Food Chemistry 127 (2011) 1186–1192

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Food Chemistry
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Flavonoids and phenolic acids from Labisia pumila (Kacip Fatimah)

Lee Suan Chua a,⇑, Norliza Abdul Latiff a, Sze Yean Lee a, Chew Tin Lee b, Mohamad Roji Sarmidi a,
Ramlan Abdul Aziz a
a
Metabolites Profiling Laboratory, Chemical Engineering Pilot Plant, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia
b
Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia

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

Article history: Both total phenolic content (TPC) and total flavonoid content (TFC) of Labisia pumila extracts were deter-
Received 27 October 2010 mined spectrophotometrically. L. pumila leaves extracted in 60% methanol (MeOH) were fractionated on
Received in revised form 6 January 2011 C18 cartridge and the antioxidant property of each fraction was determined by measuring free radical
Accepted 26 January 2011
1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity. The 40% MeOH fraction exhibited the highest
Available online 1 February 2011
scavenging activity. Nine flavonols (quercetin, myricetin and kaempferol), two flavanols (catechin and
epigallocatechin) and nine phenolic acids were identified from this active fraction by UPLC–ESI-MS/
Keywords:
MS, and confirmed by comparison with the mass spectra of standard aglycones, theoretical fragments
Labisia pumila
Flavonols
generated from MS Fragmenter software, and literature values.
Flavanols Ó 2011 Elsevier Ltd. All rights reserved.
Phenolic acids
UPLC–ESI-MS/MS

1. Introduction Labisia pumila (Myrsinaceae), also called Kacip Fatimah, is an

Flavonoids are polyphenolic compounds that contain a C15 fla-
vone skeleton (diphenylpropane) and are collectively known as
vitamin P. They consist of flavones, flavonols, flavanols, flavanone
and flavanonols, and represent the majority of plant secondary
metabolites. Harborne (1993) reported that more than 2000 flavo-
noids have been identified and the number continues to grow. The
chemistry and physiological action of polyphenolics are clearly de-
scribed by Haslam (1998). Flavonoids are thought to play a role in
protection of plants from microbial and insect attack. Moreover,
flavonoids have remarkable health promoting effects, such as
anti-inflammatory (Yamamoto & Gaynor, 2001), anti-microbial
(Tim Cushnie & Lamb, 2005), antioxidant (Shahidi & Wanasundara,
1992), anti-cancer (Wei, Tye, Bresnick, & Birt, 1990) activity as well
as the prevention of osteoporosis (Migliaccio & Anderson, 2003).
Apart from polyphenols, phenolic acids, including benzoic acids
and cinnamic acids, constitute another major group of plant sec-
ondary metabolites. They are frequently associated with sugar
moieties or occur as esters that further complicate the phenolic
profiles of plants (Inbaraj, Lu, Kao, & Chen, 2010). Nowadays, phe-
nolic acids receive considerable attention because of their protec-
tive role against cancer and heart disease. This role may be
attributed to their antioxidant activity which was reported to be
higher than vitamins C and E against reactive oxygen species (Tsao
& Deng, 2004).
⇑ Corresponding author. Tel.: +60 7 5532595; fax: +60 7 5569706.
E-mail address: chualeesuan@yahoo.com (L.S. Chua).
herb that has been widely applied by decoction in South East Asian
communities for a variety of illnesses and also used as health sup-
plements. Both phenolic acids and flavonoids are believed to be
responsible for the wide spectrum of pharmacological activities
attributed to the herb (Zhang & Ye, 2008). However, positive re-
sults in pharmacological studies are not enough to provide sub-
stantial physiological explanation. This is mainly due to the lack
of information on the types of flavonoids and phenolic acids pres-
ent in the herb. The antioxidant activity of the aqueous L. pumila
extract has been reported as providing significant protection to hu-
man dermal fibroblasts, from cell damage caused by UV irradiation
(Choi et al., 2010), most likely due to the presence of flavonoids
(Norhaiza, Maziah, & Hakiman, 2009). To the best of our knowl-
edge, there is no literature report regarding the determination of
flavonoids and phenolic acids from L. pumila extract.
In the present study, ultra-performance liquid chromatography
coupled with electrospray ionisation tandem mass spectrometry
(UPLC–ESI-MS/MS) was used for the determination of flavonoids
and phenolic acids from L. pumila extracts. UPLC provides im-
proved separation in a shorter analysis time without compromis-
ing peak capacity and sensitivity (Liu et al., 2007; Yang, Guan, &
Li, 2007). The ESI technique provides information on the structure
of aglycones and glycosides of flavonoids without time-consuming
pre-purification or derivatisation steps (Hakkinen & Auriola, 1998).
Extracts of plant origin are generally very complicated and con-
tain a considerably large number of peaks. The comparison of such
complex data is practically impossible with traditional visual
methods. Principle component analysis (PCA) is an unsupervised

