractive

extractive
% Yield of

% Yield of

The total phenolic content of the extracts as evaluated by the

Folin-Ciocalteu method is shown in Table 1. Among the rind
extracts, RDM showed the highest phenolic content with
Extract*

Extract*

84.9 mg GAE/g DE followed by RDP (79.5 mg GAE/g DE) and

RDM

RCM
Table 1

RDP
RDS

RCP
RCS

RDS with 79.0 mg GAE/g DE. The rachis extracts showed the
*

**

same trend as the rind in the order of RCM > RCP > RCS with
 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632 625

Fig. 1. Total ion chromatogram (TIC) and LC profiles at k 254, 280 and 310 nm of RDM (a) and RCM (b).

77.2, 74.0 and 73.5 mg GAE/g DE, respectively. As shown in
Table 1, a very strong correlation was indeed observed between
TPC-DPPH, indicated by high Pearsons correlation coefficient val-
ues of r > 0.8 for both rind and rachis. The DPPH scavenging
activity showed the highest correlation coefficient values with
TPC for both rind (r > 0.9999) and rachis (r > 0.9931) compared
to others. These findings are supported by a study by
McDonald, Prenzler, Antolovich, and Robards (2001) which found
that in general, extracts with high amount of phenolic content
could significantly contribute to high antioxidant activity. The
higher TPC values obtained for the rind may be explained by
the higher accumulation of phenolic compounds in the external
part compared to inner part since their formation is a light-
dependent process (Guanghou & Lai, 2006; Ribera, Reyes-Daz,
Alberdi, Zuiga, & Mora, 2010). It has also been reported that
the highest levels of phenolic compounds especially phenolic
acids group are often found in the external parts of the ripe fruit
and these could contribute to high antioxidant capacity (Hidalgo
& Almajano, 2017).
3.4. Total flavonoid content (TFC)
Flavonoids are a family of polyphenolic compounds almost
ubiquitous in plants and play a major role for human health
especially as antioxidant agent. Previous studies on A. heterophyl-
lus reported the isolation of flavonoids from the leaves, stems,
barks, heartwoods and pulp. This warrants an evaluation of the
total flavonoid content of the rind and rachis crude extracts.
The TFC values obtained from three different extraction methods
are shown in Table 1, showing the same trend as the TPC values.
626 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632

(a)
RDM

a b d
c e

RDP

d
e

RDS

d
e

(b)
Rind extracts

RDM

RDP

e
RDS

Fig. 2. (a) LC profiles of RDM, RDS and RDP at k 280 nm (b) comparison of intensity strengths for peak d and e from rind extracts.

For the rind extracts, RDM showed the highest TFC value with that contain these functional groups (such as phenolic acids) will
871.4 mg QE/g DE compared to RDP and RDS with 511.6 and also yield positive results due to complex formation as shown
381.4 mg QE/g DE, respectively. For the rachis extracts, RCM below (Cornard, Lapouge, & Andre, 2013; Hirpaye & Rao, 2013).
exhibited the highest TFC value of 660.9 mg QE/g DE, followed
by RCP and RCS with values of 429.4 and 300 mg QE/g DE, O O
respectively. The TFC values for both rind and rachis extracts
showed very strong correlations with antioxidant activities, indi- OH
OH
cated by their high correlation coefficients (r > 0.8) between TFC-
DPPH, and TFC-FRAP, as shown in Table 1. As in the TPC assay,
the DPPH scavenging activity also showed the highest correlation HO O
coefficient values TFC values for both rind (r > 0.9911) and rachis OH Al+ O
(r > 0.9931) compared to others.
In principle, the mechanism for the detection in both assays is O O

based on functional groups such as carbonyl and hydroxyl

(Blainski, Lopes, & de Mello, 2013) which are usually present in OH O
the flavonoids skeleton (Liu & Guo, 2015; Pontis, da Costa, da
Al+
Silva, & Flach, 2014). The values are based on the formation of a OH O
complex between Al(III) and carbonyl and hydroxyl groups that
are usually present in flavonoids skeleton. However, compounds OH OH
 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632 627

Fig. 3. (a) LC profiles of RCM, RCS and RCP at k 280 nm (b) comparison of intensity strengths for peak d from rachis extracts.

