LWT - Food Science and Technology 50 (2013) 634e641

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LWT - Food Science and Technology

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Antioxidant and antimicrobial activities of various solvent extracts, piperine and

piperic acid from Piper nigrum
Zied Zarai*, Emna Boujelbene, Nadia Ben Salem, Youssef Gargouri, Adel Sayari
Laboratoire de Biochimie et de Gnie Enzymatique des Lipases, ENIS, BP 1173, University of Sfax, 3038 Sfax, Tunisia

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

Article history: Black pepper (Piper nigrum L.) has long been regarded as a spice added to many foods and it is also
Received 30 July 2011 considered as a medicinal plant. The predominant compound obtained from ethanolic extract of P. nig-
Received in revised form rum, the piperine, was puried and identied by HPLC, 13C NMR and by FT-IR analysis. Piperic acid was
30 June 2012
synthesized by alkaline hydrolysis of the puried piperine. The antioxidant and the antibacterial activ-
Accepted 24 July 2012
ities of different solvent extracts from P. nigrum and puried piperine and piperic acid were determined
by using various in vitro tests. In all ours experiments, synthesized piperic acid was found to have the
Keywords:
highest antioxidant power and was the most effective with the minimum inhibitory concentration
Piper nigrum L.
Piperic acid
(<325 mg/ml) against all strains tested. This comparative report indicates that these compounds, piperine
Alkaline hydrolysis and piperic acid, could be used as natural antioxidant and antibacterial agents in both food preservation
Antioxidant activity and human health.
Antimicrobial activity 2012 Elsevier Ltd. All rights reserved.
The minimum inhibitory concentration

1. Introduction
Herbs and spices, which are important part of the human diet,
have been used for thousands of years in traditional medicine and
to enhance the avour, colour and aroma of foods. In addition to
boosting avour, herbs and spices are also known for their
preservative (Neilsen & Rios, 2000), antioxidative (Shobana &
Naidu, 2000), and antimicrobial (Salie, Eagles, & Leng, 1996) roles.
Black pepper or Piper nigrum is one of the most popular spice
products in oriental countries (mostly in Southeast Asia). P. nigrum
is a plant of the piperaceae family, largely used as a avouring agent
in foods. Its characteristic aromatic odour is due to the volatile oils
in the cells of the pericap (Murthy & Bhattacharya, 2008). It has
been traditionally used for the treatment of malaria in India and the
epilepsy in China (Majeed & Prakash, 2000). Moreover, black
pepper has an anti-inammatory activity manifested by stimu-
lating the production of an anti-inammatory cytokine like (IL-10).
On the other hand, black pepper inhibits the expansion of genes
encoding the nitric oxide synthase (iNOS) and the cyclooxygenase-
Abbreviations: DPPH, 1,1 diphenyl-2-picrylhydrazyl; TCA, trichloroacetic acid;
BHT, butylated hydroxytoluene; HPLC, High Performance Liquid Chromatography;
Na2CO3, sodium carbonate; CDCl3, deuterated chloroform; MTT, 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.
* Corresponding author. Tel./fax: 216 74 675 055.
E-mail address: zaraizied@hotmail.fr (Z. Zarai).
2 (COX-2) (Mueller, Hobiger, & Jungbauer, 2010). The iNOS and COX-
2 stimulate the production of many pro-inammatory mediators
such as (Interleukin-4 (IL-4), Interleukin-10 (IL-10), Interleukin-13
(IL-13), interferon-alpha (a-IFN)), and the transformation growth
factor b-TGF (Hanada & Yoshimura, 2002; Makarov, 2000).
Piperine (1-piperoyliperidine), which belongs to the alkaloid
family, represents the major component in the dry fruit of P. nigrum.
Piperine has been reported to have several pharmacological effects
such as anti-diarrhoeal and hepatoprotective (Bajad, Bedi, Singla, &
Johri, 2001; Koul & Kapil, 1993). Some studies have shown that
piperine possesses an anti-inammatory and an analgesic effect
(Gupta, Bansal, Bhardwaj, & Velpandian, 2000). In addition, it has a
high antioxidant activity and is used for the treatment of Alzheimer
diseases (Chonpathompikunlert, Wattanathorn, & Muchimapura,
2010; Selvendiran, Vijeya Singh, Krishnan, & Sakthisekaran, 2003).
The chemical modication in the structure of piperine to piperic acid
was conrmed by the appearance of the carboxyl group during the
hydrolysis. Piperic acid has a high anti-hyperlipidemic activity (Han
et al., 2008). Recently, piperine and its derivatives have been eval-
uated for their inhibitory effects against epimastigote and amasti-
gote (Ribeiro et al., 2004; da Silva Ferreira et al., 2008).
The aim of this work was to examine the efciency of different
solvents for the extraction of the major compounds from P. nigrum,
traditionally used for their medicinal properties, and evaluate the
antioxidant and antimicrobial activities of the different extracts and
the puried molecules (piperine and piperic acid).

