International Food Research Journal 20(1): 495-500 (2013)

Journal homepage: http://www.ifrj.upm.edu.my

Total phenolic compounds and scavenging activity in Clitoria ternatea and Vitex
negundo linn

*
Rabeta, M. S. and An Nabil, Z.

Food Science and Technology Division, School of Industrial Technology,

University Science Malaysia, 11800 Minden, Penang,
Malaysia

Article history Abstract

Received: 14 April 2012 This study was done to assess the total phenolic compounds (TPC) and 1, 1-diphenyl-2-
Received in revised form: picrylhydrazyl (DPPH) scavenging activity in the flowers and leaves of Clitoria ternatea
11 August 2012 and Vitex negundo Linn. by using methanol and water extraction. TPC were evaluated using
Accepted: 17 September 2012
Folin-Ciocalteau method. Methanol was more efficient in extracting phenolic compounds
compared with water in measuring TPC. Vitex negundo Linn. contained higher amount of TPC
compared to Clitoria ternatea. Besides that, leaves for both plants showed higher amount of
TPC compared to the flowers. Methanol extracted Vitex negundo Linn. showed higher DPPH
scavenging activity compared with Clitoria ternatea. In contrast, DPPH scavenging activity
Keywords for water extracted Clitoria ternatea showed higher value in compare with water extracted
Vitex negundo Linn.. The type of solvent used to extract the plant material and concentration
Total phenolic content of extracts used showed significance difference (P < 0.05) on the amount of DPPH scavenged
scavenging activity
by the plant extract. The presence of antioxidant activity in both leaves and flowers showed
DPPH
Clitorea ternatea that Clitoria ternatea and Vitex negundo Linn. have the potential to be an alternative source of
Vitex negundo Linn natural antioxidants. In vivo study is needed for successful commercialization and to benefit
both the food and pharmaceutical industries.

© All Rights Reserved

Introduction oxygen species (ROS) especially in the process of

ageing.
Lipid peroxidation is one of the factors that may Plants and herbs have been an important
affect appearance, flavour, and nutritional quality in contributor to the quality of human life for thousands
many processed foods. The process is also important of years. Some of them are well known medicinal
for the stability of processed food (Aruoma, 1999). herbs. Butterfly pea or blue pea (Clitoria ternatea)
In the process of lipid peroxidation, many radical from family of Fabaceae is a vine with vivid blue
species such as lipid alkoxyl radical, are generated flowers 1 to 2 inches long, having wavy-rimmed
and cause not only food deterioration but also DNA, standard and white centre, which is rather common
cell, and tissue damage (Nakatani, 1994). in gardens of Hawaii (Neal, 1968). Clitoria ternatea
In response, natural or synthetic antioxidants flower are commercially known as Bunga telang by
have been widely used to delay or prevent lipid the locals and are widely used as the food dyes in
peroxidation process in a variety of food products. Nasi kerabu (the local dish in Kelantan, Malaysia)
These naturally occurring compounds impart bright and a Baba and Nyonya kueh known as kueh tekan.
colour to fruits and vegetables and act as antioxidants The flowers are used in those products just for the
in the body by scavenging harmful free radicals, purplish blue colour from its petals without knowing
which are implicated in most degenerative diseases their health benefit.
(Kaur and Kapoor, 2001). During lipid peroxidation, Leggundi (Vitex Negundo Linn.) commonly
antioxidants may act in various ways such as binding known as ‘‘lemuni hitam’’ in Malaysia is from family
metal ions, scavenging free radicals, and decomposing of Verbenaceae. Verbenaceae family commonly
peroxides (Moure et al., 2001). Antioxidants also has been known as having quadrangular branches
defend our immune system to fight against reactive (Neal, 1968). Recently, indigenous Vitex negundo’s

*Corresponding author.
Email: [email protected]
496 Rabeta, M. S. and An Nabil, Z./IFRJ 20(1): 495-500

