Kinetics of light-induced cis-trans isomerization of four piperines and their levels in ground black peppers as determined by HPLC and LC/MS

Article abstract of DOI:10.1021/jf070831p

The pungent compounds piperine and isomers thereof, secondary metabolites present in black and white pepper fruit, undergo light-induced isomerizations. To facilitate studies in this area, an HPLC method has been developed for analysis and isolation of the following four possible piperine-derived photoinduced isomers: piperine, isopiperine, chavicine, and isochavicine. The limits of detection (LOD) estimated from calibration plots were ～15-30 ng for each isomer. Reproducibilities of the analyses were excellent, and recoveries of spiked samples were as follows (average ± SD; n = 3): chavicine, 98.4 ± 2.1%; isopiperine, 96.2 ± 3.2%; piperine, 104 ± 3.8%; isochavicine, 98.9 ± 3.0%. To determine the kinetics of these isomerizations, fluorescent light, sunlight, and UV radiation at 254 nm was used to induce cis-trans geometric isomerization as a function of light intensities and time of exposure determined with the aid of high-performance liquid chromatography (HPLC) and liquid chromatography with diode array UV detection-mass spectrometry (LC-DAD/MS). HPLC was also used to determine the distribution of the isomers in four commercial ground black pepper products used as spices in culinary practice. Isomerization increased with light intensities and time of exposure and leveled off at the so-called photostationary phases. The piperine levels of the four products were quite similar, ranging (in wt %) from 10.17 to 11.68. The amounts of the other three isomers ranged from 0.01 to 0.07 of the total for chavicine; from 0.15 to 0.23 for isopiperine; and from 0.37 to 0.42 for isochavicine. The results establish the utility of the HPLC method for simultaneous analysis of the four isomers both in pure form and in black pepper extracts. The dietary significance of the results is discussed.

Full text of DOI:10.1021/jf070831p

J. Agric. Food Chem. 2007, 55, 7131−7139 7131
Kinetics of Light-Induced Cis−Trans Isomerization of Four
Piperines and Their Levels in Ground Black Peppers as
Determined by HPLC and LC/MS
NOBUYUKI KOZUKUE,† MAL-SUN PARK,† SUK-HYUN CHOI,‡ SEUNG-UN LEE,†
MAYUMI OHNISHI-KAMEYAMA,§ CAROL E. LEVIN,# AND MENDEL FRIEDMAN*,#
Department of Food Service Industry, Uiduk University, San 50 Yugeom, Gangdong, Gyeongju,
Gyongbuk 780-713, Korea, Department of Food & Beverage, Kyungdong College of
Techno-Information, Geongsan City, Gyongbuk 712-904, Korea, Mass Analysis Laboratory, National
Food Research Institute, Tsukuba, Japan 305-8642, and Western Regional Research Center,
Agricultural Research Service, United States Department of Agriculture, Albany, California 94710
The pungent compounds piperine and isomers thereof, secondary metabolites present in black and
white pepper fruit, undergo light-induced isomerizations. To facilitate studies in this area, an HPLC
method has been developed for analysis and isolation of the following four possible piperine-derived
photoinduced isomers: piperine, isopiperine, chavicine, and isochavicine. The limits of detection (LOD)
estimated from calibration plots were ∼15-30 ng for each isomer. Reproducibilities of the analyses
were excellent, and recoveries of spiked samples were as follows (average ( SD; n ) 3): chavicine,
98.4 ( 2.1%; isopiperine, 96.2 ( 3.2%; piperine, 104 ( 3.8%; isochavicine, 98.9 ( 3.0%. To determine
the kinetics of these isomerizations, fluorescent light, sunlight, and UV radiation at 254 nm was used
to induce cis-trans geometric isomerization as a function of light intensities and time of exposure
determined with the aid of high-performance liquid chromatography (HPLC) and liquid chromatography
with diode array UV detection-mass spectrometry (LC-DAD/MS). HPLC was also used to determine
the distribution of the isomers in four commercial ground black pepper products used as spices in
culinary practice. Isomerization increased with light intensities and time of exposure and leveled off
at the so-called photostationary phases. The piperine levels of the four products were quite similar,
ranging (in wt %) from 10.17 to 11.68. The amounts of the other three isomers ranged from 0.01 to
0.07 of the total for chavicine; from 0.15 to 0.23 for isopiperine; and from 0.37 to 0.42 for isochavicine.