0023-6438/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2012.07.036
 Z. Zarai et al. / LWT - Food Science and Technology 50 (2013) 634e641
2. Materials and methods
2.1. Chemical reagents and standards
To perform experiments, several materials were provided. In
fact, 1,1 diphenyl-2-picrylhydrazyl (DPPH), b-carotene, linoleic
acid, tween 40, potassium ferricyanide, MTT [3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide], trichloroacetic
acid (TCA), ferric chloride and FolineCiocalteu reagents were
purchased from Sigma chemicals (Steinheim, Germany). Butyl-
ated hydroxytoluene (BHT) was purchased from Prolabo (Paris,
France). Gallic acid and quercetin were obtained from Fluka
(Buchs, Switzerland). All other chemicals and solvents were of
analytical grade.
2.2. Polyphenol extraction
2.2.1. Preparation of crude extracts
The material used in this work consisted of berries of
P. nigrum purchased from the local market. Black pepper
(P. nigrum) powder (5 g) was mixed with 100 ml of solvents at
different polarities (chloroform, ethyl acetate, ethanol, methanol
and water). The mixture was boiled for 4 h under the water-bath
reux. After extraction, the suspensions were cooled and ltered
through a buchner funnel. The various solvent extracts were
concentrated using a rotary evaporator (Buchi Rotavapor R-200)
at 50  C. The water extract was lyophilized and the extraction
yield was calculated based on the dry weight of the spice
P. nigrum. After lyophilization the extract was ground and the
resulting powder was packed in a glass bottle and stored at 4  C,
until needed.
2.2.2. Total phenolic content
The total amount of phenolics was determined by the
FolineCiocalteu reagent as previously described (Slinkard &
Singleton, 1977). A volume of 0.5 ml of each sample extract was
mixed with 0.5 ml of the FolineCiocalteu reagent. After 5 min,
0.5 ml of 20% sodium carbonate (Na2CO3) solution was added and
the solution was brought up to 5 ml by adding distilled water.
After a 90 min-incubation at room temperature and under
conditions of darkness, the absorbance was measured at 760 nm.
Gallic acid was used as standard reference. The concentration of
total phenolic compounds on the different extracts of black
pepper was determined as micrograms of gallic acid equivalent/g
of P. nigrum. The equation obtained from the standard gallic acid
graph is as follows:


Absorbance 0:0074 mg gallic acid 0:023 R2 0:99
2.2.3. Total avonoid content
The total avonoid content was determined as previously
described (Zhishen, Mengcheng, & Jianming, 1999). An aliquot of
each sample (250 ml) was mixed with 1 ml of distilled water and
subsequently with 150 ml of 150 mg/ml sodium nitrite solution.
After a 6 min-incubation, 75 ml of 100 mg/ml aluminium chlo-
ride solution was added, and then the mixture was left for
a further 5 min before 1 ml of 40 mg/ml sodium hydroxide
(NaOH) solution was added. Distilled water was immediately
added to the mixture until the total volume reached 2.5 ml. The
absorbance was measured at 510 nm. Quercetin was used as
a reference compound. The total avonoid content was calcu-
lated and expressed as mg quercetin equivalent (QE)/g of
P. nigrum. The calibration recorded for this standard was
expressed as follows:
635


