PaNADaARRAAKCLBN1.docx

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Colloids and Surfaces B: Biointerfaces 101 (2013) 353– 360
Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c
a t e / c o l s u r f b
harmacokinetics of curcumin-loaded PLGA and PLGA–PEG
blend nanoparticles
fter oral administration in rats
ajeh Maissar Khalil , Thuane Castro Frabel do Nascimento
, Diani Meza Casa , Luciana Facco Dalmolin ,
na Cristina de Mattos, Ivonete Hoss, Marco Aurélio
Romano, Rubiana Mara Mainardes ∗
epartment of Pharmacy, Universidade Estadual do Centro-
Oeste/UNICENTRO, Rua Simeão Camargo Varela de Sá 03,
85040-080 Guarapuava, PR, Brazil
r t i c l e i n f o
rticle history:
eceived 2 March 2012
eceived in revised form 10 June 2012
ccepted 12 June 2012
vailable online 28 June 2012

eywords:
urcumin
C–MS/MS
ioavailability
anoparticles
a b s t r a c t
The aim of this study was to assess the potential of
nanoparticles to improve the pharmacokinetics of
curcumin, with a primary goal of enhancing its
bioavailability. Polylactic-co-glycolic acid (PLGA) and
PLGA–polyethylene glycol (PEG) (PLGA–PEG) blend
nanoparticles containing curcumin were obtained
by a single-emulsion solvent-evaporation technique,
resulting in particles size smaller than 200 nm. The
encapsulation efficiency was over 70% for both
formulations. The in vitro release study showed that cur-
cumin was released more slowly from the PLGA
nanoparticles than from the PLGA–PEG nanoparticles. A
LC–MS/MS method was developed and validated to quantify
curcumin in rat plasma. The nanoparticles
were orally administered at a single dose in rats, and the
pharmacokinetic parameters were evaluated
and compared with the curcumin aqueous suspension. It was
observed that both nanoparticles formu-
lations were able to sustain the curcumin delivery over
time, but greater efficiency was obtained with
the PLGA–PEG nanoparticles, which showed better results
in all of the pharmacokinetic parameters ana-
lyzed. The PLGA and PLGA–PEG nanoparticles increased
the curcumin mean half-life in approximately 4
and 6 h, respectively, and the Cmax of curcumin increased
2.9- and 7.4-fold, respectively. The distribution
and metabolism of curcumin decreased when it was carried

by nanoparticles, particularly PLGA–PEG
nanoparticles. The bioavailability of curcumin-loaded
PLGA–PEG nanoparticles was 3.5-fold greater than
the curcumin from PLGA nanoparticles. Compared to the
curcumin aqueous suspension, the PLGA and
PLGA–PEG nanoparticles increased the curcumin
bioavailability by 15.6- and 55.4-fold, respectively.
These results suggest that PLGA and, in particular,
PLGA–PEG blend nanoparticles are potential carriers
for the oral delivery of curcumin.
. Introduction
Curcumin is a polyphenol compound extracted from the
oot of Curcuma longa Linn, commonly known as turmeric.
hemically, curcumin is 1,7-bis(4-hydroxy-3-methoxyphenyl)-
,6-heptadiene-3,5-dione, commonly called diferuloylmethane.
urcumin has been used for centuries in Chinese and Indian
edicine to treat a variety of disorders [1]. Several studies have
hown that curcumin presents anti-inflammatory [2,3],
antioxidant
4–6] and antimicrobial activities [7], but the most important
effect
s its potential use against cancer due to its ability to suppresses
the
roliferation of a wide variety of tumor cells [8–10]. Curcumin is
ble to modulate numerous targets including transcription
factors,
eceptors, kinases, cytokines, enzymes and growth factors,
affect-
ng numerous molecular and biochemical cascades [1,11].
∗ Corresponding author. Tel.: +55 42 3629 8160; fax: +55 42

3629 8102.
E-mail address: [email protected] (R.M. Mainardes).
927-7765/$ – see front matter © 2012 Elsevier B.V. All rights
reserved.
ttp://dx.doi.org/10.1016/j.colsurfb.2012.06.024
© 2012 Elsevier B.V. All rights reserved.
The great pharmacological potential of curcumin and its ther-
apeutic applications are restricted because the molecule presents
some drawbacks, including low aqueous solubility at acidic and
physiological pH conditions, rapid hydrolysis in alkaline media
and light instability, inherent to its chemical composition. The
hydrophobic character of curcumin results in pharmacokinetic
restrictions such as low absorption and bioavailability by oral
route,
extensive metabolism and rapid elimination [12,13]. The main
strategies used to overcome the physicochemical limitations of
cur-
cumin and to increase its bioavailability are based on loading
the
compound in nanocarriers, such as liposomes [14],
cyclodextrins
[15], solid lipids [16,17] and polymeric nanoparticles [18–20].
Biodegradable polymeric nanoparticles are extensively used to
improve the therapeutic properties of various drugs and bioac-
tive compounds. Nanoencapsulation protects the molecules from
premature degradation, improves their solubility, and promotes
controlled drug release and drug targeting. Nanoparticles
present
low risk of toxicity, and the drug efficacy, specificity, tolerabil-
ity and the therapeutic index are enhanced with their use. The
pharmacokinetic parameters of the drug are modified when it