The results establish the utility of the HPLC method for simultaneous analysis of the four isomers
both in pure form and in black pepper extracts. The dietary significance of the results is discussed.
KEYWORDS: Piperine; isopiperine; chavicine; isochavicine; black peppers; photoisomerization; HPLC;
LC-MS
INTRODUCTION
antidepressant (12, 13), antidiarrheal (14), antioxidative (15, 16),
insecticidal (17), antiulcer (18), and insulin-resistance (19)
activities. Piperine is also reported to inhibit enzymes (cyto-
chrome P450, UDP-glucoronyltransferase) that catalyze the
biotransformation of nutrients and drugs, thus enhancing their
bioavailability and efficacies in ViVo (20-23).
Piperines exist as the following four distinct geometric
isomers whose structures are shown in Figure 1: E,E-(trans-
trans)-piperine (piperine), Z,E-(cis-trans)-piperine (isopiperine),
E,Z-(trans-cis)-piperine (isochavicine), and Z,Z-(cis-cis)-piperine
(chavicine) (24). The E,E-(trans-trans)-piperine appears to be
the main constituent that is largely responsible for the pungency
of black and white peppers. The other three isomers appear to
be formed by light-induced and/or enzyme-catalyzed cis-trans
(geometric) isomerizations (photoisomerizations) of the double
bonds of the parent piperine molecule.
Piperines are secondary pungent metabolites present in the
outer part of the fruits and in the seeds peppers (Piper nigrum
L.) (1, 2). Black pepper is produced from green unripe berries
of the pepper plant, whereas white pepper is obtained when the
seeds of fully ripe berries are dried (3). Interest in piperine arises
from the fact that, in addition to its sensory properties that impact
the taste of food, the compound is reported to possess several
nonsensory beneficial biological/pharmacological properties that
may also benefit human health. These include antibacterial/
antiprotozoan (4-7), anticarcinogenic/antigenotoxic (8-11),
* Author to whom correspondence should be addressed. E-mail:
mfried@pw.usda.gov. Fax: (510) 559-5777. Phone: (510) 559-5615.
† Uiduk University.
‡ Kyungdong College of Techno-Information.
§ National Food Research Institute.
# United States Department of Agriculture.
10.1021/jf070831p CCC: $37.00
© 2007 American Chemical Society
Published on Web 07/28/2007

7132 J. Agric. Food Chem., Vol. 55, No. 17, 2007
Kozukue et al.
100 g of diet. Although the hen diet contained only piperine,
and precautions were taken to avoid light-induced isomerization
during the trial, an egg yolk sample contained all four isomers.
These observations suggest that after consumption, piperine
probably undergoes enzyme-catalyzed cis-trans isomerizations
in ViVo.
Although several studies have described the photoisomeriza-
tion of piperine to form a mixture of cis-trans isomers (24, 31,
33, 34), to our knowledge, no previous study has investigated
the analogous individual photoinduced isomerizations of pure
isomers of piperine, namely, isopiperine, chavicine, and iso-
chavicine exposed to light over a long time period. Therefore,
the main objectives of the present study were (a) to validate an
HPLC method for the analysis of the composition of mixtures
of four piperine isomers; (b) to determine whether LC-MS can
be used to characterize the isomers; (c) to determine the time-
course (kinetics) of conversion of individual pure isomers to
mixtures of all four isomers as function of light intensities; (d)
to define the time-course of analogous isomerization of isopi-
perine, chavicine, and isochavicine induced by fluorescent light
of a single intensity; and (e) to demonstrate the applicability of
the HPLC method to measure the content of the four isomers
in commercial ground black peppers products.
MATERIALS AND METHODS
Materials. Piperine (97.89% pure) was obtained from Aldrich
(Milwaukee, WI). HPLC grade acetonitrile, methanol, and analytical
grade formic acid were obtained from commercial sources. The solvents
were filtered through a 0.45 µm membrane filter (Millipore, Bedford,
MA) and then degassed in an ultrasonic bath before use. Because
chavicine, isopiperine, and isochavicine standards were not available
from commercial sources, these three compounds were isolated by
HPLC from photoisomerized piperine as described below. Four
commercial ground black peppers were obtained from local stores, two
in the United States and two in Korea.