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.01.122
 L.S. Chua et al. / Food Chemistry 127 (2011) 1186–1192
clustering method which requires little prior knowledge of the
data set and offers a unique possibility for the elucidation of sim-
ilarities and dissimilarities to facilitate sample classification. It acts
to reduce the dimensionality of multivariate data without losing
important information (Eriksson, Johansson, Kettaneh-Wold, &
Wold, 2001; Kemsley, 1996). When coupled with discriminant
analysis (PCA-DA), it becomes a supervised technique to further
compress high dimensional data by maximising the ratio of be-
tween-class variance and minimising the ratio of within-class var-
iance (Tozer, Davies, Altmann, Miller, & Tofts, 2006).
2. Experimental
2.1. Chemicals
Formic acid, sodium carbonate and aluminium chloride were
obtained from Fisher Scientific (Pittsburgh, PA). HPLC-grade meth-
anol (MeOH) and acetonitrile (CH3CN) were obtained from Merck
(Darmstadt, Germany). 18.2 MX-cm water was produced from a
Barnstead NANOpure Diamond water purification system (Thermo,
Waltham, MA). The standards quercetin (P98%), myricetin (>96%),
kaempferol (P90%) and catechin (P98%), as well as Folin–Ciocal-
teau reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ascorbic
acid, were sourced from Sigma–Aldrich (St. Louis, MO). The stan-
dards fumaric acid and succinic acid were obtained from Supelco
Analytical (Bellefonte, PA); gallic acid (98%) and rutin (97%) were
obtained from Acros Organics (Pittsburgh, PA). Strata C18-E SPE
with silica-based sorbents (100 mg) was supplied by Phenomenex
(Torrance, CA).
2.2. Plant material
The plant material, L. pumila (FRI 59810) was procured from
Forest Research Institute Malaysia (FRIM). The leaves of the plant
were dried in oven at 40 °C and finely ground using a blender.
The samples were stored at 20 °C until further analysis.
2.3. Total phenolic content
The total phenolic content (TPC) of the leaves extracted with
100% MeOH, 100% water, 60% MeOH and 100% CH3CN was analysed
using Folin–Ciocalteu reagent, based on the method described by
Kumaran and Karunakaran (2007), with some modification. Sam-
ples (100 ll) were mixed with 500 ll of Folin–Ciocalteu reagent
and 1.5 ml of 20% sodium carbonate. The mixture was shaken thor-
oughly and made up to 10 ml with distiled water. The mixture was
incubated statically for 2 h at 30 °C, before the absorbance was
measured at 765 nm using a UV–Vis spectrophotometer (Perkin–
Elmer Lambda 25, Waltham, MA). Gallic acid (0–1000 mg/l) was
used as a standard for calibration curve preparation. The TPC was
expressed as lg of gallic acid equivalent (GAE) in mg of dry weight
plant extract. All assays were carried out in triplicate.
2.4. Total flavonoid content
The total flavonoid content (TFC) of the leaves extracted with
100% MeOH, 100% water, 60% MeOH and 100% CH3CN was deter-
mined based on the method of Djeridane et al. (2006), with some
modification. Sample (1 ml) was mixed with 1 ml methanolic solu-
tion containing 2% aluminium chloride. A flavonoid-aluminium
complex was formed after 15 min incubation at 30 °C. The forma-
tion of the complex was measured at 430 nm by using UV–Vis
spectrophotometer. Rutin (0–100 mg/l) was used as a standard
for calibration curve preparation. The TFC was expressed as lg of
rutin equivalent (RE) in mg of dry weight plant extract.
1187
2.5. Sample preparation for the 60% MeOH extract and its fractions
The dried and finely powdered leaves of L. pumila (5 g) were ex-
tracted with hexane (20 ml) to remove fatty substances and soni-
cated at 30 °C. The samples were filtered and air dried before
suspension in 60% MeOH (100 ml) and sonication. The methanolic
supernatant was then filtered and evaporated to dryness in vacuo.
The extract (1.69 g) was dissolved in MeOH (170 ml), loaded onto a
preconditioned SPE column and eluted with 40%, 60%, 80% and
100% MeOH. The four fractions were dried in oven overnight at
40 °C, to give 210.2, 332.2, 128.8 and 291.5 mg of sample,
respectively.
2.6. Free radical-scavenging activity of the 60% MeOH extract and its
fractions
The free radical-scavenging activity of L. pumila extracts was
determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.
This method measured the reduction of purple DPPH to yellow-
coloured 2,2-diphenyl-1-picrylhydrazine (DPPH–H). The remain-
ing DPPH showed maximum absorption at 517 nm. The DPPH
solution (40 ppm) was prepared by dissolving 4 mg of DPPH in
