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journal of functional foods 12 (2015) 498508

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Determination of phenolic acids and flavonoids


in Rhinacanthus nasutus (L.) kurz by highperformance-liquid-chromatography with
photodiode-array detection and tandem
mass spectrometry
R.T. Huang a, Y.F. Lu b, B. Stephen Inbaraj a, B.H. Chen a,*
a
b

Department of Food Science, Fu Jen University, Taipei 242, Taiwan


Department of Nutrition, Fu Jen University, Taipei 242, Taiwan

A R T I C L E

I N F O

A B S T R A C T

Article history:

The variety and content of phenolic acids and flavonoids in a Chinese herb Rhinacanthus

Received 29 August 2014

nasutus were determined by developing an HPLC method coupled with photodiode-array-

Received in revised form 28

detection and tandem-mass-spectrometry. A high yield of phenolic acids and flavonoids was

November 2014

obtained using 30% ethanol-in-water and shaking in 60 C water-bath for 3 h. By employ-

Accepted 1 December 2014

ing a C18 column and gradient mobile phase of 0.1% formic acid-in-water and acetonitrile,

Available online 25 December 2014

a total of 20 phenolic acids and 3 flavonoids were separated within 45 min with detection
at 280 nm, flow rate at 0.8 mL/min and column temperature at 35 C. Internal standards va-

Keywords:

nillic acid and quercetin were used for quantitation of phenolic acids and flavonoids in

Rhinacanthus nasutus

R. nasutus, respectively, which included caffeic acid derivatives (6.40 mg/g), quercetin de-

Chinese herb

rivatives (4.43 mg/g), ferulic acid derivatives (3.20 mg/g), p-coumaric acid derivatives (1.63 mg/

Phenolic acids

g), sinapic acid hexoside (1.11 mg/g), kaempferol-3-O-rutinoside (1.02 mg/g), hydroxycinnamic

Flavonoids

acid derivatives (0.44 mg/g) and protocatechuic acid (0.39 mg/g).

HPLC-PADMS/MS

1.

Introduction

Rhinacanthus nasutus (L.) Kurz (R. nasutus), a popular Chinese


herb mainly distributed in China and Taiwan, is often consumed as tea or healthy drink on the market (Kao & Chen, 2011).
It has been well documented that the intake of R. nasutus can
protect against inflammation (Siriwatanametanon, Fiebich,
Efferth, Prieto, & Heinrich, 2010), cancer (Siripong et al., 2006)
and bacteria (Puttarak, Charoonratana, & Panichayupakaranant,
2010). For instance, the methanolic extract of R. nasutus was
shown to be effective in inhibiting growth of leukemia cell

2014 Elsevier Ltd. All rights reserved.

Hl-60 and oral cancer cell HSC-2, HSC-3 and HSC-4 (Horri et al.,
2011), as well as inflammation (Siriwatanametanon et al., 2010).
Furthermore, the liposomal naphthoquinone esters isolated
from R. nasutus were efficient in retarding tumor growth in
meth-A sarcoma-bearing BALB/c mice at a dose of 5.0 mg/kg
(Siripong et al., 2006). In a later study Puttarak et al. (2010) used
a microdilution assay to determine potent bacteriostatic activity of R. nasutus extract and reported that the minimum
inhibitory concentrations toward Streptococcus mutans, Streptococcus epidermidis, propionibacterium acnes and Staphylococcus
aureus ranged from 4 to 16 g/mL. All these biological activities are believed to be closely associated with the presence of

* Corresponding author. Department of Food Science, Fu Jen University, Taipei 242, Taiwan. Tel.: +886 2 29053626; fax: +886 2 29051215.
E-mail address: 002622@mail.fju.edu.tw (B.H. Chen).
http://dx.doi.org/10.1016/j.jff.2014.12.002
1756-4646/ 2014 Elsevier Ltd. All rights reserved.

journal of functional foods 12 (2015) 498508

various functional components such as naphthoquinones, carotenoids, chlorophylls, flavonoids and phenolic acids (Kao &
Chen, 2011; Wu, Hsu, Wu, Teng, & Wu, 1998). However, the composition of flavonoids and phenolic acids in R. nasutus remains
uncertain.
Phenolic acids are widely present in plant material as secondary metabolites in the form of free, soluble ester and
glucosides, and insoluble bound compounds. The main types
of phenolic acids in plants include hydroxybenzoic acid derivatives such as vanillic acid and gallic acid as well as
hydroxycinnamic acid derivatives such as caffeic acid and
ferulic acid (Chen, Inbaraj, & Chen, 2012). Most importantly,
phenolic acids have been demonstrated to possess vital biological activities including anti-cancer (Spilioti et al., 2014), antibacteria (Sanchez-Maldonado, Schieber, & Ganzle, 2011) and
antioxidation (John & Shahidi, 2010).
Like phenolic acids, flavonoids being a class of polyphenol
compounds, are widely distributed in plants, especially fruits,
vegetables and flowers, among others. More than 6000 flavonoids have been characterized in nature and most are present
in glycosidic form (Chen et al., 2012). From the structural point
of view, flavonoids can be divided into flavones, flavonols, flavanones, flavonols, isoflavones, anthocyanidins, dihydroflavonols
and chalcones (Cook & Samman, 1996). Numerous reports have
shown that flavonoids exhibit important physiological functions such as antioxidation (Chandrasekara & Shahidi, 2011),
anti-cancer (Tsai, Lin, & Chen, 2010), antibacterial (Orhan,
Ozcelik, Ozgen, & Ergun, 2010) and anti-inflammation (Kao, Wu,
Hong, Wu, & Chen, 2007). In addition, the antioxidant activity of flavonoids can be dependent upon the number and
position of sugar moiety, hydroxyl and methoxy groups (John
& Shahidi, 2010).
Due to the highly-polar nature of phenolic acid and flavonoid, both are often extracted with polar solvents including
hot water, methanol, ethanol, acetone, or a combination of these
solvents in different proportions (Bae, Jayaprakasha, Jifong, &
Patil, 2012; Chen et al., 2012). However, due to the formation
of large complexes between polyphenol compounds and proteins or carbohydrates in plant matrices, many impurities such
as wax, lipid and chlorophylls have to be removed during the
extraction (Naczk & Shahidi, 2004). Many recent studies have
shown a combination of ethanol and water in an appropriate
proportion to be the most efficient solvent system for simultaneous extraction of phenolic acids and flavonoids (Bae et al.,
2012; Chen et al., 2012; Qiao et al., 2011). After extraction, both
phenolic acids and flavonoids are often subjected to separation, identification and quantitation by HPLC coupled with
photodiode-array detection (PAD) and mass spectrometry (MS),
with the C-18 column and gradient mobile phase being the most
frequently used (Chen et al., 2012; Kao, Huang, Inbaraj, & Chen,
2008; Qiao et al., 2011). As most published methods suffer a
major drawback of long separation time or inadequate resolution, it is necessary to develop an improved HPLC method
for simultaneous determination of phenolic acids and flavonoids in R. nasutus.
The objectives of this study were to develop an appropriate method for simultaneous extraction, separation,
identification and quantitation of phenolics and flavonoids from
R. nasutus extract by HPLC coupled with a photodiode-array detector and a mass spectrometer.