leaves are used to make ‘‘Nasi Lemuni’’, a famous shape were selected, while for Vitex negundo Linn.,
indigenous food in the northern area of Peninsular the leaves chosen were basically the young shoots,
Malaysia. In addition, the young leaves of the plant that happened to have completely purple colour on
are used as one of the traditional remedy for the the underneath part of the leaves.
postnatal women. An essential oil of Vitex negundo
Linn. is found to be useful for outer layer of wounds Sample preparation
and ulcers (Khokra et al., 2008). In brief, the collected flowers and leaves were
The addition of antioxidants is required to preserve separated from their stems and dried in a vacuum
flavour and colour and to avoid vitamin destruction oven (Binder Vacuum Oven Model VD53) at 30°C
in processed food (Moure et al., 2001). Now, as then, for 12 hours. Dried raw materials were left in
recent increasing interest in natural antioxidant found desiccators for 24 hours. All the dried flowers and
in plant sources has become a trend, because people leaves were ground using normal grinder and sieved.
worldwide are focusing on using natural additives, Plant powders were stored in an air-tight polyester
not only in food, but also in cosmetics industry. As a container and stored at -20°C (Lab Freezer Protech,
whole, antioxidant has become such popular topic to Model CR400) prior to the analysis.
be discussed mainly on their effect on preventing the
risk of chronic disease in many types of cancer. As Methanol extract of leaves and flowers
the global cancer incidence is increasing, researchers Based on the method proposed by Fu et al. (2009),
and health leaders around the world are scratching 5 g of powder were added to 100 ml of 70% methanol.
their heads and trying to understand the mechanisms Mixture was kept in orbital shakers (Lab Companion,
to reduce the risk of this frightening disease. There Model SI600R) overnight and then filtered using
has been a lot of interest in natural antioxidants since Whatman No. 1. Filtrate was vaporized in a rotary
the possible side effects of synthetic antioxidant such vacuum evaporator (Heildoph VV2200) by using a
as butylated hydroxyanisole (BHA) (Munday et al., water bath (Büchl Waterbath Model B-480) at 45°C
1998; Munday et al., 1999). Therefore, there have and vacuum pump to obtain dry extract.
been numerous researches on crude extraction and
bioactive compound of potential plants for natural Aqueous extract of leaves and flowers
and possibly economic and effective antioxidants to Aqueous extraction of plant material was done
replace the synthetic ones. Plants are great alternatives based on the method proposed by Wong et al.,
in many local communities in Malaysia and Asia in (2006). Briefly, 0.5 g of plant powder was extracted
general, used as health supplements and therapeutic using 25 ml of deionised water. The mixture was
(Khatijah and Mohamad Ruzi, 2006) allowed to stand in dark area at room temperature
These plants have not received much attention for 1 hour, with occasional agitation. By using filter
as antioxidant sources compared to others plants in paper (Whatman No.1), the mixture was filtered and
Malaysia due to the lack of information about total supernatant obtained was used for analysis without
phenolic compounds (TPC) and scavenging activity of further treatment.
Clitoria ternatea and Vitex negundo Linn.. Therefore
this study aimed to assess the TPC in the flowers and Total phenolic content determination
leaves of Clitoria ternatea and Vitex negundo Linn. by Total phenolics content in methanol extract of
using two types of extraction, namely methanol and flowers and leaves of both plants were determined
aqueous. This study was also undertaken to evaluate using the method proposed by Kaur et al. (2006)
the antioxidant activity possessed by both plants, by with slight modification. A total of 100 mg of dry
using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical extract was leached using 250 ml of methanol/water
scavenging assay. (60:0, v/v, 0.3% HCl) and then filtered using filter
paper (Whatman No 1). For every 100 μl of filtrate,
Materials and Methods about 100 μl of 50% Folin-Ciocalteau reagent and
2 ml of 2.5% sodium carbonate were added. The
Plant material mixture was mixed completely and allowed to stand
Fresh flowers and leaves of both Clitoria ternatea for 2 hours. Then, the absorbance of the solution at
and Vitex negundo Linn. were collected form Kg. 750 nm was measured. Quantification of TPC was
Seronok, Bayan Lepas, Pulau Pinang, Malaysia. done using standard curve of gallic acid as a standard
Fully bloomed flower of both Clitoria ternatea and phenolic compound (0.01-0.05 mg/ml), which
Vitex negundo Linn. were collected for the study. As was dissolved in methanol/water (60:40, v/v, 0.3%
for Clitoria ternatea leaves, only those with good HCl) and expressed as mg gallic acid per gram of
plant material. Sample measurements were done in
 Rabeta, M. S. and An Nabil, Z./IFRJ 20(1): 495-500 497