Absorbance 0:504 mg quercetin 0:014 R2 0:99
2.2.4. Isolation of piperine from crude extracts
The isolation of piperine, a major component of P. nigrum, was
carried out, as previously described (Han et al., 2008), under heat-
reux extraction using the ethanol extract from P. nigrum. The pale
yellow product obtained was dissolved in ethanol. A 20 ml of the
obtained solution (0.02 mg/ml) was injected into High Performance
Liquid Chromatography (HPLC). During this analysis, the column C-
18 (250 mm  4.6 mm, Shimadzu) was kept at a temperature of
35  C and the separation of piperine was performed by using an
isocratic gradient of aqueous solution of potassium dihy-
drogenphosphate (KH2PO4) (50 mM, pH 3.5) and acetonitrile
(40:60 v/v) at a ow rate of 0.6 ml/min.
2.2.5. Preparation of piperic acid from piperine extract
Piperic acid was prepared by alkaline hydrolysis of piperine
according to a previously described protocol (Mishra et al., 2005).
Piperine (1 g) was dissolved in 30 ml of anhydrous ethanol con-
taining 20% potassium hydroxide (KOH) in a 50 ml reaction ask.
The mixture was heated at reux with stirring for 10 h to give the
precipitate of potassium piperate. The precipitate was washed with
ethanol, then dissolved in distilled water and puried by adding
HCl solution (0.1 M). Piperic acid was identied by HPLC. During the
hydrolysis the decrease of the peak intensity corresponding to
piperine extract was accompanied by the appearance of a new peak
corresponding to the piperic acid.
2.3. Piperine and piperic acid analysis
2.3.1. TLC and HPLC analysis
The product obtained after different times of alkaline hydrolysis
of piperine was analysed by Thin Layer Chromatography (TLC) on
silica gel previously activated at 60  C during 30 min. The migration
was performed with a mixture of acetone/hexane (3/2, v/v). After
that, the spots were visualized with iodine vapour. The piperic acid
obtained after alkaline hydrolysis was identied by HPLC system.
2.3.2. NMR and FT-IR analysis
The evidence of transformation of piperine into piperic acid was
checked by 13C NMR (Madison, USA). Samples were dissolved in
Deuterated chloroform (CDCl3) containing trace amounts of tetra-
methylsilane, which was used as an internal chemical shift refer-
ence to indicate, in parts per million (ppm), the difference of the
resonance frequency.
The structure of piperine and piperic acid was also determined
by FT-IR NEXUS spectrophotometer (Nicoleit, Madison, WIS, USA)
showing the appearance of the carboxyl group of piperic acid and
the disappearance of the amide group of piperine. The sample was
mixed with KBr. FT-IR spectra were acquired after 32 scans between
4000 and 400 cm1, with spectral resolution of 400 cm1.
2.4. Antioxidant activity of solvent extracts, piperine and
piperic acid
To evaluate the antioxidant rather of the 5 solvent extracts of
P. nigrum, puried piperine and piperic acid, several in vitro tests
have been used. BHT was used as a positive control.
2.4.1. DPPH radical-scavenging assay
1,1 Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay
was determined as previously described (Bersuder, Hole, & Smith,
1998) with some modications. A stock solution (10 mg/ml each)
636 Z. Zarai et al. / LWT - Food Science and Technology 50 (2013) 634e641
of solvent extracts, puried piperine and piperic acid were
prepared in absolute ethanol. All samples were used at concen-
trations in the range of 20e300 mg/ml. A volume of 500 ml of each
sample was mixed with 500 ml ethanol and 125 ml of freshly
prepared solution of 0.02% DPPH in 99.5% ethanol. The mixture
was shaken vigorously and incubated at room temperature in
darkness during 60 min. The absorbance of the remaining DPPH
radicals was read at 519 nm using a Shimadzu UV mini-1240
UVeVIS spectrophotometer (Paris, France). The scavenging of
DPPH radical was calculated according to the following equation:
DPPH Radical  scavenging activity%
h
. i
Acontrol  Asample Acontrol  100
where Acontrol and Asample are the absorbance of the control and of
the sample respectively. The values are presented as the means of
triplicate analysis.
2.4.2. Reducing power assay
The reducing power was investigated based on the method
previously described (Yildirim, Mavi, & Kara, 2001) with some
modications. An aliquot of each sample (0.5 ml) was mixed
with phosphate buffer (1.25 ml, 0.2 M, pH 6.6) and potassium
ferracyanide (1.25 ml, 1%). The mixture was incubated in a water
bath at 50  C for 30 min. After that, TCA (1.25 ml, 20%) was
added. Then, the samples were centrifuged for 10 min at 1650 g.
An aliquot of 1.25 ml of the upper layer was mixed with 1.25 ml
of distilled water and 0.25 ml of 0.1% (w/v) ferric chloride
solution. After 10 min incubation, the absorbance of the reaction
mixture was measured at 700 nm. A higher absorbance of the
reaction mixture indicates greater reducing power of the
sample. The values were presented as the means of triplicate
analysis.
2.4.3. b-Caroteneelinoleic acid assay
The antioxidant activity of the ve solvent extracts, the
piperine and the piperic acid was evaluated in b-
caroteneelinoleic acid system, according to the protocol previ-
ously described (Koleva, van Beek, Linssen, de Groot, &
Evstatieva, 2001). A stock solution was prepared as follows:
0.5 mg b-carotene, 25 ml of linoleic acid and 200 ml of tween 40
were dissolved in 1 ml of chloroform (HPLC grade). Chloroform
was completely evaporated under vacuum in rotavapory at 40  C,
then, 100 ml of distilled water was added with vigorous shaking.
The reaction medium contained 500 ml of each sample and 2.5 ml
of the freshly prepared emulsion. All the test tubes were imme-
diately placed in a water bath at 50  C for 2 h. The absorbance
was measured at 470 nm before and after heat treatment. A
control containing 0.5 ml of ethanol instead of the sample solu-
tion was carried out in parallel. The values were presented as the
means of triplicate analysis.
2.4.4. Phosphomolybdate assay
The phosphomolybdate method (Prieto, Pineda, & Aguilar, 1999)
has been used to evaluate the antioxidant capacity of the ve
solvent extracts, the piperine and the piperic acid. Aqueous sample
(0.1 ml) was added to 1 ml of reagent solution (0.6 M sulphuric acid,
28 mM sodium phosphate and 4 mM ammonium molybdate). The
tubes were capped and incubated in a water bath at 90  C for
90 min. After that, the mixture of each sample was cooled to room
temperature and the absorbance was measured at 695 nm against
a blank. BHT was used as standard. The activity of each sample was
expressed as a-tocopherol equivalent using the following linear
equation:


Absorbance 0:011 C 0:0049; R2 0:987
where C is the concentration of a-tocopherol equivalent (mmol/ml).
The values are presented as the means of triplicate analysis.
2.5. Antibacterial activity
In order to determine the antimicrobial activity of the different
solvent extracts, piperic acid and piperine, various Gram-positive
and Gram-negative bacteria were used: Escherichia coli (ATCC
25922), Klebsiella pneumonia (ATCC 27853), Salmonella enterica
(ATCC 43972), Staphylococcus aureus (ATCC 25923), Staphylococcus
epidermidis (ATCC 14990), Enterococcus faecalis (ATCC 29122), and
Bacillus subtilis (ATCC 6633). Staphylococcus xylosus was isolated in
LBGEL from waste water (Mosbah, Sayari, Mejdoud, Dhouib, &
Gargouri, 2005).
2.5.1. Agar diffusion method
The agar diffusion method was employed for the determina-
tion of antibacterial activities of the solvent extracts, piperine
and piperic acid according to the method described by Berghe
and Vlietinck (1991). The dried extracts were dissolved in 100%
dimethyl sulfoxide (DMSO) to a nal concentration of 10 mg/ml
and sterilized by ltration trough 0.22 mm Nylon membrane lter.
The bacterial strains were cultured in a nutriment broth for 24 h.
Then, 200 ml of each suspension bacteria (106 colony-forming
unit estimated by absorbance at 600 nm) was spread on Luria
Broth agar. Bores were made by using a sterile borer and were
loaded with 10 ml of each sample extract. Dimethyl sulfoxide
(DMSO) was used as negative control and ampicillin (10 mg/well)
as positive reference standard. All the plates were incubated at
37  C for 24 h. Antibacterial activity was evaluated by measuring
the zone of inhibition in millimetres. All experiments were done
in triplicates.
2.5.2. Determination of the minimal inhibitory concentration (MIC)
The minimal inhibitory concentration (MIC) values, which
represent the lowest plant extracts concentration that completely
inhibits the growth of microorganisms, were determined by
a micro-well dilution method (Wade et al., 2001). The inoculum
of each bacteria was prepared and the suspensions were adjusted
to 106 CFU/ml. All the extracts were dissolved in 100% DMSO and
then dilutions series were prepared in a 96-well plate, ranging
from 7.8 mg/ml to 5 mg/ml. Each well of the microplate included
40 ml of the growth medium, 10 ml of inoculums and 50 ml of the
diluted sample extract. The ampicillin and DMSO are used as
positive and negative controls, respectively. The plates were then
covered with the sterile plate and incubated at 37  C for 24 h.
After that, 40 ml of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) at a nal concentration 0.5 mg/ml
freshly prepared in water was added to each well and incubated
for 30 min. The change to red colour indicated that the bacteria
were biologically active. The MIC was taken to the well, where no
change of colour of MTT was observed. The MIC values were done
in triplicate.
2.6. Statistical analysis
All the experiments were carried out in triplicates. Tests of
signicant differences between means were determined by Dun-
cans multiple range tests at a signicance level of 0.05 using
statistical package for the social sciences SPSS 11.5 (SPSS Inc., Chi-
cago, IL, USA).
 Z. Zarai et al. / LWT - Food Science and Technology 50 (2013) 634e641
3. Results and discussion
3.1. Extraction yield and total phenolic and avonoid contents
The P. nigrum components were extracted by traditional reux
method using solvents with different polarities (chloroform, ethyl
acetate, ethanol, methanol and water). The yields of the different
solvent extracts as well as the total phenolic and avonoid contents
obtained are presented in Table 1. From this table, we note that the
extraction yields with methanol and ethanol resulted in the highest
amount of total extractable compounds whereas the lowest yields
were obtained with ethyl acetate, chloroform, water. In fact, this
variation in the yields can be attributed to the polarities of the
different compounds present in the spice. One can also note from
the data presented in Table 1 that there are differences in the total
phenolic and avonoid contents of the different extracts. The
highest levels of total phenolic content were found in the ethanol
extract (45.08 mg of gallic acid equivalent/g of P. nigrum) followed by
the methanol, chloroform and ethyl acetate, while total phenolic
content of the water extract was the lowest (101.09 mg of gallic acid
equivalent/g of P. nigrum). This result is in agreement with this
previously obtained by Han et al. (2008) who reported that ethanol
was the suitable solvent for extraction of phenolic compounds from
P. nigrum.
The content of total avonoids from P. nigrum, varied from 0.29