HPLC. HPLC was carried out on a Hitachi liquid chromatograph
model 655-II equipped with an autosampler (model 655A- 40). The
stainless steel column (250 mm × 4.0 mm inner i.d.) was packed with
Inertsil ODS-3v (5 µm particle diameter) (GL Sciences, Tokyo, Japan).
The column temperature was maintained constant with a Shimadzu
column oven CTO-10vp (Shimadzu, Kyoto, Japan). Separation was
achieved using a mixture of acetonitrile/0.5% formic acid in distilled
water (30:70, v/v) as eluent with an isocratic flow rate with 0.8 mL/
min at 25 °C. A Shimadzu UV-vis detector (model SPD-10Avp) was
set at 220 to 340 nm.
Photoisomerization of Piperine by White Fluorescent and UV
Light. Commercial piperine (3.14 mg/mL of methanol) was placed in
a 5 mL glass vial. The vials were irradiated with the three fluorescent
light intensities (1000, 3000, and 6000 lux) and UV light (2 lux at 254
nm) for nine photoperiods (from 5 to 2880 min) at 25 °C. After
irradiation, each solution (10 µL) was directly injected into HPLC for
the separation of isomers produced by photochemical isomerization.
A Hitachi Chromato-integrator model D-2500 recorded the peak areas
of each individual piperine isomer. The glass vial absorbed 3.4% of
the incident UV light.
LC-MS/MS. Liquid chromatography/mass spectrometry experi-
ments were performed by an LCQ mass spectrometer (Thermo Fisher
Scientific Inc., MA) equipped with an HPLC system (Agilent, CA)
connected with a DAD (G1315A). The piperine mixture solution (5
µL) was applied on an Inertsil ODS-3 column (2.1 × 150 mm, 3 µm,
GL Sciences Inc., Tokyo, Japan) and was separated to piperine isomer
peaks using isocratic solvent system consisting of 30% acetonitrile (LC/
MS grade, Wako Pure Chemical Industries, Ltd., Osaka, Japan)
containing 0.5% formic acid (amino acids sequence analysis grade) at
the flow rate of 200 µL/min. UV-vis spectra were recorded in the
wavelength range from 220 to 500 nm. The mass spectrometer was
initially tuned using a solution containing the mixture of piperine
isomers. The HPLC eluate was introduced into the mass spectrometer
Figure 1. Structure of piperine isomers.
Reported analytical methods for piperine include NMR (25),
vibrational spectroscopy (26), UV spectroscopy (27), HPLC (28,
29), LC/MS (30, 31), and TLC/GC (32). Verzele et al. (33)
found that piperine in solution is the principal pungent substance
of black peppers; the other three isomers have low pungency.
These authors used the internal standard p-bromoacetanilide to
achieve good separation of the four isomers on a nitro-silica
gel stationary phase, but with poor precision. However, the
analytical results were difficult to reproduce. A later study (34)
describes improved separation by micro-liquid chromatography.
Ternes and Krause (24) developed two HPLC methods for
the piperine isomers based on separation on ODS-Hypersil and
on a second column, whose packing consisted of silver-modified
cation-exchange ligand-covered spherical silica material. These
authors related the structures of the individual isomers to the
respective observed UV, NMR, and FT-IR spectra. The method
was successfully applied to the analysis of piperine isomers in
eggs produced by Leghorn laying hens fed 80 mg of piperine/

Light-Induced Piperine Isomerizations
J. Agric. Food Chem., Vol. 55, No. 17, 2007 7133
from 5 to 115 min. Mass and multiple tandem mass spectrometry (MSn)
were operated in the positive-ion mode in the mass range of m/z 50-
500.
Helium was used as the collision gas for the MSn spectrometric
procedures, followed by the isolation of ions over a selected mass
window of 2 Da. MSn represents multiple stage of precursor ion m/z
selection followed by product ion detection for successive progeny ions.
Mass selection of the analyte by m/z is followed by fragmentation and
analysis of the fragments.
Preparative Conversion of Piperine to Chavicine, Isopiperine,
and Isochavicine. Chavicine, isopiperine, and isochavicine were
produced from commercial piperine (3.14 mg/mL in methanol) by light
exposure (6000 lux for 72 h). The individual compounds were isolated
by repetitive high performance liquid chromatography using UV
detection (280 nm). The isomer solutions (20 µL) produced by light
exposure were each applied to HPLC column and eluted with
acetonitrile/0.5% formic acid (30:70, v/v) at 25 °C. The individual
isomers were monitored at 280 nm and collected from the UV detector.