Hence, the TFC value could instead represent the content of
phenolic acids instead of the flavonoids. The good correlation
between the TFC value and antioxidant activity in this study indi-
cated that this type of compound could be responsible for the
strong antioxidant activity observed (Dian et al., 2015) although
the value ordinarily gives an estimation of flavonoid content.
3.5. Profiling
In this study, LCMS was used in the negative mode for profiling
constituents since it has illustrated better resolution compared to
LCMS run in the positive mode. As reported by previous studies
(Mena et al., 2012; Plazonic et al., 2009), LCMS run in the negative
mode was more sensitive for profiling phenolic and polyphenolic
group of constituents, and this due to its capability to form nega-
tive ions readily especially in ESI (Hua & Jenke, 2012).
3.5.1. Rind extracts
Fig. 1 (a) and (b) shows the total ion chromatogram (TIC) along
with the LC chromatograms at k 254, 280 and 310 nm of RDM and
RCM, respectively. Since phenolics mostly absorb at k 280 nm
(Zhang et al., 2013), we shall base our following discussion for
RDM, RCM and other extracts on the LC profile at this wavelength
rather than on the TIC profiles. As shown in Fig. 2 (a), the LC profile
of RDM, RDP and RDS at 280 nm shows the presence of phenolic
constituents in the extracts with some notable differences. RDM
628 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632

Table 2
MS data analysis for peaks a, b, c, d and e in the chromatogram of rind and rachis crude extracts of AhJ33 fruit waste.

Peak a
Molecular Formula m/z (Observed) m/z (Calculated) Diff (ppm) % Score Structure and Name
C6 H12 O7 195.0508 195.051 1.15 99.75

Gluconic acid
C18 H18O9 377.0843 377.0878 9.30 86.59

Vaccihein A
C25 H30 O13 537.1654 537.1614 7.50 77.29

Grandifloroside
C24 H44 O22 683.2238 683.2228 1.97 97.53

Stachyose hydrate
Peak b
Molecular Formula m/z (Observed) m/z (Calculated) Diff (ppm) % Score Structure and Name
C7 H12 O6 191.0567 191.0561 3.06 98.29

Quinic acid
Peak c
Molecular Formula m/z (Observed) m/z (Calculated) Diff (ppm) % Score Structure and Name
C 5 H 4 O3 111.0091 111.0088 2.97 99.24