(water extract) to 5.02 mg (chloroform extract) of quercetin equiv-
alents/g of P. nigrum (Table 1). The results clearly showed that the
highest content of avonoid was obtained with a decrease in the
polarity of the solvent used. The variations in the phenolic and
avonoid compounds of the different extracts may be attributed to
the polarities of compounds present in the raw material.
3.2. Piperine and piperic acid: preparation and analysis
3.2.1. Piperine and piperic acid preparation
One of the natural products that forms a major constituent of
P. nigrum is commonly known as piperine just like many other
amides both from natural and synthetic sources which possess
many biological activities. Most reported methods (Kanaki, Dave,
Padh, & Rajani, 2008; Stahl & Schild, 1981, pp. 410e411;
Staudinger & Schnieider, 1923) for the isolation of piperine are
based on several processing steps, often under difcult operating
conditions, which results in a high cost of production. Here, we
have used traditional reux method, one of the most widely used
traditional techniques for piperine extraction, using solvents with
different polarities (chloroform, ethyl acetate, ethanol, methanol
and water) (data not shown). The reported method was found to
give a high yield of piperine of fairly pure quality in a very rapid and
economical way. The highest purication yield obtained by this
method, using ethanol as extraction solvent, was 3.78% (data not
shown). Similar results were previously obtained by Kanaki et al.
Table 1
Extraction yield and total phenolics and avonoids contents from Piper nigrum.
Extraction solvent Extraction Total phenolics* Total avonoids*
yield (%)* (mg gallic acid/g (mg quercetin/g
Piper nigrum) Piper nigrum)
Chloroform 35.95  0.65c 25.03  0.38c 5.02
Ethyl acetate 16.14  0.78d 22.69  0.58d 3.65
Methanol 49.09  0.98b 37.48  0.67b 3.01
Ethanol 55.91  1.42a 45.08  2.21a 1.79
Water 8.72  1.05e 1.09  0.10e 0.29
* Data expressed as mean of three data points (n 3).
a, b, c, d and e: different letters within same column means signicant different at
p < 0.05.
637
(2008) who developed a new method for the extraction of
piperine from P. nigrum and showed that the highest yield obtained
was 4%.
3.2.2. HPLC and TLC analysis
The puried piperine from ethanol extract was analysed and
identied by HPLC. Pure commercial piperine was used as reference
for HPLC identication. The obtained results showed the presence
of a single peak corresponding to piperine emerging at the same
time as the pure piperine reference.
Similar to the standards, the extracted piperine was eluted at
a retention time of 6.45 min and corresponds to the principle
compound found in P. nigrum.
The crude powder obtained after ethanol extraction was also
used to prepare piperic acid. After different times of alkaline
hydrolysis of piperine extract, the precipitated piperic acid ob-
tained was analysed by Thin Layer Chromatography on silica gel.
One can note that the intensity of the spot of the initial piperine
used decreased and a new spot appeared during the hydrolysis
process (data not shown).
After a washing step with HCl solution and water, the yellow
precipitate of piperic acid was ltrated and analysed by HPLC.
Similar to the piperic acid standard, the obtained piperic acid was
eluted at a retention time of 4.9 min (data not shown). The results
obtained showed the presence of a major peak corresponding to
piperic acid and a small peak corresponding to the residual
piperine.
3.2.3. 13C NMR and FT-IR analysis
The chemical structures of commercial piperine (Fig. 1A),
piperine puried from P. nigrum (Fig. 1B) and piperic acid (Fig. 1C)
were determined by 13C NMR.
Compared to the spectrum of standard and extracted piperine,
which presented the same bands, a new strong band appeared
above 168.86 ppm, corresponding to the carboxyl group of piperic
acid. The intensity of the band at 165.53 ppm corresponding to the
amide group decreased and disappeared after transformation of the
piperine into piperic acid. The appearance of the band at
168.86 ppm and the disappearance of the one at 165.53 ppm could
be considered as an argument for the transformation of piperine
into to piperic acid because of the vibration of the carboxyl group
and the amide group used to reside in this region.
The transformation of piperine into piperic acid was also
conrmed by FT-IR analysis. From Fig. 2 we can see the appearance
 0.58a
 0.62b
 0.35c
 0.05d
 0.03e
Fig. 1. 13C NMR spectra of commercial piperine (A), piperine puried from P. nigrum (B)
and piperic acid (C). The peaks corresponding to the carboxyl and amide groups are
marked by arrows.
638 Z. Zarai et al. / LWT - Food Science and Technology 50 (2013) 634e641