This procedure was repeated four times. Solutions containing each of
the isolated isomers were stored in the dark at -30 °C. Because piperine
isomers are sensitive to light, all experimental operations were
performed in the dark.
Photoinduced Isomerization Kinetics of Chavicine, Isopiperine,
and Isochavicine. Each solution (1 mL) of pure chavicine, isopiperine,
and isochavicine isolated by HPLC from irradiated piperine was placed
into a 5 mL vial. Each sample was then irradiated at a photointensity
of 3000 lux for eight photoperiods ranging from 15 to 4320 min at 25
°C. After irradiation, each sample (80 µL) was injected into the HPLC
column.
Figure 2. Time course of the photoisomerization of piperine at 3000 lux:
HPLC chromatograms of piperine isomers produced from piperine before
Quantification of Piperines. To establish the linearity of the
response by HPLC, piperine (17.2 mg) was placed into a 10 mL vial
to which was added to 5 mL of ethanol. The sample was then irradiated
with white fluorescent light at an intensity of 3000 lux for 4 days at 25
°C. The reaction mixture containing the four isomers was then subject
to 9-fold serial dilution series with ethanol. Each of the nine diluents
(10 µL) was then directly injected onto the HPLC column in order to
determine the concentration-dependent linear response of the integrated
peak areas for each isomer. The calibration plot of each compound
was obtained by plotting the integrated peak area against the amount
injected.
and after light exposure. Column: Inertsil ODS-3v (5 µm, 4.0 × 250 mm).
Detector: UV at 280 nm. Flow rate, 0.8 mL/min. Chart speed: 1.25 mm/
min.
The HPLC system we previously used to determine both
capsaicinoid levels derived from red peppers and piperine levels
derived from black peppers in pepper-containing foods was
further refined and adapted in the present study to the analysis
of the four piperine isomers by defining how composition of
the mobile phase and column temperature affected retention
times. The elution times of the four isomers on HPLC
chromatograms were determined with ratios of the mobile phase
of acetonitrile/0.5% formic ranging from 28/72 (v/v) to 40/60
(v/v), each at four temperatures ranging from 25 °C to 40 °C.
The separation of peaks and retention times decreases with
increasing acetonitrile/0.5% formic acid ratios and column
temperatures. We selected the 30/70 (v/v) ratio for the mobile
phase at 25 °C as conditions for optimum separation of the
isomers, as illustrated in Figure 2. The retention times ranged
as follows: chavicine, 129.5 min; isopiperine, 133.9 min;
piperine, 141.0 min; isochavicine, 148.8 min.
Because only one standard (piperine) was available to us, the
parameter we selected to quantitate the data was the ratio of the
integrated peak area for each compound as a percent of the sum of all
the peak areas obtained for each mixture of the four isomers. These
values were automatically calculated with the aid of a Hitachi
Chromato-integrator model D-2500.
Recovery of Piperine Isomers after Spiking. Piperine isomers were
analyzed before and after addition of known amounts of each isomer
to the mixture of isomers after exposure to fluorescent light of 3000
lux for 30 h at 25 °C. Recovery (%) ) (concentration of each compound
in spiked sample)/concentration of endogenous compound + spike) ×
100.
Extraction-Analysis of Piperines from Commercial Ground
Black Peppers. All operations were carried out in the dark. Each dry
black pepper powder (0.1-0.15 g) was placed into a 5 mL vial to which
was added 2 mL of 80% ethanol. The suspension was sonicated for 60
min in an ultrasonic bath and then centrifuged at 18000g for 10 min at
1 °C. The supernatants were then passed through a 0.45 µm Millipore
nylon filter (Bedford, MA). The filtrates were analyzed by HPLC.
Plots of concentration versus peak area (calibration plots)
were linear from 0 to 5000 ng. The limit of detection (LOD)
estimated from calibration plots was ∼15-30 ng. Recoveries
of spiked samples were as follows (average ( SD; n ) 3):
chavicine, 98.4 ( 2.1%; isopiperine, 96.2 ( 3.2%; piperine,
104 ( 3.8%; isochavicine, 98.9 ( 3.0%.