2.

Materials and methods

2.1.

Materials

499

A total of 6 kg Rhinacanthus nasutus (L.) Kurz (R. nasutus) was


purchased from a Chinese drug store located in Wan-Hua district, Taipei City, Taiwan. Then R. nasutus samples were washed
with tap water, stemmed, freeze-dried, ground into a powder
(10 m), and placed into 10 vacuum bags separately with each
weighing about 20 g for storage at 20 C until further use.
Phenolic acid standards including protocatechuic acid (purity
97%), p-coumaric acid (purity 98%), ferulic acid (purity 99%)
and vanillic acid (purity = 97%), were all procured from Fluka
Chemical Co. (Buchs, Switzerland). Caffeic acid (purity = 98%)
and sinapic acid (purity 98%) were from Sigma-Aldrich Co. (St.
Louis, MO, USA). Flavonoid standards including quercetin-3O-rutinoside (purity 94%) and quercetin (purity = 98%) were
also from Sigma-Aldrich Co. Kaempferol-3-O-rutinoside
(purity = 98.8%) was from Chromadex Co. (Santa Ana, CA, USA).
HPLC-grade solvents methanol and acetonitrile were obtained from Merck Co. (Darmstadt, Germany), while ethanol
and formic acid were from Sigma-Aldrich Co. Deionized water
was made using a Milli-Q water purification system from
Millipore Co. (Bedford, MA, USA).

2.2.

Instrumentation

The HPLC instrument was composed of G1311A quaternary


pump, G1379A on-line degasser, G1316A column temperature controller, G1328B injector, G1315B photodiode-array
detector, and 6130 single quadrupole mass spectrometer (MS)
with multi-mode ion source (ESI and APCI), all of which were
from Agilent Technologies Co. (Palo Alto, CA, USA). Furthermore, the Accela 600 series HPLC instrument composed of
column temperature controller, quaternary pump and LTQ
Orbitrap XL mass spectrometer (MSMS) was from Thermo
Fisher Scientific Co. (San Jose, CA, USA). Three HPLC columns,
including Phenomenex Gemini C18 (250 4.6 mm ID, particle
size 5 m, Torrance, CA, USA), Vydac 201TP54 C18 (250 4.6 mm
ID, particle size 5 m, Hesperia, CA, USA), and Cosmosil 5C18AR-II (250 4.6 mm ID, particle size 5 m, Kyoto, Japan), were
used for comparison of separation efficiency of phenolic acids
and flavonoids. The Eyela N-1 rotary evaporator and A-3S
vacuum pump were from Tokyo, Japan. The low-temperature
circulation water bath (Firstek B402L) was from Taoyuan, Taiwan.
The high-speed centrifuge was from DuPont Co. (Wilmington, DL, USA). The sonicator (2210R-DTH) was from Branson
Co. (Danbury, CT, USA). The freeze-dryer (FD24) was from JinMin Co. (Taipei, Taiwan). The shaker (V-U type) was from HsianTai Co. (Taipei, Taiwan). The ELISA reader (VersaMax) was from
Molecular Devices Co. (Sunnyvale, CA, USA).

2.3.

Methods

2.3.1.

Extraction

Initially 3 solvent systems of ethanolwater in different proportions were compared with respect to extraction efficiency
of total phenolic acids and total flavonoids. In brief, 0.5 g of
R. nasutus powder were mixed with 30 mL of 30, 50 or 70%

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journal of functional foods 12 (2015) 498508

ethanol in water, after which each mixture was shaken at 60 C


in a water bath for 3 h, followed by centrifuging at 1700 g at
4 C for 30 min, collecting supernatant, evaporating to dryness
and diluting to 10 mL with acetonitrile and water (1:1, v/v).

2.3.2.

Determination of total phenolic acids

A method described by Kao et al. (2012) was used to determine total phenolic acids in R. nasutus samples. Briefly, 5
concentrations of 50, 100, 200, 300 and 400 g/mL of gallic acid
standard in ethanol were prepared. Then 50 L each was collected and mixed with 200 L FolinCiocalteu reagent, after
which the mixture was stirred homogeneously, followed by
standing in the dark for 5 min, adding 1 mL aqueous solution
of sodium carbonate (15%) for reaction for 1 h at room temperature, and measuring absorbance at 750 nm. The gallic acid
standard curve was thus prepared by plotting concentration
against absorbance, while both the linear regression equation and correlation coefficient were obtained. Then 50 L
sample of R. nasutus extract was collected for absorbance measurement at 750 nm for calculation of gallic acid equivalents
based on the linear regression equation.

2.3.3.

Determination of total flavonoids

A method based on Kao et al. (2012) was used to determine


total flavonoids in R. nasutus samples. In brief, 6 concentrations of 25, 50, 100, 200, 300 and 400 g/mL of quercetin standard
in ethanol solution were prepared. Then 200 L of each was
collected and mixed with 30 L of 5% aqueous solution of
sodium nitrite, after which the mixture was allowed to stand
at room temperature for 5 min, followed by addition of 60 L
of 10% aluminum chloride aqueous solution. After mixing homogeneously for 5 min, 300 L of 1 M NaOH solution and 200 L
chloroform were added. Then the mixture was mixed again,
followed by centrifuging, collecting supernatant, and measuring the absorbance at 510 nm. The quercetin standard curve
was thus prepared by plotting concentration against absorbance, while both the linear regression equation and correlation
coefficient were obtained. Then 200 L of R. nasutus sample
extract was collected for absorbance measurement at 510 nm,
which was used for calculation of quercetin equivalents based
on the regression equation.