triplicates and the mean and standard deviations were Results and Discussion
calculated in each case.
For aqueous extracts, the TPC was determined Total phenolic compounds
based on the method by Wong et al., (2006). About The amount of TPC in plant extract was based
100 μl (20 μg/ml) of the extract was mixed with on the absorbance of sample and Folin-Ciocalteau
2.5 ml Folin-Ciocalteau reagent (10 times dilution) reagent mixture at 725 nm. The amount of total
and 2.5 ml of 2.5% sodium carbonate solution. The phenolic compounds that present in each of the
mixture was allowed to stand for 1 hour and the samples was reported as mg GAE per gram sample.
absorbance was measured at 725 nm. Total phenolics Since Folin-Ciocalteau assay measures all the
content was quantified using standard curve of gallic phenolics, the choice of gallic acid as standard is based
acid as a standard phenolic compound (0.2 to 1 mg/ on the availability of a stable and pure substance, and
ml), which was dissolved in deionised water and gallic acid is both, and it is less expensive than other
expressed as mg gallic acid equivalent (GAE) per options. Two standard curves were plotted to quantify
gram plant material. the phenolic compound in samples as mg GAE per
gram samples, one for methanol extract and another
1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical one for water extraction. The total amount of TPC in
scavenging activity determination each sample was represented as mean mg GAE per
Scavenging activity of extracts against DPPH gram sample (Table 1).
radical was measured based on the method proposed Generally, the amount of TPC exhibited by
by Gülcin et al. (2006) and by Fu et al. (2009). Briefly, methanol plant extract (61.7 – 93.7 mg GAE per gram
0.1 mM solution of DPPH in methanol was prepared plant parts) was higher compared with aqueous plant
and 1 ml of this solution was added with 3 ml of extract (18.5 – 25.8 mg GAE per gram plant parts).
respective extracts solution (aqueous or methanolic Based on the results obtained, phenolic compounds
extract) at five different concentrations. The mixture in both plants are more soluble in methanol
was shaken vigorously and allowed to stand at room compared with water. Phenolic compounds and
temperature for 30 min. Then the absorbance was their derivatives are secondary metabolites in many
measured at 517 nm using UV-VIS spectrophotometer plants. They are safe to be consumed by human and
(Shimadzu UV Visible Recording Spectrophotometer animal (Chew et al., 2009). Basically, the chemical
Model UV-160A). Lower absorbance of the mixture forms of natural phenolic acids are distinguishable
indicates higher free radical scavenging activity. by hydroxycinnamic and hydroxybenzoic acids
Positive control was prepared by mixing 2 ml structures (Qiu et al., 2010).
of ascorbic acid (0.05mg/ml) and 1 ml of DPPH The amount of TPC for methanol extract in
(0.04mg/ml), whereas negative control was prepared Clitoria ternatea were 61.7 mg GAE per gram flower
by mixing distilled water with 1 ml of DPPH. About and 64.8 mg GAE per gram leaves while for water
3 ml of the mixture (final concentration of 25, extract, 20.7 mg GAE per gram flower and 18.5 mg
50,100, 125 and 150 µg/ml) prepared from working GAE per gram leaves. Comparing this result with
solution was added to 1 ml of DPPH. The mixture that of Vitex negundo Linn., the amount of TPC
was gently homogenized and left to stand at the room from methanol extract of flowers and leaves were
temperature for 30 min. Absorbance was read using 83.3 mg GAE per gram and 93.7 mg GAE per gram,
spectrophotometer (Shimadzu UV Visible Recording respectively. Lower TPC was obtained from water
Spectrophotometer Model UV-160A) at 517 nm. extract of both samples, which were 18.8 mg GAE
per gram and 25.8 mg GAE per gram, respectively.
Statistical analysis Both amount were lower compared to total phenolic
All experiments were repeated three times. compounds found in C. siamea flower with 257 mg
Results for total phenolic contents were reported GAE per gram sample (Kaur et al., 2006) but higher
as means ± SD. Significant differences for multiple compared to TPC found in pineapple (34.7 mg GAE
comparisons were determined by one-way analysis per 100 gram) and guava (153 mg GAE 100 gram) as
of variance (ANOVA) followed by Duncan test reported by Alothman et al. (2009).
with p value less than 0.05 which was considered as This study found that Vitex negundo Linn.
statistically significant by SPSS statistical package contains higher amount of TPC compared to Clitoria
(ver.17.0). ternatea. For Vitex negundo Linn., TPC in methanol
and water extract ranged from 83.3 to 97.3 mg GAE
per gram sample and 18.8 to 25.8 mg GAE per gram,
respectively. While TPC obtained were 61.7 – 64.8
498 Rabeta, M. S. and An Nabil, Z./IFRJ 20(1): 495-500