Pyromeconic acid
C 6 H 4 O5 154.9985 154.9986 0.62 99.95

Comenic acid
 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632
Table 2 (continued)
Peak a
Molecular Formula m/z (Observed) m/z (Calculated)
C7 H12 O6 191.0559 191.0561
Peak d
Molecular Formula m/z (Observed) m/z (Calculated)
C 7 H 6 O4 153.0191 153.0193
Peak e
Molecular Formula m/z (Observed) m/z (Calculated)
C7 H12 O6 191.0559 191.0561
C16 H18 O9 353.0871 353.0878
showed five peaks while RDP and RDS showed two peaks only. The
two peaks labeled as d and e represent the common constituents
present in the extracts. In RDM, three additional peaks (a, b and
c) were also observed in the range 4.57.0 min. Fig. 2 (b) compares
the intensity strength of the major peaks d and e for RDM, RDP and
RDS extracts. For all extracts, peak d displayed higher intensity
than peak e and in RDS, peak e is hardly seen. For both peaks,
RDM showed the highest intensity compared to RDP and RDS. In
addition, for peak d, the intensity strength of RDM is double that
of RDP and RDS. However, there is only a slight difference in the
intensity strength between RDP and RDS for both peaks. The differ-
ence in the intensity strength of isolated peaks d and e could justify
the order of the antioxidant activities observed for the three rind
extracts, i.e. RDM > RDP > RDS. The lowest antioxidant activity for
RDS may be due to the degradation of the active constituents rep-
resented by peaks d and e. The low intensity strength of peaks d
and e for RDS compared to RDM and RDP (besides the absence of
peaks a, b and c), indicated that some constituents may be
degraded to a certain extent during the extraction period due to
high temperatures. These findings are supported by the work of
Sarker et al. (2006). This is also observed in the LC profile and
intensity strength of peaks a, b and c although it could not be ascer-
tained as to whether these constituents contributed significantly to
the antioxidant activities of the extracts.
3.5.2. Rachis extracts
Fig. 3 (a) shows the LC profile of RCM extract produced from the
maceration technique at k 280 nm. Unlike in RDM, only one major
phenolic compound could be observed in RCM. The absence of
629
Diff (ppm) % Score Structure and Name
1.10 99.78
Quinic acid
Diff (ppm) % Score Structure and Name
1.51 99.69
Protocatechuic acid
Diff (ppm) % Score Structure and Name
1.10 99.78
Quinic acid
1.99 98.53
Chlorogenic acid
other phenolic compounds could justify the significantly lower
antioxidant activity observed for RCM as compared to RDM.
Fig. 3 (b) shows the comparison of intensity strength for peak d
which is in the order of RCM > RCP  RCS extracts. Thus justify
the order of antioxidant activity observed for the extracts:
RCM > RCP  RCS.
3.6. Identification of phenolic constituents by mass spectrometry
In mass spectrometry, the identity of compounds is usually based
on m/z values of molecular ion peaks and other fragment ions includ-
ing the base peak which is the most intense ion peak in the mass
spectrum (Banerjee & Mazumdar, 2012). However, in the absence
of standards and due to unavailable database, it is not easy to iden-
tify compounds based on their m/z values alone. Furthermore, in cer-
tain cases, the molecular ion may be unstable or can fragment into
very stable species and hence may not be observed at all. Since the
base peak represents the most stable ion, it is useful for identifying
compounds (Khan, Kennedy, & Christian, 2012).
Identification of compounds represented by peaks a-e were car-
ried out for RDM, RDP, RDS, RCM, RCP and RCS extracts. Fig. 4
showed the ESI-MS for peaks a, b, c, d and e. The m/z values and
the corresponding molecular formulas for major peaks a-e of the
different extracts are tabulated in Table 2. These values were sub-
jected to data mining via online database as described in 2.9 and
possible molecular formulas and corresponding error/accuracy
are also tabulated along with tentative structures and names
(Table 2). The acceptable accuracy used in this study is 7 ppm.
630 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632

For peak a, three fragment ion peaks with relatively high inten-
sities at m/z 683, 537, 377 were observed. Data mining identified
the former to be stachyose hydrate (error 1.97 ppm). Possible
structures associated with m/z values of 537 and 377 are shown
in Table 2. However, an error of 7.5 and 9.3 ppm, respectively
may indicate an inaccurate identification of the compound. The
existence of peaks at m/z 537 and 377 may be due to the fragmen-
tation of stachyose hydrate (m/z 683) but could not be positively
identified due to lack of available database. The base peak at m/z
195 was positively identified as gluconic acid which is a common
product of the conversion of carbohydarates. For peak b, the only
intense peak is the base peak at m/z 191 (error of 3.06 ppm) which
is identified as quinic acid. Other peaks which may represent the
molecular ion peak are present in low intensities. Interestingly,
data mining on these peaks yielded possible structures (as shown
in Table 2) with error of less than 7 ppm. For peak c, the base peak
at m/z 191 also suggests it to be a quinic acid derivative. In addi-
tion, the presence of peaks at m/z 154 and 111 in the mass spec-
trum for peak c may indicate rearrangement of quinic acid to
comenic acid and pyromeconic acid.
Peaks d and e were two isolated peaks observed at acquisition
time of 911 min. Peak e is barely seen for the rachis extracts. Peak
d is identified as protocatechuic acid from the fragment ion at m/z
153. This is supported by the base peak at m/z 109

Fig. 4. ESI-MS for peaks a, b, c, d and e.

