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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 2 (COX-2) (Mueller, Hobiger, & Jungbauer, 2010). The iNOS and COX-
2 stimulate the production of many pro-inammatory mediators
Herbs and spices, which are important part of the human diet, such as (Interleukin-4 (IL-4), Interleukin-10 (IL-10), Interleukin-13
have been used for thousands of years in traditional medicine and (IL-13), interferon-alpha (a-IFN)), and the transformation growth
to enhance the avour, colour and aroma of foods. In addition to factor b-TGF (Hanada & Yoshimura, 2002; Makarov, 2000).
boosting avour, herbs and spices are also known for their Piperine (1-piperoyliperidine), which belongs to the alkaloid
preservative (Neilsen & Rios, 2000), antioxidative (Shobana & family, represents the major component in the dry fruit of P. nigrum.
Naidu, 2000), and antimicrobial (Salie, Eagles, & Leng, 1996) roles. Piperine has been reported to have several pharmacological effects
Black pepper or Piper nigrum is one of the most popular spice such as anti-diarrhoeal and hepatoprotective (Bajad, Bedi, Singla, &
products in oriental countries (mostly in Southeast Asia). P. nigrum Johri, 2001; Koul & Kapil, 1993). Some studies have shown that
is a plant of the piperaceae family, largely used as a avouring agent piperine possesses an anti-inammatory and an analgesic effect
in foods. Its characteristic aromatic odour is due to the volatile oils (Gupta, Bansal, Bhardwaj, & Velpandian, 2000). In addition, it has a
in the cells of the pericap (Murthy & Bhattacharya, 2008). It has high antioxidant activity and is used for the treatment of Alzheimer
been traditionally used for the treatment of malaria in India and the diseases (Chonpathompikunlert, Wattanathorn, & Muchimapura,
epilepsy in China (Majeed & Prakash, 2000). Moreover, black 2010; Selvendiran, Vijeya Singh, Krishnan, & Sakthisekaran, 2003).
pepper has an anti-inammatory activity manifested by stimu- The chemical modication in the structure of piperine to piperic acid
lating the production of an anti-inammatory cytokine like (IL-10). was conrmed by the appearance of the carboxyl group during the
On the other hand, black pepper inhibits the expansion of genes hydrolysis. Piperic acid has a high anti-hyperlipidemic activity (Han
encoding the nitric oxide synthase (iNOS) and the cyclooxygenase- 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).
Abbreviations: DPPH, 1,1 diphenyl-2-picrylhydrazyl; TCA, trichloroacetic acid; The aim of this work was to examine the efciency of different
BHT, butylated hydroxytoluene; HPLC, High Performance Liquid Chromatography; solvents for the extraction of the major compounds from P. nigrum,
Na2CO3, sodium carbonate; CDCl3, deuterated chloroform; MTT, 3-(4,5-dimethyl- traditionally used for their medicinal properties, and evaluate the
thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.
antioxidant and antimicrobial activities of the different extracts and
* Corresponding author. Tel./fax: 216 74 675 055.
E-mail address: zaraizied@hotmail.fr (Z. Zarai). 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 635
2. Materials and methods Absorbance 0:504 mg quercetin 0:014 R2 0:99
of solvent extracts, puried piperine and piperic acid were Absorbance 0:011 C 0:0049; R2 0:987
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
where C is the concentration of a-tocopherol equivalent (mmol/ml).
sample was mixed with 500 ml ethanol and 125 ml of freshly
The values are presented as the means of triplicate analysis.
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 2.5. Antibacterial activity
radicals was read at 519 nm using a Shimadzu UV mini-1240
UVeVIS spectrophotometer (Paris, France). The scavenging of In order to determine the antimicrobial activity of the different
DPPH radical was calculated according to the following equation: solvent extracts, piperic acid and piperine, various Gram-positive
and Gram-negative bacteria were used: Escherichia coli (ATCC
DPPH Radical scavenging activity% 25922), Klebsiella pneumonia (ATCC 27853), Salmonella enterica
h . i
(ATCC 43972), Staphylococcus aureus (ATCC 25923), Staphylococcus
Acontrol Asample Acontrol 100
epidermidis (ATCC 14990), Enterococcus faecalis (ATCC 29122), and
Bacillus subtilis (ATCC 6633). Staphylococcus xylosus was isolated in
where Acontrol and Asample are the absorbance of the control and of LBGEL from waste water (Mosbah, Sayari, Mejdoud, Dhouib, &
the sample respectively. The values are presented as the means of Gargouri, 2005).
triplicate analysis.