UltraViolet and Mass Spectra of Piperines. In the present
study, structural identification of individual piperine isomers
following irradiation was performed by associating the HPLC
peak for each isomer with its corresponding UV spectrum that
was previously associated by Ternes and Krause (24) with the
corresponding NMR spectrum. We also assessed the potential
of LC/MS to differentiate among the isomers.
Figures 3 and 4 depict the UV and mass spectra of the four
piperine isomers determined in the present study by LC-DAD/
MS. The UV absorbance shown in the three-dimensional plot
RESULTS AND DISCUSSION
Analytical Aspects. Identification of ChaVicine, Isopiperine,
Piperine, and IsochaVicine. We had previously developed and
validated HPLC methods for analysis of extracts of fresh peppers
containing capsaicinoids and of both capsaicinoids and piperines
in pepper-containing foods (35, 36). This method was used to
quantify the distribution of capsaicinoids in 11 Korean whole
peppers and in 12 commercial pepper-containing foods.

7134 J. Agric. Food Chem., Vol. 55, No. 17, 2007
Kozukue et al.
Figure 3. UV
−
vis spectra/chromatogram plot by HPLC-photodiode array detection (HPLC-DAD): Three-dimensional chromatogram/spectra plot of four piperine
isomers determined by diode array UV detection (DAD). The UV absorbance was monitored at wavelengths 220
−400 nm throughout the chromatographic run.
Figure 4. Identification of HPLC/LC peaks 1−4 by UV and mass spectra: UV and mass spectra of four piperine isomers. Peak 1: piperine. Peak 2: isochavicine.
Peak 3: isopiperine. Peak 4: chavicine. MS spectra show only identical parent ion peaks at 286.1 Da. The major peaks in the MS/MS and MS3 spectra may be
due to the mass fragments shown in Figure 8. Table 2 summarizes relative abundance of peaks in the MS3 spectra.
of Figure 3 was monitored at wavelengths 220-400 nm
throughout the chromatographic run. To visualize the chro-
matogram, look along the x-axis: note gray trace. To visualize
the UV spectrum, take a slice at any particular time (for
example, 92 min for the first peak) along the z-axis (wavelength)
and mentally rotate that clockwise. These spectra can be checked

Light-Induced Piperine Isomerizations
J. Agric. Food Chem., Vol. 55, No. 17, 2007 7135
Figure 5. Piperine mass fragments: Possible structures of major mass spectral fragments of piperines.
Table 1. Reproducibility as Determined by HPLC of Four Separate Light-Induced Isomerizationsa
retention time (min)
peak area (µV)
experiment
chavicine
isopiperine
piperine
isochavicine
chavicine
isopiperine
piperine
isochavicine
1
2
3
4
122.70
122.63
123.23
123.28
122.96
0.34
126.43
126.59
127.07
126.50
126.70
0.33
132.40
132.67
133.10
132.96
132.72
0.35
139.92
140.19
140.62
140.28
140.24
0.35
406128
404816
396294
399139
401594
4657
1213859
1202798
1175316
1189382
1195339
16684
1927683
1936908
1900063
1887995
1913162
22947
2249932
2243921
2174519
2225244
2223404
34243
average
SD
RSD (%)
0.28
0.26
0.27
0.25
1.16
1.40
1.20
1.54
a Piperine was exposed to 3000 lux for 30 h.
against known spectra for purity. The following observed λmax
values in the UV spectra (in nm) are consistent with corre-
sponding values reported in the literature (24, 33): chavicine,
316; isopiperine, 332; piperine, 340; isochavicine, 332.
Table 2. Relative Intensities (in %) of the Four Major Mass Spectra
Peaks of the Four Piperine Isomers Detected by MS3 (Positive
Ionization Mode)
+
mass spectra (MS3), M m/z:
Previously, Ternes and Krause (24) found that the fragment
patterns of the MS spectra of the piperine isomers did not differ
[(m/z 285 (M+), 201, 173, 143, 115, 84)]. Scott et al. (30)
observed the following mass spectra M+ m/z (relative intensities)
for piperine: 287.1 (20), 286.1 (100), 201.0 (35), 135.0 (5).
Hashimoto (31) found that the spectra of the four isomers
determined by HPLC/APCIMS appeared identical, each with a
single major [MH+] ion at m/z 286. By monitoring the increased
relative abundance of the ions, these authors attempted to
identify piperine isomers in natural pepper before and after
exposure to sunlight. Finally, Marutolu et al. (32) identified
piperine and chavicine in black pepper by TLC and GC-MS.