of a band emerging at 1678.76 cm1 (Fig. 2B) which corresponds to

the carboxyl group of piperic acid. Fig. 2A shows the disappearance
of the band at 1633.95 cm1, corresponding to the amide group of
initial piperine (Fig. 2A). Bands between 2500 and 3000 cm1
correspond to the eCeH aromatic.

3.3. Antioxidant activity

Several in vitro tests were adopted to evaluate the antioxidant

activity of solvents extracts, piperine and piperic acid at different
concentrations and the results were compared to BHT used control
(Fig. 3).

3.3.1. DPPH radical scavenging activity

Fig. 2. FT-IR spectra of piperine before (A) and after (B) its transformation to piperic
acid. The peaks corresponding to the carboxyl and amide groups are marked by arrows.
The 1,1 diphenyl-2-picrylhydrazyl (DPPH) is a stable free radical
with a characteristic absorbance at 517 nm. It is one of the most
commonly substrates used to evaluate the antioxidant activity
(Yamaguchi, Takamura, Matoba, & Terao, 1998). This assay is based
on the ability of DPPH to react with a proton-donor substrate. The
decrease of the absorbance of reduced DPPH is used to evaluate the
radical-scavenging potential of the samples.
Fig. 3A shows the DPPH radical scavenging activity of various
solvent extracts from P. nigrum. As it can be deduced from this
gure, the scavenging activity of all samples was concentration
dependant. The result indicated clearly that the ethanol extract,
which contained the highest amount of total phenolics, showed
a high antioxidant activity 65.59% at a nal concentration of 50 mg/
ml. At the same concentration, 60% of radical scavenging activity

Fig. 3. Antioxidant activities of solvent extracts: methanol (), ethanol (:), ethyl acetate (6) choloroform (A) and water (>), puried piperine (B) and piperic acid (C) tested with
four methods: (A) DPPH radical-scavenging, (B) reducing power activity, (C) b-caroteneelinoleic acid assay and (D) Phosphomolybdate assay. For each method, a positive control
using BHT (-).
 Z. Zarai et al. / LWT - Food Science and Technology 50 (2013) 634e641 639

was observed with the BHT, used as standard antioxidant. The

156.25b
156.25b
78.12c
312.5b

312.5b
lowest radical scavenging activity was observed with water

312.5c
625b
625a
extract.