2.3.4. HPLC-PADMS/MS analysis of phenolic acids


and flavonoids
Three C18 columns, as described in Section 2.3, were selected for comparison of separation efficiency of phenolic acids
and flavonoids in R. nasutus samples. The separation efficiency was evaluated based on retention factor (k) and
separation factor (), with the k value ranging from 2 to 10 and
value higher than 1 required to attain an optimum resolution (Skoog, Holler, & Crouch, 2007). In addition, the polarity
index of mobile phase has to be controlled carefully to obtain
a suitable k value (Chen et al., 2012). Determination of polarity index was based on mixing two or more solvents together
to attain a mobile phase of optimum polarity according to the
formula, PAB = VAPA + VBPB, where PA and PB are polarity indices
of solvents A and B, respectively, and VA and VB are the volume
fractions of the two solvents. Accordingly, several gradient
mobile phases, as reported by Kao et al. (2008) and Chen et al.
(2012), were used for separation of phenolic acids and

flavonoids in R. nasutus samples, after which some modifications were made so that a satisfactory separation could be
achieved. The peak purity of each phenolic acid and flavonoid on the HPLC chromatogram was automatically determined
by using an Agilent G2180A spectral evaluation software data
management system through comparison of absorption spectra
of unknown peaks with reference standards. However, for
unknown peaks without commercially available standards, the
purity was calculated based on the degree of overlapping
through determination of absorption spectra of left tip, apex
and right tip of the peak.
Identification was carried out by comparing retention times,
absorption spectra and mass spectra of various phenolic acid
and flavonoid peaks with reference standards and values reported in the literature. As mentioned in Section 2.3, two HPLC
MS systems were employed for mass spectra determination
for positive identification. One was a single quadrupole mass
spectrometer with ESI in negative mode for detection with the
scanning range of 1001000, drying gas flow 13 L/min, nebulizer pressure 60 psi, drying gas temperature 350 C, vaporizer
temperature 250 C, capillary voltage 2500 V, charging voltage
2000 V, and fragmentor voltage 200 V. The other was a ultrahigh resolution LTQ Orbitrap XL mass spectrometer (MSMS)
with ESI in negative mode for detection with the scanning range
of 1001000, heater temperature 250 C, sheath gas flow rate
30 arbitrary units, auxiliary gas flow rate 5 arbitrary units, spray
voltage 4 kV, capillary temperature 275 C, capillary voltage 35 V,
and tube lens voltage 100 V.
The internal standard, vanillic acid, was subsequently dissolved in acetonitrile/water (1:1, v/v) at a concentration of 20 g/
mL for phenolic acid quantitation, while quercetin dissolved
in methanol at the same concentration for flavonoid
quantitation. For preparation of standard curves, phenolic acid
standards including protocatechuic acid, caffeic acid, ferulic
acid and sinapic acid were dissolved in acetonitrile/water (1:1,
v/v) separately at 6 concentrations of 0.5, 1.0, 5.0, 10, 20 and
40 g/mL. Likewise, 7 concentrations of 0.1, 0.5, 1.0, 5.0, 10, 20
and 40 g/mL were prepared for p-coumaric acid. For flavonoids, both quercetin-3-O-rutinoside and kaempferol-3-Orutinoside were dissolved in methanol to obtain 6
concentrations of 0.5, 1.0, 5.0, 10, 20 and 40 g/mL. Then all the
phenolic acid and flavonoid standard solutions were mixed with
vanillic acid and quercetin, respectively, so that each solution contained internal standard at 20 g/mL. Each standard
concentration was injected into HPLC twice and all the standard curves were prepared by plotting the concentration ratios
(standard versus internal standard) against the area ratios (standard versus internal standard). The linear regression equations
and coefficients of determination (R2) were then obtained automatically for each standard curve.

2.3.5. Method validation and quantitation of phenolic


acids and flavonoids
R. nasutus sample extracts were mixed with a known concentration of internal standard and subjected to extraction and
HPLC-PADMS/MS analysis for quantitation of phenolic acids
and flavonoids. Sample extracts were injected into HPLC 3 times
each in the morning, afternoon and evening on the same day
for a total of 9 injections. Then the intra-day variability (repeatability) was obtained by calculating mean standard

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journal of functional foods 12 (2015) 498508

deviation and relative standard deviation (RSD %). For interday variability determination, sample extracts were injected
into HPLC on day 1, day 2 and day 3 with 3 injections each in
the morning, afternoon and evening for calculation of
mean standard deviation and relative standard deviation.
Both limit of detection (LOD) and limit of quantitation (LOQ)
of various phenolic acids and flavonoids were determined based
on the method as described by International Conference of
Harmonization (ICH) (1996). Three concentrations of 200, 300
and 400 ng/mL were prepared for procatechuic acid; 100, 150
and 200 ng/mL for caffeic acid; 20, 50 and 100 ng/mL for
p-coumaric acid and ferulic acid; 100, 200 and 300 ng/mL for
sinapic acid; 100, 200 and 400 ng/mL for quercetin-3-Orutinoside; 50, 100 and 200 ng/mL for kaempferol-3-O-rutinoside.
Each standard concentration was injected into HPLC three times
and the standard curves were prepared by plotting concentration against peak height. Then the linear regression equations
were obtained for calculation of slope (s) and standard deviation () for each standard curve. Both LOD and LOQ of phenolic
acid and flavonoid standards were calculated using the following formula: LOD = 3.3 /s and LOQ = 10 /s.
For recovery study, two concentrations of various phenolic acid and flavonoid standards were added to 0.5 g of R. nasutus
sample extracts: protocatechuic acid (400 and 1000 g), caffeic
acid (500 and 1000 g), p-coumaric acid and ferulic acid (800
and 2000 g each), sinapic acid (500 and 1500 g), quercetin3-O-rutinoside (1500 and 3000 g), kaempferol-3-O-glucoside
(1000 and 2500 g). After extraction and HPLC analysis, the recovery of various phenolic acids and flavonoids were obtained
based on the relative ratio of the amount of each standard after
HPLC to that before HPLC (original amount).
A fixed amount of internal standards vanillic acid and quercetin was added to R. nasutus sample separately for extraction
and HPLC analysis for quantitation of phenolic acid and flavonoid respectively. By using the linear regression equation
derived from each standard curve as shown earlier, the amounts
of various phenolic acids and flavonoids were determined based
on a formula reported by Chen et al. (2012).

2.3.6.

Statistical analysis

Triplicate extractions were performed for each R. nasutus sample


and the mean data were subjected to analysis of variance and
Duncans multiple test for comparison of significant difference (P < 0.05) by ANOVA and SAS (2011).

3.

Results and discussion

3.1.