95% ethanol (100 ml). The preparation was carried out with mini-
mal exposure to light. The solution was vortexed and stored in a
dark place. Samples (2 ml) with different concentrations, ranging
from 100 to 5000 ppm, were added to 2 ml of DPPH solution
(0.1 mM). The absorbance was measured at 517 nm after 30 min
of incubation at 30 °C. Ascorbic acid was used as positive control.
The ability to scavenge the DPPH was calculated using Eq. (1),
where Acontrol and Asample are the absorbance of control and sample.
The concentration of sample required to scavenge 50% of DPPH
was determined. The experiment was performed in triplicate.
Acontrol  Asample
DPPH  scavenging activity ð%Þ ¼  100 ð1Þ
Acontrol
2.7. UPLC–ESI-MS/MS
The analytical UPLC, Waters Acquity (Milford, MA) system was
coupled with a triple quadrupole-linear ion trap tandem mass
spectrometer (Applied Biosystems 4000 Q TRAP; Life Technologies
Corporation, Carlsbad, CA) with an electrospray ionisation (ESI)
source. A C18 reserved phase Acquity column (150  4.6 mm,
1.7 lm) protected by a guard column was used throughout this
study.
The mobile phase was a binary solvent system consisting of sol-
vent A (water with 0.1% formic acid) and solvent B (CH3CN). The
UPLC gradient was: 0–5 min, 10% B; 5–15 min, 10–90% B; 15–
20 min, 90% B; 20–25 min, 90–10% B; 25–30 min, 10% B for final
washing and equilibration of the column for the next run. The flow
rate was 0.25 ml/min and the injection volume was 5 ll. All sam-
ples were filtered with 0.2-lm nylon membrane filter prior to
injection.
The mass spectra were acquired from m/z 100–1500 with a 20-
ms ion accumulation time. All mass spectrometric data were ac-
quired in negative ionisation mode. Positive ion mode was also
done when necessary, in order to compare with the literature data.
The capillary and voltage of the ESI source were maintained at
400 °C and 4.5 kV, respectively. All other parameters were as fol-
lows: nitrogen was used as ion source gas for nebulisation, 40 psi;
for drying solvent, 40 psi; curtain gas, 10 psi; collision gas, high;
declustering potential, 40 V, and collision exit energy, 10 V.
The scan rate was 1000 amu/s. Data acquisition and data process-
ing were performed using Analyst 1.4.2.
The scan mode of enhanced mass spectra (EMS) was used to
screen the sample profile. Enhanced product ion (EPI) scan was
1188 L.S. Chua et al. / Food Chemistry 127 (2011) 1186–1192
used to determine the characteristic ions and to confirm the pres-
ence of aglycone peaks. Three parallel EPI runs with different col-
lision energies ranging from 10 to 50 V were carried out, to
obtain the most information-rich fragmentation pattern. The pre-
cursor ion (PI) scan was used to identify the parent ion of the agly-
cone and hence to determine the molecular weight of the unknown
compounds. Low energy collision-induced dissociation (CID) was
used to confirm the aglycone peak in multiple reactions monitor-
ing (MRM) mode.
2.8. Data processing and interpretation
The data processing software, MarkerView 1.1 (Applied Biosys-
tems/MDS Sciex) was used to perform sample classification by car-
rying out principle component analysis (PCA) and principle
component analysis-discriminant analysis (PCA-DA).
MS Fragmenter 12.0 (Advanced Chemistry Development, Toron-
to, Canada) was used to predict compound fragmentation in both
positive and negative ionisation modes.
3. Results and discussion
3.1. Determination of TPC and TFC
The 100% MeOH and 60% MeOH crude extracts of L. pumila
leaves showed the highest TPC, 468 and 427 lg GAE/mg extract,
respectively (Table 1). The small difference in TPC between these
two extracts can be explained by the similar polarity of the extrac-
tion solvents. These values are measured in gallic acid equivalence
not only because of the acid stability, but also due to its response to
Folin–Ciocalteu reagent, which has been shown to be equivalent to
most other phenolic compounds. The CH3CN extract showed the
highest TFC (71.8 lg RE/mg extract), followed by the 100% MeOH,
aqueous and 60% MeOH extracts (Table 1). Overall, the TFC values
of all extracts were lower than the TPC results. The ratios of TFC/
TPC for the four extracts are also presented in Table 1. The 60%
MeOH extract might contain about 10% of flavonoids from the total
phenolic compounds.
The 60% MeOH crude extract of the leaves was then fractionated
on a C18 SPE column. The TPC of each fraction was found to de-