The results of the present study are shown in Figures 4 and
5 and in Table 2. Figure 4 shows that the peak patterns of the
MS and MS/MS spectra of the piperine isomers appear identical.
isomer
115.1
142.9
171.0
173.0
chavicine
isopiperine
piperine
22.4
19.3
21.8
23.5
67.4
69.1
70.9
68.9
100.0
100.0
100.0
100.0
63.5
54.3
59.2
65.0
isochavicine
By contrast, Figure 4 and Table 2 also show that the fragment
patterns in terms of relative abundance obtained with MS3
(positive ionization mode) appear somewhat different from each
other. Thus, setting the intensity of the 171.0 m/z peak at 100%,
the relative intensities (in %) for the 115.1 m/z peak for the
four isomers ranged from 19.3 to 23.5; for the 142.9 m/z peak,
from 67.4 to 70.9; and for the 173.0 peak, from 54.3 to 65.0.
Because the differences in relative abundances of the fragments

7136 J. Agric. Food Chem., Vol. 55, No. 17, 2007
Kozukue et al.
Figure 7. Identical composition of four isomers resulting from the
photochemical isomerization of piperine induced by (1) fluorescent light
(3000 lux) for 20 days at 25
temperature.
°C; and (2) sunlight for 20 days at room
Figure 6. Effect of quality of light on piperine degradation and isomer
formation: Effect of light intensities and exposure times on losses of
piperine (A) and formation of isopiperine (B), chavicine (C), and
isochavicine (D). Plotted values are averages from two separate
experiments
visible.
± SD (n ) 2). SD values for most points are too small to be
are probably within the method’s normal variation, the elec-
trospray MS/MS data may not show significant enough differ-
ences to differentiate between the isomers.
Figure 8. Percentage of each piperine isomer produced from isolated
piperine isomers by timed exposure to white fluorescent light (3000 lux).
Time-course of the photoisomerization by fluorescent light (3000 lux) of
three isolated isomers: isopiperine (A), chavicine (B), and isochavicine
Figure 5 shows postulated structures of mass spectral
fragments of the isomers. Although the fragment of mass 201
must be formed by the indicated cleavage of the carboxyamide
moiety, the mechanisms of formation of the structures with
masses of 171 and 143 are not obvious to us. Note that three
possible structures can account for the 143 m/z peak. Because
piperine and its isomers possess the same molecular weight and
similar mass fragments in the mass spectra, our results suggest
that it may not be possible to concurrently identify all four
isomers by LC/MS.
(C). Plotted values are averages from two separate experiments
± SD.
SD values for most points are too small to be visible.
izations of the four individual isomers as a function of light
intensities and sources. The data are plotted in Figures 6-8
for a visual depiction of the observed trends.
Figure 6 shows the percent decrease in piperine concentra-
tions and the concurrent formation of the other three isomers
as a function of both intensity of light source and exposure time
Piperine Photoisomerizations. A main objective of the
present study was to determine the kinetics of the photoisomer-

Light-Induced Piperine Isomerizations
J. Agric. Food Chem., Vol. 55, No. 17, 2007 7137
-
c
Table 3. Piperine Isomer Content in Four Commercial Brands of Pure Ground Black Peppersa
pepper
A
piperine
isopiperine
chavicine
36
0.03
70
0.07
12
0.01
40
0.04
isochavicine
total (sum)
µ
g/g
% total
g/g
% total
g/g
% total
g/g
% total
116,807
±
±
±
±
1338
270
0.23
158
0.15
194
0.18
177
0.17
±
±
±
±
45
2
±
6
555
0.47
455
0.43
401
0.37
426
0.42
±
±
±
±
13
6
117,669
±
±
±
±
1402
99.27
B
C
D
µ
105,158
99.35
107,727
99.44
1245
594
±
12
2
105,841
108,335
102,367
1240
556
µ
32
42
±
4
µ
101,724
99.37
1005
±
3
12
1038
a Pepper source: A, Albertson’s (USA); B, McCormick (USA); C, Daesaug (Korea); D, Ottogi (Korea). b Values in
of total of all four isomers.