Evaluation of extraction method

Three solvent systems containing ethanol and water in different proportions as mentioned in Section 2.3 were used for
evaluation of extraction efficiency based on total phenolic acids
and total flavonoids expressed as gallic acid and quercetin
equivalents, respectively, which are shown in Table 1. A solvent
system of 30% ethanol in water was found to obtain the highest
yield (2.16 mg/g) of total phenolic acids, followed by 50% ethanol
in water (1.98 mg/g) and 70% ethanol in water (1.94 mg/g).
However, there was no significant difference (P > 0.05) between

Table 1 The changes in content of total phenolics (mg/


g) and total flavonoids (mg/g)a in R. nasutus as affected
by extraction using different ethanolwater mixturesb.
EtOH

Total phenolicsc

Total flavonoidsd

30%
50%
70%

2.16 0.04A
1.98 0.05B
1.94 0.02B

4.93 0.18A
4.58 0.19B
3.81 0.09C

a
b

c
d

Average of triplicate analyses standard deviation.


Symbols bearing different letters (AC) in the same column are
significantly different (p < 0.05).
Data expressed as mg/g of gallic acid equivalent.
Data expressed as mg/g of quercetin equivalent.

50% ethanol in water and 70% ethanol in water. The same trend
was observed for total flavonoids, with 30% ethanol in water
generating the highest yield (4.93 mg/g), followed by 50% ethanol
in water (4.58 mg/g) and 70% ethanol in water (3.81 mg/g). Apparently these outcomes demonstrated that a solvent system
of 30% ethanol in water should be the most appropriate for
simultaneous extraction of phenolic acids and flavonoids in
R. nasutus.

3.2.

Evaluation of HPLC column and mobile phase system

In many published reports the mobile phases used for separation of phenolic acids and flavonoids by HPLC often include
a combination of water and methanol or acetonitrile with acidic
modifiers such as formic acid, acetic acid or phosphoric acid
to retard ionization, reduce interaction between polyphenol
compounds and column stationary phase, and improve peak
tailing as well as broadening (Chen et al., 2012; Inbaraj, Lu, Kao,
& Chen, 2010; Luo et al., 2011). In addition, Wang and Huang
(2004) pointed out that with tetrahydrofuran (THF) as modifier the selectivity of mobile phase toward phenolic compounds
could be enhanced. Nevertheless, THF being an aprotic solvent
would be unable to provide proton for ionization of target compounds during MS analysis of phenolic acids and flavonoids,
which could induce polymerization with APCI mode to contaminate corona needle and thus lower sensitivity. Therefore
in this study we choose a combination of water and methanol or acetonitrile as mobile phase with 0.1% formic acid as
modifier for evaluation of separation efficiency of phenolic acids
and flavonoids.
Prior to mobile phase evaluation, three C18 columns as mentioned in Section 2.3 were compared for separation efficiency
by employing a gradient mobile phase developed by Chen et al.
(2012); 92% A (0.1% formic acid in water) and 8% B (acetonitrile) initially, maintained for 10 min, increased to 14% B in
24 min, 23% B in 35 min, 24% B in 44 min, maintained for 12 min,
32% B in 60 min, 37% B in 66 min and returned to 8% B in
68 min. Of the 3 columns, the Phenomenex Gemini C18 column
showed a better resolution of phenolic acid and flavonoid peaks
than the other two columns, and thus was selected for mobile
phase evaluation. After numerous studies, we found that acetonitrile was superior to methanol in resolving phenolic acid
and flavonoid peaks for R. nasutus samples as the separation
number of both increased substantially. However, the resolution of both phenolic acids and flavonoids has still to be

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journal of functional foods 12 (2015) 498508

Fig. 1 HPLC-DAD chromatogram of phenolic acids and flavonoids extracted from R. nasutus. Column, Gemini C18; mobile
phase, 0.1% formic acid in water (A) and ACN (B); flow rate, 0.8 mL/min; detection wavelength, 280 nm. Peak identification:
peak 1, hydroxycinnamic acid derivative (1); peak 2, hydroxycinnamic acid derivative (2); peak 3, hydroxyferulic acid
hexoside (1); peak 4, protocatechuic acid hexoside; peak 5, 5-hydroxydihydroferulic acid derivative; peak 6, caffeic acid
hexoside (1); peak 7, dihydrocaffeic acid hexoside; peak 8, sinapic acid hexoside (1); peak 9, hydroxyferulic acid hexoside
(2); peak 10, caffeic acid hexoside pentoside; peak 11, p-coumaric acid hexoside (1); peak 12, caffeic acid hexoside (2); peak
13, hydroxyferulic acid deoxyhexoside; peak 14, p-coumaric acid hexoside (2); peak 15, sinapic acid hexoside (2); peak 16,
caffeic acid hexoside pentoside (2); peak 17, dihydro- p-coumaric acid hexoside; peak 18, p-coumaric acid hexoside (3);
peak 19, p-coumaric acid glucuronide; peak 20, quercetin-3-O-rutinoside; peak 21, kaempferol-3-O-rutinoside; peak 22,
dihydrocaffeic acid hexoside pentoside; peak 23, quercetin pentoside.

improved by reducing flow rate and changing the gradient


mobile phase as follows: 96% A (0.1% formic acid in water) and
4% B (acetonitrile) in the beginning, maintained for 4 min, increased to 6% B in 10 min, 7% B in 12 min, 8% B in 15 min, 13%
B in 18 min, 15% B in 25 min, 20% B in 28 min, 28% B in 32 min,
maintained until 50 min, with flow rate at 0.8 mL/min and detection at 280 nm. With this HPLC condition a total of 20
phenolic acids and 3 flavonoids in R. nasutus samples were adequately separated within 45 min (Fig. 1).
Table 2 shows retention time (tR), retention factor (k), separation factor () and peak purity of phenolic acids and flavonoids
in R. nasutus extracts based on the HPLC chromatogram shown
in Fig. 1. The retention times ranged from 9.83 min for
hydroxycinnamic acid derivative (1) to 44.21 min for quercetin pentoside and retention factor from 1.46 to 10.10, implying
a proper mobile phase solvent strength was controlled. The
separation factor ranged from 1.01 for caffeic acid hexoside (1)
to 1.30 for hydroxyferulic acid hexoside (1), indicating a suitable selectivity of mobile phase toward phenolic acids and
flavonoids was attained. The purities were ranged from 84.4%
for dihydro-p-coumaric acid hexoside to 99.9% for dihydrocaffeic
acid hexoside pentoside. With the exception of dihydrocaffeic
acid hexoside and dihydro-p-coumaric acid hexoside, the purities of the other phenolic acids and flavonoids were all higher
than 92.2%.
Several reported HPLC methods for simultaneous determination of phenolic acids and flavonoids suffer a major drawback
of long separation time and/or poor separation efficiency. In