crease from 266 to 165, 164 and 65.4 lg GAE/mg extract as the
MeOH content increased from 40 to 60, 80 and 100%. The 40%
MeOH fraction contained the most polar phenols and strongly
inhibited the oxidation of the Folin–Ciocalteu reagent.
3.2. Determination of scavenging activity
The scavenging activity of the 60% MeOH extract and its frac-
tions (40–100% MeOH) was determined using the DPPH assay.
The 40% MeOH fraction was found to have an SC50 of 1060 ppm
and accounted for the activity observed in the 60% MeOH extract
(Table 2). The scavenging activities of the extracts and fractions
were also compared to several standards known for their antioxi-
Table 1
Ratio of total flavonoid content (TFC) to total phenol content (TPC) in rutin
equivalents (RE) and gallic acid equivalents (GAE).
Solvent TPC ± SE* TFC ± SE* Ratio of TFC/TPC
(lg GAE/mg (lg RE/mg (lg RE/lg GAE)
extract) extract)
100% methanol 468 ± 57.5 25.2 ± 5.06 0.05
100% water 274 ± 14.3 6.33 ± 0.25 0.02
100% acetonitrile 103 ± 22.4 71.8 ± 6.81 0.70
60% methanol 427 ± 25.0 5.08 ± 2.57 0.01
*
SE, standard error.
Table 2
DPPH scavenging activity of L. pumila samples and standards.
Sample Concentration at SC50* (ppm)
60% MeOH extract 1058
40% MeOH fraction 1062
60% MeOH fraction 1743
80% MeOH fraction 1386
100% MeOH fraction 18,060
Standards
Quercetin 145.6
Myricetin 197.1
Catechin 227.8
Rutin 296.4
Ascorbic acid 298.5
*
SC50, scavenging activity at 50%.
dant properties. The results showed that the free radical-scaveng-
ing activity of the most active fraction (40% MeOH fraction) was
about seven times lower than quercetin, five times lower than
myricetin, four times lower than catechin, and three times lower
than rutin and ascorbic acid (Table 2). The essential requirement
for effective radical scavenging activity is the 30 ,40 -orthodihydroxy
configuration in ring B and a carbonyl group at C4 in ring C, impart-
ing better electron donating properties (Fig. 1, Mustafa, Abdul Ha-
mid, Mohamed, Abu Bakar, 2010). The presence of a C2–C3 double
bond conjugated to the C4 carbonyl in flavonols is responsible for
electron delocalisation from ring B, which would indirectly in-
crease the radical-scavenging activity. The antioxidant activity of
flavonoids also depends on the number and position of the hydro-
xyl groups. Therefore, the antioxidant properties, via the mecha-
nism of DPPH scavenging activity of the flavonols (quercetin and
myricetin) were higher than that of the flavanols (catechin).
Although rutin belongs to the flavonol group, glycosylation of its
aglycone reduces its scavenging ability making its value compara-
ble to ascorbic acid.
3.3. Determination of flavonols and flavanols in the 40% MeOH fraction
Nine flavonol and two flavanol derivatives were identified from
the 40% MeOH fraction. It is important to note that this paper does
not give a complete identification of phenolic compounds present
in this plant. These compounds were elucidated based on the mass
spectral analysis from various functional scans, namely EPI, PI, NL
and MRM. The chemical structure of the phytochemicals was con-
firmed by comparison with standard aglycones (quercetin, myrice-
tin, kaempferol and catechin) and with literature values. Their
retention times and product ions from the negative ion mode EPI
scan are listed in Table 3.
Four quercetin glycosides were detected based on the fragment
ion m/z 301, which is the [MH] of the standard quercetin. The
fragmentation pattern of these four compounds concurred with
the findings of Van Elswijk, Schobel, Lansky, Irth, and van der Greef
(2004) and Hollecker et al. (2009). The positive mode mass spectral
data also contained the fragment ions m/z 303[M+H]+, 285(–H2O),
257(–CO), 229(–CO), 211, 165, 153 and 137, which were reported
by Hakkinen and Auriola (1998), and Dehkharghanian, Adenier,
and Vijayalakshmi (2010).
In subsequent analysis, PI scan was carried out to determine the
parent ions of these quercetin compounds. The results showed that
the quercetin derivatives have parent ions of m/z 600, 609, 615 and
761. The EPI scan of m/z 600 produced fragment ions such as m/z
448, 301, 179 and 151. The loss of 152 from m/z 600 to 448 and
146 Da from m/z 447 to 301 is most likely due to the breakdown
of pentose and deoxyhexose, respectively. It is also worth noting
that flavonoids are naturally present in various forms as a result
of hydroxylation, methylation and, most commonly, glycosylation
 L.S. Chua et al. / Food Chemistry 127 (2011) 1186–1192 1189