 M.N.H. Daud et al. / Food Chemistry 232 (2017) 621632
(3,4-hydroxyphenol) which resulted from the loss of the carboxyl
moiety.
The mass spectrum of peak e showed two distinct peaks with m/
z 191 and m/z 353. Data mining found the identity to be quinic acid
and chlorogenic acid, respectively. The loss of the dihydroxycin-
namic acid moiety from chlorogenic acid possibly resulted in qui-
nic acid as its base peak at m/z 191.
Peaks a, b and c which were identified as gluconic acid, quinic
acid and a quinic acid derivative was only seen in RDM. Quinic acid
and its derivative are widely distributed in many plant parts, espe-
cially fruits and it exists naturally in form of a cyclic polyol and
usually forms phenolic acid derivatives (Herrmann, 1995). As
reported by previous studies, quinic acid plays a potent role as
an antioxidant agent (Bhandari & Kawabata, 2004). The presence
of a carboxylic group and a hydroxyl group in quinic acid main
chemical structure could promote antioxidant ability (Bendary,
Francis, Ali, Sarwat, & El Hady, 2013). The presence of the quinic
acid moiety could justify the highest antioxidant activity recorded
for RDM as compared to RDP and RDS.
For the rachis extracts, only RCM shows the presence of peak d
in high intensity while peak e was found to be in low intensities in
all three rachis extracts. This indicates the low amount of protocat-
echuic acid (peak d) and chlorogenic acid (peak e) in RCP and RCS
which could explain their relatively lower antioxidant activity. Pro-
tocatechuic acid or 3,4-dihydroxybenzoic acid are natural phenolic
acids that are widely distributed in more than 500 edible plants
especially in fruits and nuts (Kakkar & Bais, 2014). This compound
is known as an active natural antioxidant agent that scavenges free
radical with an IC50 of 1.88 lg/ml, as reported by Li, Wang, Chen,
and Chen (2011). Similarly, chlorogenic acid, also a phenolic acid
could directly interact with reactive oxygen species and demon-
strated effective radical scavenging properties (Zang, Cosma,
Gardner, Castranova, & Vallyathan, 2003). Application of chloro-
genic acid as a water-soluble antioxidant due to its hydrophilic
side groups, such as OH or COOH function has been distributed
in medicine, food processing and cosmetic chemical industry
(Xiang & Ning, 2008). These reports further justify that the lower
antioxidant activity observed for rachis crude extracts compared
to the rind crude extracts could be due to low intensities of proto-
catechuic acid and chlorogenic acid derivatives in both RDP and
RDS extracts.
4. Conclusions
This study revealed that for both rind and rachis, crude
extracts prepared via percolation (RDP and RCP) showed the
highest percent yield of extracted substances compared to other
extracts. However, the rind and rachis extracts prepared via
maceration (RDM and RCM) showed the highest antioxidant
activities compared to other rind and rachis crude extracts. In
the TPC and TFC assays, RDM and RCM showed the highest
TPC and TFC values for both rind and rachis extracts, indicating
a strong correlation with antioxidant activities, which are sup-
ported by high correlation coefficient values. TOF LCMS analyses
suggested that protocatechuic acid and chlorogenic acid deriva-
tives could be the two major constituents responsible for the
observed antioxidant activities of RDM and RCM extracts. Fur-
thermore, the presence of gluconic acid, quinic acid and its
derivative could also contribute to the antioxidant activity of
RDM. Based on these findings, it can be concluded that macera-
tion is the best extraction method for extracting antioxidant con-
stituents of AhJ33 variety fruit waste compared to percolation
and Soxhlet methods. Hence, maceration would be the recom-
mended technique in the utilization and conversion of the
AhJ33 variety fruit waste to a useful economic food product.
631
Acknowledgements
The authors would like to thank the Faculty of Pharmacy, UiTM
for permission to use the TOF LC/MS instrument. MNHD would like
to acknowledge MARDI for PhD scholarship award and for facilitat-
ing the research project (MOSTI-060308-SF0411).
The authors declare no conflict of interest.
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