PA
Fig. 3A showed the DPPH radical scavenging of puried
piperine and piperic acid at different concentrations. The results

312.5a

312.5a
312.5a
clearly indicated that piperic acid presented a higher radical

625a

625a

625a
625b
625b
scavenger than piperine. In fact, at a nal concentration of 50 mg/

Pip
ml, the percentage of radical scavenging activity of piperine and
piperic acid were 10.28% and 29.5%, respectively. These results

Water
show that the increase in scavenging activity after transformation

NS
NS
NS

NS
NS
NS
NS
NS
of piperine into piperic acid can be related to the appearance of
the eCOOH radical.

>2500
>2500
>2500

>1250
>1250
>2500
>2500
>2500
CHCl3
3.3.2. Reducing power assay
The reducing power assay of various solvent extracts, piperine

>2500
>2500
>2500

>625
>1250
>2500
>2500
>2500
and piperic acid may serve as a signicant index of their potential

EtOAc
antioxidant activity. In fact, the presence of reductants (antioxi-
dant) in samples causes the reduction of Fe3/ferracyanide
complex to ferrous form (Yildirim et al., 2000). Therefore Fe2

312.5a

>312.5
>312.5
complex can be monitored by measuring the formation of perls

>1250
>1250
>2500

>625
>625
MeOH
percussion blue at 700 nm (Chung, Chang, Chao, Lin, & Chou,
2002). Fig. 3B revealed that the reducing power activity of the

MIC (mg/ml)*
ve solvent extracts, piperine and piperic acid at various

156.25b

156.25b
156.25c

312.5b
312.5a
concentrations. It can be observed that their reducing power

1250a
1250a
625a
EtOH
increased with the increasing of the concentration of each extract

Inhibition zone (mm) and minimal inhibitory concentration (mg/ml) of solvent extracts, puried piperine and piperic acid of Piper nigrum.
(25e75 mg/ml). A difference in the reducing power is observed
between the ethanol extract and puried piperine which sug-

16.7  0.6a

0.6a
1.0a

1.3a
0.6c
1.0c
9.0  05a
gested the presence of other compounds acting in synergy with

10.51a
piperine to give a higher antioxidant power. However, no signi-






14.1
12.7
8.3
10.0
7.2
cant difference was observed between piperine and piperic acid

PA
(Fig. 3B).

0.8d
8.0  0.6b

1.2b

0.5a
9.7  0.1c

1.0c

1.0c
3.3.3. b-Caroteneelinoleic acid assay

10.21a






In this assay, an aqueous emulsion of b-carotene and linoleic

11.2
10.7
8.2
7.7
7.5
Pip

acid were used as a test to measure the antioxidant activity of

various solvent extracts, piperine and piperic acid (Fig. 3C). The
presence of antioxidant can reduce the extent of b-carotene
0.4e
0.5c
0.6c
0.6c
0.5c
1.8  0.5f

destruction by reacting with the linoleic acid free radical or any

Water






other free radical formed within the system (Amarowicz, Pegg,
7.5
8.5
5.3
9.3
5.3
NS
NS

a, b, c, d, e and f: different letters within same line means signicant different at p < 0.05.
Rahimi-Moghaddam, Barl, & Weil, 2004; Jayaprakasha, Singh, &
Sakariah, 2001). As shown in Fig. 3C, all solvent extracts inhibi-
10.7  0.6d

11.7  0.6b

ted the oxidation of b-carotene at various degrees. The ethanol

4.8  0.8d

2.3  0.6d
6.0  0.8e

8.7  1.0c
5.0  1.0c

extract showed a higher ability to prevent the bleaching of b-

CHCl3

carotene than all other solvent extracts. The discolouration of b-

NS

carotene is probably due to the higher amount of total phenolics

and avonoids present in the ethanol extract which could prevent
10.5  0.8d
10.7  1.5b
13.0  1.0a
13.0  0.1a
6.8  0.8d
4.1  0.4e

5.0  0.4c

the oxidation of b-carotene.

EtOAc

Its also noteworthy from the data presented in Fig. 3C that the
NS

transformation of piperine into piperic acid was accompanied by

Inhibition zone diameter (mm)*

an increase in the inhibition of the b-carotene oxidation. Thus, one

* Data expressed as mean of three data points (n 3).
1.5d
8.7  0.8b

0.5b
1.0b

0.8a

can conclude that the presence of carboxylic group in piperic acid

7.5  0.9c

9.5  0.5c

03a

probably protects the oxidation of b-carotene more than the






MeOH

12.0
11.0
7.3
13.8
7.8

amide group of piperine.