Table 1. Comparison between total phenolic compound in Clitoria acid, flavonoids, and anthocyanins. Commonly,
Ternatea and Vitex Negundo Linn. Of Methanol and Water Extract
flavonoids will be extracted from plant materials
Plant species Parts used
mg GAE per g samples together with methanol, ethanol, or water that was
Water extraction Methanolic extraction
used as extractor solvent.
Clitoria ternatea Flowers 20.7 ± 0.1b 61.7 ± 0.2a
Anthocyanin may contribute to the amount of
Leaves 18.5 ± 0.4a 64.8 ± 0.1b
Vitex negundo Linn. Flowers 18.8 ± 0.1a 83.3 ± 0.1c TPC obtained from the plant samples as it is one
Leaves 25.8 ± 0.2c 93.7 ± 0.4d of the important group of water-soluble pigments
*
Means with different superscript in any column are significantly different at p < 0.05,
according to Duncan’s Multiple-Range Test (n = 3)
in plants (Clifford, 2000). This pigment is very
important in both Clitoria ternatea flower and Vitex
negundo Linn. leaves as their colour were blue and
mg GAE per gram sample and 18.5- 20.7 mg GAE purple. Acidic methanol has been used to leach the
per gram samples for methanol and water extraction dry methanol extract. Naczk and Shahidi (2004),
of Clitoria ternatea, respectively. stated that anthocyanin is usually extracted from
Phenolic compounds are water soluble natural plant material by using acidified organic solvent
antioxidants which normally an aromatic ring bearing especially methanol. Acidified organic solvent
one or more hydroxyl substituent. They are usually used will destroy cell membranes, simultaneously
located in the vacuole of the plant cells and may dissolves anthocyanin thus stabilizing them.
combine with sugar, to be glycosides (Waterhouse,
2002). When comparing TPC based on the parts of DPPH free radical-scavenging activity
plant used, Clitoria ternatea flower exhibited higher Determination of DPPH scavenging activity in
TPC content compared with the leaves part in water both plants, methanol and aqueous extract, was done
extract sample. This result obtained were supported in triplicates. DPPH is a compound that consists of a
by statements by Terahara et al., (1998) and Sethiya nitrogen free radical which is easily destroyed by a
et al., (2009) even though pigments present in Clitoria free radical scavenger (Chew et al., 2009). Scavenging
ternatea are low but it has high stability in water. activity of the mixture was measured using UV-
Because of its high stability in aqueous solution, the Vis at 517 nm after being incubated for 30 min in a
flower pigment is used in South-East Asia as food dark place. Five different concentrations of samples
colorant (Terahara et al., 1998; Mukherjee et al., 2008) extracts were tested to identify the significance of
had also reported the wide use of the blue flowers of extract concentration on the scavenging activity.
the plant due to its stability. Going further, according Water and DPPH mixture was used as negative control
to Sethiya et al., (2009) the extractive value (w/w, while ascorbic acid was used as positive control (Fu
%) of Clitoria ternatea is higher in water (18.21%) et al., 2009).
compared to its extractive value in alcohol (16.14%). The amount of scavenging activity in each plant
However, in methanol extracted sample, Clitoria extract of both extractions is shown in Table 2.
ternatea leaves part contained higher TPC. This From the table, it can be observed that, percentage
situation may happen due to the solubility nature of of DPPH scavenging activity for all plant extract
plant phenolics that has been enhanced by the organic increased with the concentration of plant extract
solvent (methanol), which facilitates solubilisation used. This statement is true for all methanol plant
through penetration in plant cell structure (Moure et extract with concentration; 25, 50, 100 and 125 μg/
al., 2001). ml. However, at 150 μg/ml extract concentration, the
Folin-Ciocalteau assay only gave crude estimate scavenging activity of the extract decreased (like in
of the total phenolic compound present in an extract. Clitoria ternatea flowers, Vitex negundo Linn. flowers
It does not measure specific polyphenols, but many and leaves) or maintained at the same amount like for
interfering compounds may react with the reagent, thus the 125 μg/ml extracts.
giving elevated apparent phenolics concentrations It can be seen that Vitex negundo Linn. extracts
(Prior et al., 2005). Moreover, Tawaha et al., (2007), exhibited more scavenging activity towards DPPH
stated that various phenolic compounds will respond radicals compared to Clitoria ternatea extract in
differently to the reagent, depending on the number methanol extracted sample solution. A study done by
of phenolic groups they have and total phenolic does Tiwari and Tripathi (2006) has shown that all fraction
not incorporate necessarily all antioxidant that may of Vitex negundo Linn. have potent scavenging activity
be present in the fraction or extract. for ABTS radical cation in a dependent manner. They
Several compounds may contribute to the amount explained that this situation may be attributed to
of TPC in the plant extract. According to Naczk and the existence of polyphenols and flavonols in Vitex
Shahidi (2004), those compounds are mainly phenolic negundo Linn.
 Rabeta, M. S. and An Nabil, Z./IFRJ 20(1): 495-500 499