1,2 R1
A
1,3 3' R1
A 4' OH
2' 3'
4' OH
8 1'
B 2'
O 7H O 2 B
1 5' 8
R2 O 1'
6' 7H O 2
A 4 3
1 5' R2
6 A 4
C 3
6'
5 OH 1,2
B 6
5 OH
OH O
1,3
B OH 2,4
C
0,4
B

Fla v onols R1 R2 Fla v anols R1 R2

Quercetin OH H C atechin OH H
Myricetin OH OH E p ig allo catechin OH OH
Kaempferol H H

O OH

R 4

R 1 R 3
R 2

B e n z o ic a c id R 1 R 2 R 3 R 4

Gallic acid OH OH OH H
Protoc ate c hin a cid H OH OH H
Vanillic a cid OCH3 OH H H
Syringic a cid OCH3 OH OCH3 H
Salicylic a cid H H H OH

O O R 4

R 1 R 3

R 2

C in n a m ic a c id R 1 R 2 R 3 R 4

Coumaric a cid H OH H H
Caffeic a cid H OH OH H
Chlorogenic acid H OH OH quinic a cid

Fig. 1. Chemical structures of flavonols, flavanols, benzoic acids and cinnamic acids detected in the 40% MeOH fraction from the 60% MeOH extract of L. pumila.