2.5.1. Agar diffusion method
2.4.2. Reducing power assay The agar diffusion method was employed for the determina-
The reducing power was investigated based on the method tion of antibacterial activities of the solvent extracts, piperine
previously described (Yildirim, Mavi, & Kara, 2001) with some and piperic acid according to the method described by Berghe
modications. An aliquot of each sample (0.5 ml) was mixed and Vlietinck (1991). The dried extracts were dissolved in 100%
with phosphate buffer (1.25 ml, 0.2 M, pH 6.6) and potassium dimethyl sulfoxide (DMSO) to a nal concentration of 10 mg/ml
ferracyanide (1.25 ml, 1%). The mixture was incubated in a water and sterilized by ltration trough 0.22 mm Nylon membrane lter.
bath at 50 C for 30 min. After that, TCA (1.25 ml, 20%) was The bacterial strains were cultured in a nutriment broth for 24 h.
added. Then, the samples were centrifuged for 10 min at 1650 g. Then, 200 ml of each suspension bacteria (106 colony-forming
An aliquot of 1.25 ml of the upper layer was mixed with 1.25 ml unit estimated by absorbance at 600 nm) was spread on Luria
of distilled water and 0.25 ml of 0.1% (w/v) ferric chloride Broth agar. Bores were made by using a sterile borer and were
solution. After 10 min incubation, the absorbance of the reaction loaded with 10 ml of each sample extract. Dimethyl sulfoxide
mixture was measured at 700 nm. A higher absorbance of the (DMSO) was used as negative control and ampicillin (10 mg/well)
reaction mixture indicates greater reducing power of the as positive reference standard. All the plates were incubated at
sample. The values were presented as the means of triplicate 37 C for 24 h. Antibacterial activity was evaluated by measuring
analysis. the zone of inhibition in millimetres. All experiments were done
in triplicates.
2.4.3. b-Caroteneelinoleic acid assay
The antioxidant activity of the ve solvent extracts, the 2.5.2. Determination of the minimal inhibitory concentration (MIC)
piperine and the piperic acid was evaluated in b- The minimal inhibitory concentration (MIC) values, which
caroteneelinoleic acid system, according to the protocol previ- represent the lowest plant extracts concentration that completely
ously described (Koleva, van Beek, Linssen, de Groot, & inhibits the growth of microorganisms, were determined by
Evstatieva, 2001). A stock solution was prepared as follows: a micro-well dilution method (Wade et al., 2001). The inoculum
0.5 mg b-carotene, 25 ml of linoleic acid and 200 ml of tween 40 of each bacteria was prepared and the suspensions were adjusted
were dissolved in 1 ml of chloroform (HPLC grade). Chloroform to 106 CFU/ml. All the extracts were dissolved in 100% DMSO and
was completely evaporated under vacuum in rotavapory at 40 C, then dilutions series were prepared in a 96-well plate, ranging
then, 100 ml of distilled water was added with vigorous shaking. from 7.8 mg/ml to 5 mg/ml. Each well of the microplate included
The reaction medium contained 500 ml of each sample and 2.5 ml 40 ml of the growth medium, 10 ml of inoculums and 50 ml of the
of the freshly prepared emulsion. All the test tubes were imme- diluted sample extract. The ampicillin and DMSO are used as
diately placed in a water bath at 50 C for 2 h. The absorbance positive and negative controls, respectively. The plates were then
was measured at 470 nm before and after heat treatment. A covered with the sterile plate and incubated at 37 C for 24 h.
control containing 0.5 ml of ethanol instead of the sample solu- After that, 40 ml of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-
tion was carried out in parallel. The values were presented as the tetrazolium bromide (MTT) at a nal concentration 0.5 mg/ml
means of triplicate analysis. freshly prepared in water was added to each well and incubated
for 30 min. The change to red colour indicated that the bacteria
2.4.4. Phosphomolybdate assay were biologically active. The MIC was taken to the well, where no
The phosphomolybdate method (Prieto, Pineda, & Aguilar, 1999) change of colour of MTT was observed. The MIC values were done
has been used to evaluate the antioxidant capacity of the ve in triplicate.