addition, the methods with adequate resolution and reasonable separation time often lack simultaneous separation. For
instance, Thabti, Elfalleh, Hannachi, Ferchichi, and Campos
(2012) resolved a total of 12 phenolic acids and flavonoids in
Tunisian Morus species within 45 min, while Materska (2014)
separated only 8 compounds in Capsicum annuum with similar
retention time. Some other studies have also employed two
different HPLC mobile phase systems for separation of phenolic acids and flavonoids. For example, Gutierrez-Uribe,
Romo-Lopez, and Serna-Saldivar (2011) separated 5 phenolic
acids and 3 flavonoids using two different mobile phase systems
within 23 and 32 min, respectively, but the separation was unsatisfactory. Similarly, Andarwulan et al. (2012) separated 5
flavonoids and 3 phenolic acids in 24 medicinal vegetables
from Indonesia within 20 and 10 min, respectively, but the resolution remained insufficient. In addition to mobile phase,
different detection wavelengths are also selected for detection of phenolic acids and flavonoids for maximum quantitation
accuracy. In one study Khanam, Oba, Yanase, and Murakami
(2012) separated 6 hydroxybenzoic acid-type and 7
hydroxycinnamic acid-type phenolic acids from 8 leafy vegetables within 70 min with detection at 254 and 280 nm, while
3 flavonoids were detected at 360 nm. Likewise, two wavelengths of 280 and 360 nm were used for detection of 4 phenolic
acids and 5 flavonoids from Phoenix dactylifera, respectively, with
the separation time being within 65 min (Benmeddour,
Mehinagic, Meurlay, & Louaileche, 2013). Nevertheless, the separation efficiency has to be improved substantially. In our study

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journal of functional foods 12 (2015) 498508

Table 2 Retention time (tR), retention factor (k), separation factor (), peak purity and content of phenolic acids and
flavonoids extracted from R. nasutus.
Peak
no.

Compound

Retention
time (tR, min)

Retention
factor (k)

Separation
factor ()

Peak
purity (%)

Content
(mg/g)b

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

Hydroxycinnamic acid derivative (1)


Hydroxycinnamic acid derivative (2)
Hydroxyferulic acid hexoside (1)
Protocatechuic acid hexoside
5-Hydroxydihydroferulic acid derivative
Caffeic acid hexoside (1)
Dihydrocaffeic acid hexoside
Sinapic acid hexoside (1)
Hydroxyferulic acid hexoside (2)
Caffeic acid hexoside pentoside (1)
p-Coumaric acid hexoside (1)
Caffeic acid hexoside (2)
Hydroxyferulic acid deoxyhexoside
p-Coumaric acid hexoside (2)
Sinapic acid hexoside (2)
Caffeic acid hexoside pentoside (2)
Dihydro-p-coumaric acid hexoside
p-Coumaric acid hexoside (3)
p-Coumaric acid glucuronide
Quercetin-3-O-rutinoside
Kaempferol-3-O-rutinoside
Dihydrocaffeic acid hexoside pentoside
Quercetin pentoside

9.83
10.60
12.57
14.97
16.46
16.99
18.72
21.12
22.09
25.40
26.01
27.25
28.42
29.35
30.09
30.95
32.23
33.23
35.67
38.97
40.66
42.30
44.21

1.46
1.66
2.16
2.76
3.13
3.26
3.70
4.30
4.55
5.38
5.53
5.84
6.14
6.37
6.56
6.77
7.09
7.34
7.96
8.79
9.21
9.62
10.10

1.13 (1,2)a
1.13 (1,2)a
1.30 (2,3)a
1.28 (3,4)a
1.13 (4,5)a
1.01 (5,6)a
1.13 (6,7)a
1.16 (7,8)a
1.06 (8,9)a
1.18 (9,10)a
1.03 (10,11)a
1.06 (11,12)a
1.05 (12,13)a
1.04 (13,14)a
1.03 (14,15)a
1.03 (15,16)a
1.05 (16,17)a
1.04 (17,18)a
1.06 (18,19)a
1.10 (19,20)a
1.04 (20.21)a
1.04 (21,22)a
1.05 (22,23)a

99.8
99.3
99.4
99.7
99.6
97.1
88.7
96.9
99.6
92.2
98.2
99.6
99.7
99.8
97.0
99.8
84.4
98.7
93.2
99.4
99.7
99.9
97.8

0.403 0.007
0.038 0.002
1.032 0.017
0.389 0.009
0.155 0.002
0.693 0.006
0.287 0.002
0.429 0.012
1.686 0.013
0.115 0.003
0.123 0.009
0.276 0.009
0.323 0.005
0.133 0.006
0.680 0.012
0.096 0.006
0.065 0.005
1.069 0.013
0.244 0.006
1.612 0.113
1.017 0.081
4.933 0.068
2.813 0.049

a
b

Numbers in parentheses represent values between two neighboring peaks.


Average of triplicate analyses standard deviation.

a more comprehensive HPLC method was developed for simultaneous separation of 23 phenolic acids and flavonoids
within 45 min and detection at 280 nm. Also, two different internal standards were employed for quantitation of phenolic
acids and flavonoids separately.

3.3.

Identification of phenolic acids and flavonoids

Table 3 shows the absorption and mass spectral data for phenolic acids and flavonoids in R. nasutus. Based on the major
absorption peak obtained at 310328 nm, peaks 119 and 22
were tentatively identified as phenolic acids, while peaks 20,
21 and 23 as flavonoids based on a relatively higher absorption maximum ranging from 344 to 356 nm (Chen et al., 2012;
Inbaraj et al., 2010). However, further identification of phenolic acids and flavonoids was based on the [M-H] value and the
corresponding fragment ions obtained by tandem MS/MS. Peaks
1, 2, 3, 5, 9 and 13 were tentatively identified as derivatives of
ferulic acid based on the [M-H] value of 389, 389, 371, 255, 371,
355, respectively, all of which yielding the same fragment ions
at m/z 209211 and 191193 with the latter being consistent
with the MW of ferulic acid. Furthermore, both peaks 3 and 9
were identified as hydroxyferulic acid hexoside as the fragment ions are formed due to the elimination of hexose and
water molecule, while peak 13 was identified as hydroxyferulic
acid deoxyhexoside because of the fragment ions formed after
removal of deoxyhexose and water molecule (Simirgiotis,
Caligari, & Schmeda-Hirschmann, 2009). Based on the spectral data reported by Narvaez-Cuenca, Vincken, and Gruppen
(2012), peak 5 was identified as 5-OH-dihydroferulic acid owing