processes in plants (Abad Garcia, Berrueta, Garmon-Lobato, Gallo, For m/z 615, fragment ions at m/z 463 and 301 were detected.
Vicente, 2009). Flavonoids usually exist as O-glycosides, in which The loss of 152 Da from m/z 615 to 463 might be attributed to
one or two hydroxyl groups of the aglycone are bound to a sugar the loss of a pentose sugar. The NL scan confirmed that 152 Da
moiety with the formation of an acid-labile glycosidic oxygen–car- was a fragment of the molecular ion, m/z 615. The loss of 162 Da
bon bond (Abad Garcia et al., 2009). The molecular ion m/z 609 pro- from m/z 463 to 301 might be attributed to the loss of a hexose.
duced characteristic fragment ions at m/z 463 and 301. The loss of A fourth compound, m/z 761 was identified as a quercetin trisac-
146 Da from m/z 609 to 463, is probably due to the loss of deoxy- charide. The fragmentation pattern from its EPI scan produced sig-
hexose or rhamnose, whereas the loss of 162 Da from m/z 463 to nificant peaks at m/z 761, 615, 463 and 301. This glycoside had
301 may be due to the loss of galactose, glucose or mannose. Liter- similar fragmentation pattern to that of m/z 615 with the presence
ature studies report that most plant extracts contain quercetin 3- of an additional unit of sugar.
O-rutinoside (rutin) and that the loss of disaccharide from rutin Four myricetin glycosides were also detected in the 40% MeOH
is either rutinose (rhamnosyl-(a1 ? 1)-glucose) or neohesperidose fraction from the negative mode PI scan, m/z 463, 479, 625 and
(rhamnosyl-(a1 ? 2)-glucose) (Abad Garcia et al., 2009). Abad 778. The mass spectra of their aglycones matched well with the
Garcia et al. (2009) also reported that the 3-hydroxyl and 7-hydro- standard myricetin in both negative (m/z 317) and positive (m/z
xyl groups in flavonols are the most common glycosylation sites. 319) ion mode. The fragmentation of the myricetin moiety at the
1190 L.S. Chua et al. / Food Chemistry 127 (2011) 1186–1192
Table 3
Flavonoids from the 40% MeOH fraction of the 60% MeOH extract of L. pumila as determined by negative ion mode EPI scan.
Flavonoid, (CF, MW)* Retention time (min) Collision energy (V)
Flavonols
Quercetin 7.62 30
(C15H10O7, 302) 10.32 40
9.53 40
10.11 45
Myricetin, 9.89 40
(C15H10O8, 318) 10.90 40
4.72 40
3.71 55
Kaempferol, 5.53 50
(C15H10O6, 286) 8.00 50
Flavanols
Catechin, 3.65 30
(C15H14O6, 290)
Epigallocatechin 6.25 20
(C15H14O7, 306)
*
(CF, MW). Chemical formula and molecular weight.
O1–C2 and C3–C4 bonds of ring A (1,3A) produced m/z 151 in the
negative ion mode and m/z 153 in the positive ion mode (Fig. 1;
Dehkharghanian et al., 2010). The detection of m/z 179 was due
to the fragmentation at the O1–C2 and C2–C3 bonds of ring A
(1,2A). The molecular ion m/z 463 is most likely formed by the addi-
tion of a monosaccharide (146 Da), either deoxyhexose or rham-
nose at either the 3- or 7-hydroxyl positions (Abad Garcia et al.,
2009). The monosaccharide attached to the myricetin derivative,
m/z 479 was a hexose, based on the loss of 162 Da. The loss of
18, from m/z 479 to 461 suggests a molecule of water was lost. It
is important to note that the process of deprotonation would pref-
erentially take place at the most acidic site, the 40 -hydroxyl group,
followed by either the 30 - or 50 -hydroxyl groups. Myricetin has the
highest antioxidant potential among the flavonoids, because of the
presence of three hydroxyl groups on the B ring (Hugo, Leal,
Fernandez, Lopes, Cordeiro, 2004). However, the antioxidant prop-
erty as determined by DPPH-scavenging activity of myricetin was
found to be slightly lower than quercetin in this study (Table 2).
The third compound from the myricetin group, m/z 625 was iden-
tified as a disaccharide. This compound produced significant frag-
ment ions at m/z 463 and 317 in the negative ion EPI scan. The
sugar moieties were determined to be deoxyhexose and hexose,
due to the loss of 146 and 162 Da, respectively. Based on peak
intensity from the MRM, m/z 778 was found to be present in the
highest concentration among the detected myricetin derivatives.
The characteristic ions of m/z 778, 632, 463 and 317 showed that
this compound was bound to three additional moieties with the
loss of 146, 168 and 146 Da from its molecular ion.
This study also detected two kaempferol derivatives in the 40%
MeOH fraction. The first compound, m/z 593 was characterised as