µg/g ± SD of pepper powder; n )
2. c Values in %
up to 2880 min (2 days). At 1000 lux, the decrease in piperine
starts at about 60 min and progresses with time, resulting in a
final loss of 55.7% after 2 days. At the end of the experiment,
these decreases are accompanied by concurrent increases in the
concentrations of the three isomers in the following order:
isochavicine (38.1%) > isopiperine (16.1%) > chavicine (1.5%).
Exposure at 3000 lux for 2 days induced a 77.7% decrease in
the piperine level. The concentration of isochavicine (39.8%)
is similar to that observed at 1000 lux, and that of isopiperine
(21.6%) approaches that chavicine (16.3%). At 6000 lux, the
final loss of piperine (79.2%) is similar to that observed at 3000
lux, as are the levels of the other three isomers. Isomer
distribution resulting from exposure to UV (254 nm) radiation
parallels that observed with 3000 lux. This observation is
reinforced by our observation that identical mixtures of isomers
are formed after 20 days following exposure of piperine to both
fluorescent light at 3000 lux and sunlight that provides UV
radiation (Figure 7).
The cited observations suggest, as expected, not only that
both light intensity and exposure time favor isomerizations but
also that the formation of isopiperine reaches a maximum level
and then levels off (Figure 6B). Chavicine formation is slow
initially but then progresses linearly with time under all four
light conditions evaluated. Also note that isochavicine formation
parallels that of isopiperine (Figure 6B,D).
Isopiperine Isomerization. Loss of piperine exposed to
fluorescent and UV light is more rapid than the loss of the other
isomers. Isomerization of isopiperine is also more rapid than
that of the other isomers, reaching 77.3% after 3 h and 79.4%
after 3 days (Figure 8A). The data also show that the concurrent
formation of piperine over the prolonged time period is slower
than the formation of chavicine and isochavicine. The latter are
formed at nearly identical rates. The trends in the formation of
isomers from isopiperine as a function of light quality are similar
to those mentioned earlier for piperine.
ChaVicine Isomerization. To better define the dynamics of
the photoisomerizations, Figure 8B shows trends for the
fluorescent light (3000 lux)-induced isomerization of chavicine.
Chavicine levels decrease progressively with time up to 3 days
(67.9% loss). The decreases are accompanied by formation of
isochavicine (19.7%), piperine (20.3%), and isochavicine (19.7%).
The decrease in chavicine levels off reaching the photostationary
state at ∼500 min, as do the corresponding increases in the
amounts of three isomers formed. The leveling-off effect
suggests that none of the isomers formed is degraded during
the long exposure time to light.
IsochaVicine Isomerization. The initial loss of isochavicine
exposed to fluorescent light at 3000 lux is more rapid than losses
observed with piperine, isopiperine, and chavicine (Figure 8C).
The reduction in isochavicine concentrations level off at ∼1000
min, roughly paralleling those of isopiperine and chavicine.
Again, beginning at about that time, the photostationary phase
remains constant up to 3 days.
Piperine Content of Ground Black Peppers. In the present
study, we determined levels of all four piperine isomers in four
commercial ground black pepper products, two originating from
the United States and the other two from Korea. Table 3 shows
that (a) the piperine levels of the four products were quite
similar, ranging from 101,724 µg/g (10.17% by wt) to 116,807
µg/g (11.68% by wt), a small 13% difference from highest to
lowest level; (b) the levels of the other three isomers were quite
low, ranging from 0.01 to 0.07% of the total for chavicine; from
0.15 to 0.23% for isopiperine; and from 0.37 to 0.42% for
isochavicine; and (c) piperines comprise about 10-12% of the
weights of the dry black pepper products. These results suggest
that the described HPLC method can be used to measure very
low (0.01 wt %) to very high (11.68 wt %) levels of all four
isomers in dry pepper products.
Previously (36), we found that 4 of 12 pepper-containing
foods evaluated contained, in addition to capsaicinoids, two
piperine isomers. The piperine levels of the foods ranged from
about 0.1% to 0.8% of the levels cited above for the black
peppers. Because these foods contained both capsaicinoids and
piperines, our results suggest that they were prepared with both
red and black pepper products.
Related Photoisomerizations. Light-induced excitation of the
π-electrons of the double bond of olefin compounds (alkenes)
results in the formation of a nonplanar excited π*-transition
state of either cis or trans olefins due to promotion of an
electron, which then returns to the ground state (37). Thus,
continued exposure to light absorbed by piperine isomers over
time results in a constant ratio of the excited state being
generated and leads to a constant ratio of isomers on decay of
the light-induced excited state. Although our data show that the
individual isomers varied in their susceptibilities to light-induced
isomerization, in principle, this so-called “photostationary steady
state” is usually independent of the initial isomer or mixture of
isomers. The trends illustrated in Figures 6-8 appear generally
consistent with this concept.