to the formation of fragment ions at m/z 211 and 193 after the
removal of carbon dioxide and water molecule. Though the
same fragment ions were obtained for peaks 1 and 2, they were
categorized as hydroxycinnamic acid derivative because of their
relatively higher [M-H] value at 389. Peaks 6, 7, 10, 12, 16 and
22 were tentatively identified as derivatives of caffeic acid based
on the [M-H] values of 341, 343, 473, 341, 473 and 475, all of
which yielding the same fragments ions at m/z 179181 and
135137 with the former corresponding to the MW of caffeic
acid and the latter resulting from the loss of carbon dioxide.
More specifically, peaks 6, 7 and 12 were identified as caffeic
acid hexoside (1), dihydrocaffeic acid hexoside and caffeic acid
hexoside (2), respectively, based on the fragment ion at m/z
179181 obtained due to loss of hexose (Chen et al., 2012). Likewise, peaks 10, 16 and 22 were identified as caffeic acid hexoside
pentoside (1), caffeic acid hexoside pentoside (2) and
dihydrocaffeic acid hexoside pentoside, respectively, because
of the formation of a fragment ion at m/z 341343 after the
removal of hexose and pentose. Peaks 11, 14, 17, 18 and 19 were
tentatively identified as derivatives of p-coumaric acid based
on the same molecular ion peak ranging from 325 to 339 and
fragments ions at m/z 163165 and 119121, with the former
obtained after the removal of hexose conforming with the MW
of p-coumaric acid and the latter resulting from the loss of
carbon dioxide (Gavrilova, Kajdzanoska, Gjamovski, & Stefova,
2011). Based on these fragmentation patterns, peaks 11, 14 and
18 were identified as p-coumaric acid hexoside, while the peak
17 as dihydro-p-coumaric acid hexoside. However, peak 19 was
identified as p-coumaric acid glucuronide as the fragment ion
at m/z 163 was formed after the removal of a glucuronide

504

journal of functional foods 12 (2015) 498508

Table 3 Ultraviolet and mass spectral data for tentative identification of phenolic acids and flavonoids in R. nasutus.
Peak
no.

Compound

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

Hydroxycinnamic acid derivative (1)


Hydroxycinnamic acid derivative (2)
Hydroxyferulic acid hexoside (1)
Protocatechuic acid hexoside
5-Hydroxydihydroferulic acid derivative
Caffeic acid hexoside (1)
Dihydrocaffeic acid hexoside
Sinapic acid hexoside (1)
Hydroxyferulic acid hexoside (2)
Caffeic acid hexoside pentoside (1)
p-Coumaric acid hexoside (1)
Caffeic acid hexoside (2)
Hydroxyferulic acid deoxyhexoside
p-Coumaric acid hexoside (2)
Sinapic acid hexoside (2)
Caffeic acid hexoside pentoside (2)
Dihydro-p-coumaric acid hexoside
p-Coumaric acid hexoside (3)
p-Coumaric acid glucuronide
Quercetin-3-O-rutinosideh
Kaempferol-3-O-rutinosideh
Dihydrocaffeic acid hexoside pentoside
Quercetin pentoside

a
b
c
d
e
f
g
h

[M-H]

max (nm)

MS2 Fragment ions

Online

Reported

Online

Reported

Online

Reported

240, 292sha, 324


236, 292sh, 322
240, 298sh, 326
262, 291
278, 310
240, 296sh, 316
280
236, 300sh, 326
240 298sh, 326
240, 292sh, 318
240, 298sh, 326
238, 296sh, 325
242, 294sh, 322
242, 296sh, 328
236, 300sh, 326
236, 298sh, 324
278, 316
240, 298sh, 326
238, 298sh, 326
264, 356
264, 344
280, 312sh
254, 356

234, 293sh, 324c


257, 291d

226, 294sh, 318d

234, 293sh, 322c


234, 293sh, 324c

242, 316
226, 294sh, 318
234, 293sh, 324
242, 316e
234, 293sh, 322

242, 316e

254, 302sh, 351c


265, 290, 346f

356g

389
389
371
315
255
341
343
385
371
473
325
341
355
325
385
473
327
325
339
609
593
475
433

b
b
371c
315d
b
341d
b
385c
371c
b
325e
341d
355c
325e
385c
b
b
325e
b
609e
593f
b
433e

371, 353, 209, 191


371, 353, 209, 191
209, 191
153
211, 193
179, 135
181, 137
223, 205, 191
209, 191
341, 179, 135
163, 119
179, 135
209, 191
163, 119
223, 205, 191
341, 179, 135
165, 121
163, 119
163, 145
301
285
343, 181
301

b
b
209, 191c
153d
b
179, 135d
b
223, 205, 191c
209, 191c
b
163, 119e
179, 135d
209, 191c
163, 119
223, 205, 191c
b
b
163, 119e
b
301e
285f
b
301

Sh: Shoulder peak.


No reported data available.
Based on a reference by Simirgiotis et al. (2009).
Based on reference by Chen et al. (2012).
Based on a reference by Gavrilova et al. (2011).
Based on a reference by Engels et al. (2012).
Based on a reference by Barros et al. (2013).
Compound conclusively identified by comparison of MS spectral data of unknown peaks with authentic standards.

moiety (Algamdi, Mullen, & Crozier, 2011). Both peaks 8 and


15 were identified as sinapic acid hexoside based on the same
[M-H] value at 385 and the fragment ions at m/z 223, 205 and
191. The formation of m/z 223 and 205 is attributed to the elimination of a hexose moiety and a water molecule. Peak 4 was
identified as protocatechuic acid hexoside based on the [MH] peak at 315 with a fragment ion at m/z 153 resulting from
a loss of hexose was consistent with the MW of protocatechuic acid (Chen et al., 2012). Of the 3 flavonoids, peaks 20
and 21 were identified as quercetin-3-O-rutinoside and
kaempferol-3-O-rutinoside based on the molecular ion peaks
at 609 and 593, respectively, as well as the fragment ions at
m/z 301 and 285 obtained after the removal of a rutinoside
moiety (Engels et al., 2012; Gavrilova et al., 2011). Furthermore, both flavonoids were conclusively identified by
comparison with the reference standards. Likewise, based on
the molecular ion peak at 433 and the fragment ions at 301
formed due to removal of pentose, peak 23 was identified as
quercetin pentoside (Barros et al., 2013).

3.4.