3.3.4. Total antioxidant capacity

8.3  0.8b

12.3  0.8b

0.5b

0.6b
0.1b
0.4b
1.0a
7.5  0.5c

In the phosphomolybdenum assay, which is a quantitative







method to evaluate the antioxidant capacity (Arabshahi-Delouee

EtOH

12.5
12.7
9.6
12.0
6.0

& Urooj, 2007), all solvent extracts exhibited different degrees of

antioxidant activity as shown in Fig. 3D. As we have seen above
the ethanol extract possesses the highest antioxidative effect
Bacterial strains

(48.2 mmol/ml a-tocopherol equivalents) at a concentration of

S. epidermidis
K. pneumonia

25 mg/ml. BHT used as a positive control, was found to be more

Gram (L)

Gram (D)
S. enterica

E. faecalis
S. xylosus
B. subtilis
S. aureus

efcient (57.28 mmol/ml a-tocopherol equivalents) at the same

E. coli
Table 2

concentration. Interestingly, one can note from Fig. 3D the

increase of the antioxidant capacity of piperine after its
640 Z. Zarai et al. / LWT - Food Science and Technology 50 (2013) 634e641
transformation into piperic acid. At a concentration of 100 mg/ml,
the piperic acid has higher antioxidant power (64.1 mmol/ml a-
tocopherol equivalents) than piperine (58.8 mmol/ml a-tocopherol
equivalents).
Total phenolic and avonoid content showed that phenolic
and avonoid compounds in the tested sample contributed
signicantly to their antioxidant capacity. Major types of
phenolic constituents identied in the sample extracts were
phenolic acids, phenolic diterpenes, avonoids, and volatile. The
ethanol extracts from P. nigrum had the highest antioxidant
activity and total phenolics content. A positive correlation was
observed between percentage antioxidant and total phenolic,
which increased with the higher level of samples. The present
study conrms that various solvent extracts, piperine and
piperic acid from P. nigrum contain signicant source of
phenolic and avonoid antioxidants that may have therapeutic
potential.
Therefore, the higher antioxidant activity of compounds ob-
tained after solvent extraction and purication from P. nigrum
suggests a possible biological functionality in preventing the
oxidative degradation of membrane lipids.
3.4. Antibacterial activity
The antibacterial activity of solvent extracts, puried piperine
and piperic acid was evaluated against Gram-positive (B. subtilus,
E. faecalis, S. xylosus, S. aureus and S. epidermidis) and Gram-
negative (E. coli, K. pneumoniae, S. enterica) strains by measuring
the inhibition zone diameter and the determination of MIC values
(Table 2).
As it can be seen in Table 2, all extracts show varying degrees
of antibacterial activity against most of the Gram-positive and
Gram-negative bacteria tested. The ethanol extract was found to
be the most effective. The ethanol extract displayed a broad
antimicrobial spectrum and exerted signicant antibacterial
effect against both Gram-positive and Gram-negative bacteria
tested. The most susceptible bacteria for the ethanol extract
were S. aureus and B. subtilus with a same MIC value of
156.25 mg/ml, whereas the highest MIC value (1250 mg/ml)
occurred with E. coli and K. pneumonia. These results are of great
importance, particularly in the case of S. aureus and B. subtilus,
which are well-known for being resistant towards some antibi-
otics and for their production of several enterotoxins that cause
many enteritis types and septicaemia (Hajji et al., 2010). The
antibacterial activity of the ethanol extracts from P. nigrum
could be related to the presence of phenolic and avonoid
components.
Interestingly, one can also see from the data presented in Table 2
the difference in bacterial sensibility to piperine and piperic acid.
The latter was found to be the most active against all tested
bacteria. S. epidermidis was the most sensitive microorganism, with
a MIC value of 78.12 mg/ml.
4. Conclusion
In the present study, piperine, the predominant compound
P. nigrum, was puried from the ethanolic extract and the piperic
acid was obtained by alkaline hydrolysis of the puried piperine.
Our results show that, when using various in vitro tests, piperic acid
exhibits the highest antioxidant activity. It possesses also an anti-
bacterial activity against all Gram () bacteria tested and especially
against S. enterica. An antibacterial activity against Gram ()
bacteria was also observed. According to these results, synthesized
piperic acid may be considered as a natural preservative against
food-borne pathogens.
Acknowledgements
This work was funded by Ministry of Higher Education and
Scientic Research, Tunisia. The authors wish to thank Mr. Abdel-
majid Dammak (ENIS) for his help with the English.
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