kaempferol rhamnoglycoside. The positive PI scan produced m/z
153 as a diagnostic ion (Dehkharghanian et al., 2010). The negative
PI scan produced m/z 117 as a fragment ion and was used to differ-
entiate between kaempferol and luteolin (isomer of kaempferol)
(Dehkharghanian et al., 2010). Furthermore, this molecular ion
m/z 593 was found to match well to phytooestrogenic compounds
reported by Van Elswijk et al. (2004). A characteristic neutral loss
of 308 Da, from m/z 593 to 285, correlated to the loss of a rhamno-
glycosidic moiety (Van Elswijk et al., 2004). A second kaempferol
derivative at m/z 583 was found based on detection of the m/z
285 fragment ion.
In the 40% MeOH fraction two flavanol compounds, catechin
(m/z 289) and epigallocatechin (m/z 305), were detected in the neg-
ative ion mode EPI scan. Benavides, Montoro, Bassarello, Piacente,
and Pizza (2006) reported that catechin produced m/z 139 as a
characteristic ion in the positive ion mode with the loss of
152 Da in a retro Diels–Alder reaction. This was also observed in
Molecular ion [M](m/z) Product ion (m/z)
600 447(–pentose)/301(–deoxyhexose)/179/151
609 463(–deoxyhexose)/301(–hexose)/271/255/179/151
615 463(–pentose)/301(–hexose)/271/255/179/151
761 615(–deoxyhexose)/609/301/271/255/179/151
463 317(–deoxyhexose)/271/179/151
479 461/317(–hexose)/271/179/151
625 463(–hexose)/317(–deoxyhexose)/271/179/151
778 632(–deoxyhexose)/463/317(–deoxyhexose)/271/179/151
593 549/285/267/255/223/149/117
583 493/383/300/285/271/179/151/117
289 289/151/137/123
305 305/287/261/233/169/169/151
this study, where m/z 137 was found in the negative ion mode with
the neutral loss 152 Da from m/z 289 to 137. Epigallocatechin, with
molecular ion at m/z 305, was confirmed by detection of a gallic
acid moiety (m/z 169) in the EPI scan. The detection of m/z 261
and 233 was due to the loss of ethenol (CH2@CHOH) at 2,4C (Fig
1) and carbon monoxide (CO), respectively.
3.4. Phenolic acids from 40% MeOH fraction of L. pumila extract
In addition to the polyphenols discussed above, nine phenolic
acids and two organic acids were also identified from the 40%
MeOH fraction (Table 4); m/z 169, 315, 167, 197, 137, 163, 179,
355, 593, 115 and 117. The retention times of the compounds were
determined from the EPI scan. The phenolic acids included benzoic
acids and cinnamic acids (Fig. 1). The fragmentation patterns of the
acids were predicted using MS Fragmenter 12.0. The theoretical
fragment ions corresponded to the experimental mass spectral
data and confirmed the structures. It is worth noting that all the
acids were detected in the negative ion mode, except chlorogenic
acid. Chlorogenic acid belongs to a family of esters of quinic acid
and caffeic acid or ferulic acid. Besides chlorogenic acid, a chloro-
genic acid derivative was also detected in the negative ion mode
with a molecular ion m/z 593. The product ions m/z 353 (chloro-
genic acid), m/z 191 (quinic acid) and m/z 179 (caffeic acid) were
detected. The 240 Da difference has not been assigned.
3.5. Statistical analysis of mass spectral data
The EMS profiles of the fractions were analysed by PCA using
Pareto scaling. A total number of 12,692 m/z values were defined
using a minimum spectral peak width of 0.3 amu, a retention time
tolerance of 0.1 min and a mass tolerance of 0.1 amu. The first two
principal components (PC) represented 76.2% of the total variance
with the first PC explaining 59.5% of the total variability (Fig. 2a).
The 40% MeOH fraction (positive region) varied significantly from
the other three fractions (negative region) at the first PC. Moreover,
each fraction was separated into four clusters using PCA-DA. This
supervised discriminant analysis separated the fractions into three
discriminant groups with 33.3% of total variance for each group
(Fig. 2b). It was found that the 80% MeOH fraction of the extract
showed the highest score at the first discriminant function (posi-
tive region), but the 40% MeOH exhibited the lowest score (nega-
tive region). Therefore, the phytochemicals in the 40% MeOH
fraction were significantly different from the phytochemicals in
the 80% MeOH fraction. On the other hand, the 100% MeOH frac-
tion had the highest score at the second discriminant function (po-
sitive region). It is well separated from the other three fractions
 L.S. Chua et al. / Food Chemistry 127 (2011) 1186–1192 1191