Table 2. DPPH scavenging activity in plant extracts (methanol and water extracts)

% scavenging activity per sample extract concentration (ug/ml)

Extraction Sample and
solvent parts Sample Concentration (μg/ml)

25 50 100 125 150

Clitoria ternatea
32.67 ± 1.155a 353.33 ± 3.055 b 411.33 ± 1.155 d 422.67 ± 3.055 e 401.33 ± 2.309 c
flower

Clitoria ternatea
64.67 ± 1.155a 264.00 ± 4.00b 408.67 ± 4.163 c 472.00 ± 4.00d 472.00 ± 4.00e
leaves

Methanol
Vitex negundo
399.33 ± 3.055 a 442.00 ± 2.000 b 471.33 ± 3.055 d 491.33 ± 1.155 e 464.00 ± 4.000 c
Linn. flower

Vitex negundo
453.33 ± 2.309 d 402.67 ± 2.309 a 514.67 ± 4.619 e 437.33 ± 4.619 c 406.67 ± 2.309 b
Linn. leaves

Clitoria ternatea
390.67 ± 2.309 a 401.33 ± 3.055 b 449.33 ± 2.309 c 490.67 ± 4.619 d 506.67 ± 2.309 e
flower

Clitoria ternatea
2.67 ± 1.155a 8.67 ± 1.155b 103.33 ± 1.155 d 102.67 ± 4.619 c 130.67 ± 4.619 e
leaves

Water
Vitex negundo
145.33 ± 2.309 a 273.33 ± 2.309 b 410.67 ± 4.619 e 398.67 ± 6.110 d 394.67 ± 1.155 c
Linn. flower

Vitex negundo
184.00 ± 2.00a 284.67 ± 1.155 b 391.33 ± 1.155 c 404.67 ±3.055e 392.67 ± 3.055 d
Linn. leaves

* Means with different superscript in any column are significantly different at p < 0.05, according to Duncan’s Multiple-Range Test (n = 3)

In contrast, DPPH scavenging activity in water et al., (2009) had shown that there was a significant
extracted sample greatly deviates between both correlation between total phenolics and antioxidant
samples. Clitoria ternatea flower exhibited very high capacity of fruits extracts, namely honey pineapple,
scavenging activity by the antioxidant compared with pisang mas, and guava and this correlation confirms
the scavenging activity expressed by both parts of that the phenolic compounds are the main micro
Vitex negundo Linn. and Clitoria ternatea leaves. This constituents contributing to the antioxidant activities
complies with the result of total phenolic compounds of these fruits.
that has been discovered before. For total phenolic
compounds in water extracted Clitoria ternatea Conclusion
flower, high amount of total phenolic compounds has
been found. Thus, it can be concluded that scavenging It can be concluded that methanol is a more
activity expressed by Clitoria ternatea flower was efficient solvent in extracting phenolics compound
affected by the amount of total phenolic compound. compared with water. Total phenolics compound
In a study by Chew et al., (2009), significantly higher found in methanol extracted sample was between
phenolic content and strong free radical scavenging 61.7 to 64.8 mg GAE per gram samples for Clitoria
activity were found in B. Kockiana flower compared ternatea while 83.3 to 93.7 for Vitex negundo Linn..
with the leaf. This was due to the role of anthocyanin As for water extracted sample, range of TPC obtained
as antioxidant. was between 18.5 to 20.7 mg GAE per gram samples
Generally, it is well known that plant phenolics for Clitoria ternatea and 18.8 to 25.8 for Vitex
are highly effective free radical scavengers negundo Linn. Vitex negundo Linn. contained higher
and antioxidants. The research presented by amount of total phenolics compound compared to
Maisuthisakul et al., (2007) has reported that the Clitoria ternatea with leaves of both plants showed
phenolics compounds and its derivatives such as higher amount of phenolics compound compared with
phenolic acids and tannins were strongly correlated the flower part. As for the DPPH scavenging activity,
with antioxidants. In this study, all samples did type of solvent used to extract the plant material and
followed the trend but the only sample that totally concentration of extracts used produced significance
exhibit this trend was water extracted Clitoria difference on the amount of DPPH scavenged by
ternatea flower; which has been shown to have both the plant extract. Both Clitoria ternatea and Vitex
high TPC and scavenging activity towards DPPH negundo Linn. are readily available and can be a
radical. The purple colour in both plants could be source of natural antioxidants. Further studies are
due to anthocyanin compounds. Study by Alothman needed to evaluate the antioxidant activities in Nasi
500 Rabeta, M. S. and An Nabil, Z./IFRJ 20(1): 495-500

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