Method validation

Table 4 shows the quality control data of phenolic acids and


flavonoids in R. nasutus samples. The RSD % of the intra-day
variability were ranged from 0.8 to 8.7%, while the inter-day
variability ranged from 0.3 to 10.6%, implying that a high re-

producibility of this method was attained. Table 5 shows


recoveries of 7 standards including caffeic acid, protocatechuic acid, p-coumaric acid, ferulic acid, sinapic acid, quercetin3-O-rutinoside and kaempferol-3-O-rutinoside, which amounted
to 94.8, 92.1, 89.0, 84.4, 83.6, 88.6 and 92.9%, respectively,
revealing a high accuracy of this method. The LOD of protocatechuic acid, caffeic acid, p-coumaric acid, ferulic acid, sinapic
acid, quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside
were 98.3, 31.2, 17.7, 10.9, 74.7, 51.1, and 30.4 ng/mL, respectively, whereas the LOQ were 294.9, 93.6, 53.2, 32.7, 224.1, 153.3
and 91.1 ng/mL, respectively.
For the standard calibration curves, the linear regression
equations of protocatechuic acid, caffeic acid, p-coumaric acid,
sinapic acid, ferulic acid, quercetin-3-O-rutinoside, and
kaempferol-3-O-rutinoside were y = 0.8036x + 0.0174,
y = 1.5149x + 0.0189, y = 2.577x + 0.0738, y = 0.7297x + 0.0213,
y = 1.706x + 0.0681, y = 0.4858x + 0.0182, and y = 0.7387x + 0.0418,
respectively, with R2 being all higher than 0.99.
Compared to published reports, both LOD and LOQ shown
in this study are lower or similar. For instance, Khattab, Eskin,
Aliani, and Thiyam (2010) determined sinapic acid derivatives in canola extracts with LOD and LOQ being reported to
be 200 and 500 ng/mL, respectively. In a later study dealing with
analysis of polyphenols in honey by HPLC-DAD, Zhang et al.
(2013) reported the LOD and LOQ of quercetin-3-O-rutinoside
to be 40.5 and 123 ng/mL, respectively. Likewise, the LOD and

505

journal of functional foods 12 (2015) 498508

Table 4 Quality control data of phenolic acids and flavonoids extracted from R. nasutus.
Intra-day variabilitya

Peak
no.

Compound

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

Hydroxycinnamic acid derivative (1)


Hydroxycinnamic acid derivative (2)
Hydroxyferulic acid hexoside (1)
Protocatechuic acid hexoside
5-hydroxydihydroferulic acid derivative
Caffeic acid hexoside (1)
Dihydrocaffeic acid hexoside
Sinapic acid hexoside (1)
Hydroxyferulic acid hexoside (2)
Caffeic acid hexoside pentoside (1)
p-Coumaric acid hexoside (1)
Caffeic acid hexoside (2)
Hydroxyferulic acid deoxyhexoside
p-Coumaric acid hexoside (2)
Sinapic acid hexoside (2)
Caffeic acid hexoside pentoside (2)
Dihydro-p-coumaric acid hexoside
p-Coumaric acid hexoside (3)
p-Coumaric acid glucuronide
Quercetin-3-O-rutinoside
Kaempferol-3-O-rutinoside
Dihydrocaffeic acid hexoside pentoside
Quercetin pentoside

Inter-day variabilitya

Mean SD (mg/g)

RSD (%)

Mean SD (mg/g)

RSD (%)

0.408 0.027
0.037 0.002
1.022 0.017
0.393 0.009
0.159 0.006
0.693 0.006
0.297 0.012
0.424 0.020
1.676 0.013
0.105 0.007
0.117 0.005
0.276 0.014
0.301 0.024
0.123 0.006
0.700 0.037
0.099 0.006
0.065 0.005
1.080 0.015
0.234 0.008
1.633 0.143
1.057 0.081
4.994 0.063
2.883 0.061

6.6
6.5
1.7
2.3
4.0
0.8
4.1
4.8
0.8
7.0
4.1
5.1
7.8
5.0
5.3
6.0
7.5
1.4
3.2
8.7
7.7
1.3
2.1

0.385 0.003
0.034 0.002
1.002 0.005
0.408 0.003
0.160 0.003
0.702 0.024
0.321 0.019
0.416 0.016
1.663 0.011
0.109 0.005
0.117 0.003
0.273 0.002
0.283 0.030
0.152 0.011
0.711 0.003
0.101 0.003
0.058 0.003
1.151 0.029
0.264 0.016
1.683 0.095
1.273 0.040
5.092 0.015
3.155 0.106

0.7
6.3
0.5
0.7
2.0
3.4
5.8
3.8
0.6
4.8
2.4
0.6
10.6
7.2
0.4
2.6
5.2
2.5
6.0
5.6
3.2
0.3
3.4

Mean of triplicate analyses standard deviation.

LOQ of kaempferol-3-O-rutinoside in Gynostemma pentaphyllum


Makino were shown to be 21.0 and 63.1 ng/mL, respectively.
In addition, the recoveries of phenolic acids and flavonoids obtained in this study are similar to those reported in the
literature, as shown by the recoveries of protocatechuic acid,
caffeic acid and p-coumaric acid in honey as determined by
HPLC-DAD to be 91.7102.4, 93.498.9, and 92.9110.1%, respectively (Zhang et al., 2013). Alarcon-Flores, Romero-Gonzalez,
Vidalm, and Frenich (2013) determined recoveries of ferulic acid,
quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside in tomatoes by HPLCMS, which were shown to be in the range of

72.494.8, 78.1113.4 and 88.2104.6%, respectively. Likewise, the


recovery of sinapic acid was 88.9% in Chinese wax berry juice
as determined by HPLC-DAD (Wang, Zhao, Chen, Cheng, & Guo,
2012), with dihydrocaffeic acid hexoside pentoside being present
in the largest amount (4.93 mg/g), followed by quercetin pentoside (2.81 mg/g), hydroxyferulic acid hexoside (1,2) (2.72 mg/
g), quercetin-3-O-rutinoside (1.61 mg/g), p-coumaric acid
hexoside (1,2,3) (1.33 mg/g), sinapic acid hexoside (1,2) (1.11 mg/
g), kaempferol-3-O-rutinoside (1.02 mg/g), caffeic acid hexoside
(1,2) (0.97 mg/g), hydroxycinnamic acid derivative (1,2) (0.44 mg/
g), protocatechuic acid hexoside (0.39 mg/g), hydroxyferulic acid

Table 5 Recovery of phenolic acids and flavonoids by HPLC-PAD.


Peak no.