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 2.6. Statistical analysis
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 All the experiments were carried out in triplicates. Tests of
temperature and the absorbance was measured at 695 nm against signicant differences between means were determined by Dun-
a blank. BHT was used as standard. The activity of each sample was cans multiple range tests at a signicance level of 0.05 using
expressed as a-tocopherol equivalent using the following linear statistical package for the social sciences SPSS 11.5 (SPSS Inc., Chi-
equation: cago, IL, USA).
Z. Zarai et al. / LWT - Food Science and Technology 50 (2013) 634e641 637
3. Results and discussion (2008) who developed a new method for the extraction of
piperine from P. nigrum and showed that the highest yield obtained
3.1. Extraction yield and total phenolic and avonoid contents was 4%.
The P. nigrum components were extracted by traditional reux 3.2.2. HPLC and TLC analysis
method using solvents with different polarities (chloroform, ethyl The puried piperine from ethanol extract was analysed and
acetate, ethanol, methanol and water). The yields of the different identied by HPLC. Pure commercial piperine was used as reference
solvent extracts as well as the total phenolic and avonoid contents for HPLC identication. The obtained results showed the presence
obtained are presented in Table 1. From this table, we note that the of a single peak corresponding to piperine emerging at the same
extraction yields with methanol and ethanol resulted in the highest time as the pure piperine reference.
amount of total extractable compounds whereas the lowest yields Similar to the standards, the extracted piperine was eluted at
were obtained with ethyl acetate, chloroform, water. In fact, this a retention time of 6.45 min and corresponds to the principle
variation in the yields can be attributed to the polarities of the compound found in P. nigrum.
different compounds present in the spice. One can also note from The crude powder obtained after ethanol extraction was also
the data presented in Table 1 that there are differences in the total used to prepare piperic acid. After different times of alkaline
phenolic and avonoid contents of the different extracts. The hydrolysis of piperine extract, the precipitated piperic acid ob-
highest levels of total phenolic content were found in the ethanol tained was analysed by Thin Layer Chromatography on silica gel.
extract (45.08 mg of gallic acid equivalent/g of P. nigrum) followed by One can note that the intensity of the spot of the initial piperine
the methanol, chloroform and ethyl acetate, while total phenolic used decreased and a new spot appeared during the hydrolysis
content of the water extract was the lowest (101.09 mg of gallic acid process (data not shown).
equivalent/g of P. nigrum). This result is in agreement with this After a washing step with HCl solution and water, the yellow
previously obtained by Han et al. (2008) who reported that ethanol precipitate of piperic acid was ltrated and analysed by HPLC.
was the suitable solvent for extraction of phenolic compounds from Similar to the piperic acid standard, the obtained piperic acid was
P. nigrum. eluted at a retention time of 4.9 min (data not shown). The results
The content of total avonoids from P. nigrum, varied from 0.29 obtained showed the presence of a major peak corresponding to
(water extract) to 5.02 mg (chloroform extract) of quercetin equiv- piperic acid and a small peak corresponding to the residual
alents/g of P. nigrum (Table 1). The results clearly showed that the piperine.
highest content of avonoid was obtained with a decrease in the
polarity of the solvent used. The variations in the phenolic and 3.2.3. 13C NMR and FT-IR analysis
avonoid compounds of the different extracts may be attributed to The chemical structures of commercial piperine (Fig. 1A),
the polarities of compounds present in the raw material. piperine puried from P. nigrum (Fig. 1B) and piperic acid (Fig. 1C)
were determined by 13C NMR.