As is well-known, the described photoisomerizations of
piperines are not unique in nature. Other light-induced events
include cis-trans isomerization of carotenes (â-carotene, lutein,
lycopene, phytoein, zeaxanthin), natural polyene pigments with
11 conjugated double bonds (38, 39); cis-trans isomerizations
of double bonds of fatty acids such linoleic acid (40); cis-
trans isomerization of the 11-cis-retinal chromophore, a primary
event in human vision (41); conversion of 7-dehydrocholesterol
located under the skin to pre-vitamin D3 (42); and postharvest
synthesis of chlorophyll and glycoalkaloids in potatoes (43-
45). The dietary consequences of such light-induced changes
in food ingredients are not always apparent and merit further
study.

7138 J. Agric. Food Chem., Vol. 55, No. 17, 2007
Kozukue et al.
(12) Li, S.; Wang, C.; Wang, M.; Li, W.; Matsumoto, K.; Tang, Y.
Antidepressant like effects of piperine in chronic mild stress
treated mice and its possible mechanisms. Life Sci. 2007, 80 (15),
1373-1381.
(13) Lee, S. A.; Hong, S. S.; Han, X. H.; Hwang, J. S.; Oh, G. J.;
Lee, K. S.; Lee, M. K.; Hwang, B. Y.; Ro, J. S. Piperine from
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(14) Bajad, S.; Bedi, K. L.; Singla, A. K.; Johri, R. K. Antidiarrhoeal
activity of piperine in mice. Planta Med. 2001, 67, 284-287.
(15) Vijayakumar, R. S.; Surya, D.; Nalini, N. Antioxidant efficacy
of black pepper (Piper nigrum L.) and piperine in rats with high
fat diet induced oxidative stress. Redox Rep. 2004, 9, 105-110.
(16) Vijayakumar, R. S.; Nalini, N. Efficacy of piperine, an alkaloidal
constituent from Piper nigrum on erythrocyte antioxidant status
in high fat diet and antithyroid drug induced hyperlipidemic rats.
Cell Biochem. Funct. 2006, 24, 491-498.
Research Needs. The above-cited observations on light-
induced, time-dependent isomerization of each piperine isomer
can serve as a guide to facilitate and/or to minimize isomer-
izations, depending on need. Although, as mentioned earlier,
dietary, pharmacological, and medicinal aspects of piperine have
been extensively explored (21, 46), this is apparently not the
case for the other three isomers. For example, it would be of
interest to compare the relative insecticidal and antimicrobial
properties of isopiperine, chavicine, and isochavicine to those
reported for piperine.
Our data on the content of piperines in commercial pepper-
containing foods and in commercial black pepper products may
help consumers to select foods with desirable piperine content.
Because light may influence both the nature and amounts of
piperine isomers present, which in turn may affect sensory and
biological properties, labeling for piperine content would
undoubtedly benefit consumers.
(17) Scott, I. M.; Gagnon, N.; Lesage, L.; Philogene, B. J.; Arnason,
J. T. Efficacy of botanical insecticides from Piper species
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gastric emptying and gastrointestinal transit in rats and mice.
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(19) Vijayakumar, R. S.; Nalini, N. Piperine, an active principle from
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17, 71-86.
It is not known whether the piperine isomers we found to be
present in the four commercial ground peppers are formed
naturally or by light-induced postharvest isomerizations. We also
do not know whether and to what extent light-induced isomer-
izations of pure piperines also occur with piperine(s) located
within the complex matrices of vegetative plant tissues during
growth and/or postharvest (38). Will exposure of freshly
harvested peppers to light induce racemization? These aspects
merit further study.
(20) Shoba, G.; Joy, D.; Joseph, T.; Majeed, M.; Rajendran, R.;
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683-686.
ACKNOWLEDGMENT
We thank Leslie A. Harden and Clifton K. Fagerquist for a
helpful exchange of views about mass spectra of piperines.
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Received for review March 20, 2007. Revised manuscript received June
21, 2007. Accepted June 23, 2007.
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