Table 4
Phenolic acids from the 40% MeOH fraction of the 60% MeOH extract of L. pumila as determined by negative ion mode EPI scan.

Phenolic acid [MH] Fragment ions Retention time (min)

Benzoic acid
Gallic acid 169 125(–CO2) 2.20
Protocatechuic acid derivative 315 269(–CO2H2), 3.23
153(–C6H12O6),
136(–C6H12O6, –OH),
108(–C6H12O6, –CO2H)
Vanillic acid 167 152(–CH3), 4.39
123(–CO2),
108(–CO2, –CH3)
Syringic acid 197 182(–CH3), 4.50
180(–OH),
166(–OCH3),
152(–CO2H)
Salicylic acid 137 119(–H2O), 3.53
93(–CO2)
Cinnamic acids
Coumaric acid 163 146(–OH), 4.33
119(–CO2)
Caffeic acid 179 162(–OH), 17.12
135(–CO2),
134(–CO2H),
109(–C3O2H2),
91(–C3O2H2–H2O)
*
Chlorogenic acid 355 [M+H]+ 337(–H2O), 8.45
319(–2H2O),
301(–3H2O),
281(–2H2O–C3H2), 263(–3H2O–C3H2), 245(–4H2O, –C3H2)
Chlorogenic acid derivative 593 353(–240 Da), 191(quinic acid), 179(caffeic acid) 4.56
Organic acid
Fumaric acid 115 98(–OH), 71(–CO2) 2.00
Succinic acid 117 100(–OH), 73(–CO2) 2.07
*
Detected in the positive ion mode.

Fig. 2. Score plots of (a) PCA and (b) PCA-DA for the 40%, 60%, 80% and 100% MeOH fractions.

(negative region) at the second discriminant function. Both the
unsupervised (PCA) and supervised (PCA-DA) results showed that
SPE fractionation could produce different phytochemical profiles
for each fraction.
4. Conclusion
Preliminary screening of TPC of L. pumila extracts showed that
the MeOH extract contained the highest amount of phenolic phy-
tochemicals, followed by the 60% MeOH extract. Flavonoids consti-
tuted a large portion of the phytochemicals in the 60% MeOH leaf
extract. The 40% MeOH fraction was found to have the highest
DPPH scavenging activity. Nine flavonols, two flavanols and nine
phenolic acids were detected in this fraction using UPLC–ESI-MS/
MS coupled with powerful software for data processing and inter-
pretation. With the aid of PCA, the complex MS data were mined
for the similarities and differences in phytochemical composition
between the fractions. This information is essential for future stud-
ies in biomarker discovery.
1192 L.S. Chua et al. / Food Chemistry 127 (2011) 1186–1192
Acknowledgements
The authors would like to thank Mr. Siah Choon Loon, Mr. Yap
Ken Choy and Mr. Hong Kok Sing from Analisa Resources (M)
Sdn. Bhd. for their technical advice and support.
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