3.2. Piperine and piperic acid: preparation and analysis Compared to the spectrum of standard and extracted piperine,
which presented the same bands, a new strong band appeared
3.2.1. Piperine and piperic acid preparation above 168.86 ppm, corresponding to the carboxyl group of piperic
One of the natural products that forms a major constituent of acid. The intensity of the band at 165.53 ppm corresponding to the
P. nigrum is commonly known as piperine just like many other amide group decreased and disappeared after transformation of the
amides both from natural and synthetic sources which possess piperine into piperic acid. The appearance of the band at
many biological activities. Most reported methods (Kanaki, Dave, 168.86 ppm and the disappearance of the one at 165.53 ppm could
Padh, & Rajani, 2008; Stahl & Schild, 1981, pp. 410e411; be considered as an argument for the transformation of piperine
Staudinger & Schnieider, 1923) for the isolation of piperine are into to piperic acid because of the vibration of the carboxyl group
based on several processing steps, often under difcult operating and the amide group used to reside in this region.
conditions, which results in a high cost of production. Here, we The transformation of piperine into piperic acid was also
have used traditional reux method, one of the most widely used conrmed by FT-IR analysis. From Fig. 2 we can see the appearance
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.
* Data expressed as mean of three data points (n 3). Fig. 1. 13C NMR spectra of commercial piperine (A), piperine puried from P. nigrum (B)
a, b, c, d and e: different letters within same column means signicant different at and piperic acid (C). The peaks corresponding to the carboxyl and amide groups are
p < 0.05. marked by arrows.
638 Z. Zarai et al. / LWT - Food Science and Technology 50 (2013) 634e641
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
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
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
2.3 0.6d
6.0 0.8e
8.7 1.0c
5.0 1.0c
5.0 0.4c
Its also noteworthy from the data presented in Fig. 3C that the
NS
0.5b
1.0b
0.8a
9.5 0.5c
03a
12.0
11.0
7.3
13.8
7.8
12.3 0.8b
0.5b
0.6b
0.1b
0.4b
1.0a
7.5 0.5c
12.5
12.7
9.6
12.0
6.0
Gram (D)
S. enterica
E. faecalis
S. xylosus
B. subtilis
S. aureus
with reference to lipid peroxidation and antioxidant system in Swiss albino Wade, D., Silveira, A., Rollins-Smith, L., Bergman, T., Silberring, J., & Lankinen, H.
mice. Fitoterapia, 74, 109e115. (2001). Hematological and antifungal properties of temporin A and a cecropin
Shobana, S., & Naidu, K. A. (2000). Antioxidant activity of selected Indian spices. A-temporin A hybrid. Acta Biochimica Polonica, 48, 1185e1189.
Prostaglandins Leukotienes and Essential Fatty Acids, 62, 107e110. Yamaguchi, T., Takamura, H., Matoba, T., & Terao, J. (1998). HPLC method for eval-
da Silva Ferreira, W., Freire-de-Lima, L., Saraiva, V. B., Alisson-Silva, F., Mendona- uation of the free radical-scavenging activity of foods by using 1,1-diphenyl-2-
Previato, L., Previato, J. O., et al. (2008). Novel 1,3,4-thiadiazolium-2- picrylhydrazyl. Bioscience, Biotechnology, and Biochemistry, 62, 1201e1204.
phenylamine chlorides derived from natural piperine as trypanocidal agents: Yildirim, A., Mavi, A., & Kara, A. (2001). Determination of antioxidant and antimi-
chemical and biological studies. Bioorganic & Medicinal Chemistry, 16(6), crobial activities of Rumex crispus L. extracts. Journal of Agricultural and Food
2984e2991. Chemistry, 49, 4083e4089.
Slinkard, K., & Singleton, V. L. (1977). Total phenol analysis: automation and Yildirim, A., Mavi, A., Oktay, M., Kara, A. A., Algur, O. F., & Bilaloglu, V. (2000).
comparison with manual methods. American Journal of Enology and Viticulture, Comparison of antioxidant and antimicrobial activities of tilia (Tilia argentea
28, 49e55. Dest ex DC), sage (Salvia triloba l.), and black tea (Camellia sinensis) extracts.
Stahl, E., & Schild, W. (1981). Pharmazeutische biologie-drogenanalyse II: inhaltsstoffe Journal of Agricultural and Food Chemistry, 48, 5030e5034.
und isolierungen. New York: Gustav Fischer Verlag. Zhishen, J., Mengcheng, T., & Jianming, W. (1999). The determination of avonoid
Staudinger, H., & Schnieider, H. (1923). Relation between chemical constituents and contents in mulberry and their scavenging effects on superoxide radicals. Food
pepper taste I. Berichte der Deutschen Chemischen Gesellschaft, 56, 699e711. Chemistry, 64, 555e559.