Compound

Caffeic acid

Protocatechuic acid

p-Coumaric acid

Ferulic acid

Sinapic acid

Quercetin-3-O-rutinoside

Kaempferol-3-O-rutinoside

a
b
c

Original (g)
c

ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1486
1486
1011
1011

Spiked (g)

Found (g)

Recovery (%)a

Mean SD (%)

RSD (%)b

500
1000
500
1000
800
2000
800
2000
500
1500
1500
3000
1000
2500

464
967
452
935
734
1721
689
1653
399
1309
2792
4188
1942
3328

92.8
96.7
90.6
93.5
91.8
86.1
86.1
82.7
79.8
87.3
87.1
90.0
93.1
92.7

94.8 2.8

3.0

92.1 2.1

2.3

89.0 4.0

4.5

84.4 2.4

2.8

83.6 5.3

6.3

88.6 2.1

2.4

92.9 0.3

0.3

Recovery (%) = (amount found original amount)/amount spiked 100.


RSD (%) = (SD/mean) 100.
Not determined.

506

journal of functional foods 12 (2015) 498508

deoxyhexoside (0.32 mg/g), dihydrocaffeic acid hexoside


(0.29 mg/g), p-coumaric acid glucoronide (0.24 mg/g), caffeic acid
hexoside pentoside (1,2) (0.21 mg/g), 5-hydroxydihydroferulic
acid derivative (0.16 mg/g), and dihydro-p-coumaric acid
hexoside (0.065 mg/g).

3.5.

Quantitation of phenolic acids and flavonoids

Table 2 shows the contents of phenolic acids and flavonoids


in R. nasutus samples. Dihydrocaffeic acid hexoside pentoside was found to be present in the largest amount (4.93 mg/
g), followed by quercetin pentoside (2.81 mg/g), hydroxyferulic
acid hexoside (1,2) (2.72 mg/g), quercetin-3-O-rutinoside
(1.61 mg/g), p-coumaric acid hexoside (1,2,3) (1.33 mg/g), sinapic
acid hexoside (1,2) (1.11 mg/g), kaempferol-3-O-rutinoside
(1.02 mg/g), caffeic acid hexoside (1,2) (0.97 mg/g), hydroxycinnamic acid derivative (1,2) (0.44 mg/g), protocatechuic acid
hexoside (0.39 mg/g), hydroxyferulic acid deoxyhexoside
(0.32 mg/g), dihydrocaffeic acid hexoside (0.29 mg/g), p-coumaric
acid glucoronide (0.24 mg/g), caffeic acid hexoside pentoside
(1,2) (0.21 mg/g), 5-hydroxydihydroferulic acid derivative
(0.16 mg/g), and dihydro-p-coumaric acid hexoside (0.065 mg/
g). Due to absence of several commercial phenolic acid and
flavonoid standards, only quercetin-3-O-rutinoside and
kaempferol-3-O-rutinoside were quantified by their respective standard curves. The standard curves of protocatechuic
acid, caffeic acid, p-coumaric acid, ferulic acid and sinapic acid
were used to quantify their respective derivatives. By summarizing all the data in Table 2, the total amount of phenolic
acids was much higher than total flavonoids by 7.73 mg/g, with
the former equaled 13.17 mg/g and the latter 5.44 mg/g. Compared to some other published reports, the total amount of
phenolic acids and flavonoids shown in R. nasutus were much
greater than those reported in some other medicinal plants such
as Lycium babarum Linnaeus and Gynostemma pentaphyllum
(Inbaraj et al., 2010; Tsai et al., 2010). Similarly, the total phenolic acids and total flavonoids reported in 24 Indonesiabased medicinal vegetables were 0.00040.53 mg/g and 0.003
1.44 mg/g, respectively (Andarwulan et al., 2012), as were 0.015
1.55 and 0.0050.064 mg/g in 8 leafy vegetables (Khanam et al.,
2012), as well as 0.0810.169 and 0.0090.074 mg/g in 10 Algerian dates (Benmeddour et al., 2013). All these data are lower
than that found in R. nasutus. However, compared to the Chinese
medicinal plant Taraxacum formosanum (Chen et al., 2012), the
total amount of phenolic acids in R. nasutus is similar but flavonoids is higher. Furthermore, the total flavonoid shown in
Table 2 is similar to that in Table 1 (expressed as quercetin
equivalent), which equals 4.93 mg/g with 30% ethanol extraction, which should be due to the major flavonoid in R. nasutus
being quercetin derivative. Conversely, the total phenolic acids
in Table 1 (expressed as gallic acid equivalent) are much lower
than in Table 2, mainly because the major phenolic acids in
R. nasutus do not belong to gallic acid type.
Accordingly, herbs, being leafy or soft flowering parts of
plants, are used to add flavor and/or aroma to food and beverage. In addition, herbs are also used as medicine, cosmetics,
dyes, air fresheners, disinfectants, insect repellants, decorative materials, herbal drinks and teas, and pot pourri (Carlsen
et al., 2010). Herbs are particularly more popular among
Asians as they can be consumed as decoctions for disease

treatment as well as by adding to daily culinary preparations


for flavor enhancement and disease prevention. Therefore, over
the past decades there has been considerable interest in incorporating herbs into many medicinal preparations, functional
foods and nutraceuticals for medical purpose worldwide. Many
functional compounds found in herbs like phenolic acids and
flavonoids have been shown to be protective against a wide
range of human chronic diseases, including inflammation,
cancer and cardiovascular disease (Dai & Mumper, 2010), which
can be attributed to their strong antioxidant activities. In view
of the impact of herbs to human health, the development of
an appropriate analytical method for separation, identification and quantification of phenolic acids and flavonoids in herbs
is of great importance.

4.

Conclusion

The highest yield of total phenolic acids and flavonoids in


R. nasutus samples was obtained with 30% ethanol in water as
extraction solvent and shaken in 60 C water bath for 3 h. A
Phenomenex Gemini C18 column (250 4.6 mm ID, 5 m particle size) and a gradient mobile phase of 0.1% formic acid in
water and acetonitrile could resolve 20 phenolic acids and 3
flavonoids in R. nasutus samples within 45 min with detection at 280 nm, column temperature at 35 C and flow rate at
0.8 mL/min. The various phenolic acids and flavonoids were
identified by HPLC-PADMS/MS and two internal standards vanillic acid and quercetin were used for quantitation, respectively,
with dihydrocaffeic acid hexoside pentoside being present in
largest amount, followed by quercetin pentoside, hydroxyferulic
acid hexoside (1,2), quercetin-3-O-rutinoside, -coumaric acid
hexoside (1,2,3), sinapic acid hexoside (1,2), kaempferol-3-Orutinoside, caffeic acid hexoside (1,2), hydroxycinnamic acid
derivative (1,2), protocatechuic acid hexoside, hydroxyferulic
acid deoxyhexoside, dihydroxycaffeic acid hexoside, p-coumaric
acid glucoronide, caffeic acid hexoside pentoside (1,2),
5-hydroxydihydroxyferulic acid derivative, and dihydro-pcoumaric acid hexoside.
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