dx.doi.org/10.1016/j.colsurfb.2012.06.024
http://www.sciencedirect.com/science/journal/09277765
http://www.elsevier.com/locate/colsurfb
mailto:[email protected]
dx.doi.org/10.1016/j.colsurfb.2012.06.024
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54 N.M. Khalil et al. / Colloids and Surfa
s loaded in nanoparticles. Specifically, there are improvements
n absorption, bioavailability, and plasma circulation time, with
eduction of clearance, consequently increasing the drug’s mean
alf-life [21–25]. The physicochemical parameters of
nanoparticles
uch as particle size, surface modification charge, and hydropho-
icity influence the drug’s pharmacokinetics, impacting the
drug’s
ioavailability and biodistribution in particular. It is well docu-
ented that nanoparticles presenting a hydrophobic surface, like
he surface of poly (lactide-co-glycolide) (PLGA) nanoparticles,
resent short circulation times because they are rapidly recog-
ized by plasmatic opsonin and cleared by cells of the
mononuclear
hagocytic system (MPS). The process of opsonization is one of
he most important biological barriers to nanoparticle-based con-
rolled drug delivery. Coating of the surface of nanoparticles
with
ydrophilic polymers, such as polyethylene glycol (PEG),
polysor-
ates or poloxamers, sterically stabilizes the particles, i.e., they
re able to repel the absorption of opsonin proteins via steric
epulsion forces, and thus, the particles become “invisible” to
MPS
ells, increasing their plasmatic circulation time and resulting in
an
mprovement in drug bioavailability and half-life. Also, longer
plas-
atic circulation times increase the probability of the
nanoparticles
eaching their target [26–29].

Some recent works have demonstrated that PLGA nanoparticles
re able to improve the bioavailability of curcumin after oral
admin-
stration [18,19,30]. PLGA–PEG nanoparticles have been
developed
ecause of their great potential for having long circulation times.
lso, the potential advantage provided by the hydrophilic charac-
er of PEG can improve the biocompatibility of the delivery
system
31]. However, to the best of our knowledge, there has not yet
been
demonstrated report about the use of PLGA–PEG nanoparticles
s carriers for curcumin. Thus, polymeric nanoparticles,
especially
ong-circulating nanoparticles, were evaluated as potential
carriers
or curcumin oral delivery.
In this work, PLGA and PLGA–PEG blend nanoparticles were
btained for curcumin loading. An analytical method based on
C–MS/MS was developed and validated to quantify curcumin in
at plasma. The nanoparticles were orally administered at a
single
ose in rats, and the pharmacokinetic parameters were evaluated
nd compared with those of a curcumin aqueous suspension.
. Materials and methods
.1. Materials
Curcumin (code C1386), PLGA (Resomer RG 50:50 H; Mw
0–75 kDa, inherent viscosity 0.45–0.6 dl/g), PEG (Mw 10 kDa)
and

olyvinyl alcohol (PVA, 31 kDa, 88% hydrolyzed) were
purchased
rom Sigma–Aldrich (USA). The internal standard, salbutamol
99%), was obtained from European Pharmacopeia. Methylene
hloride and ethyl acetate were purchased from FMaia® (Brazil).
nalytical HPLC-grade ethanol, acetonitrile, methanol and acetic
cid were purchased from J.T. Baker (USA). All other solvents
and
hemicals were analytical or HPLC grade.
.2. Preparation of curcumin-loaded PLGA and PLGA–PEG
blend
anoparticles
The nanoparticles were obtained by the single-emulsion
olvent-evaporation technique, as previously described [32].
riefly, curcumin (5 mg) and PLGA (50 mg) were dissolved in a
ixture of ethyl acetate (1.5 mL) and methylene chloride (0.5
mL)
ith or without PEG (10 mg) at room temperature. This organic
hase was rapidly poured into 10 mL of PVA aqueous solution
0.5%, w/v) and emulsified by sonication for 5 min (35% of 500
W,
nique® Ultrasonic Mixing, Brazil), resulting in an oil-in-water
Biointerfaces 101 (2013) 353– 360
(O/W) emulsion. Next, the organic solvent was rapidly
eliminated
by evaporation under vacuum (20 min) at 37 ◦C. The particles
were
then recovered by centrifugation (19,975 × g, 30 min, 4 ◦C,
Cien-
tec CT-15000R centrifuge, Brazil) and washed twice with water
to

remove the surfactant. The nanoparticles were dispersed in the
cry-
oprotectant sucrose (5%, w/v), and the resulting nanosuspension
was cooled to −18 ◦C and freeze-dried (Terroni®, Brazil).
2.3. Particle size
The mean particle size and polydispersity index were deter-
mined by dynamic light scattering (BIC 90 plus – Brookhaven
Instruments Corp., USA). The analyses were performed at a
scat-
tering angle of 90◦ and a temperature of 25 ◦C. For each sample,
the
mean particle diameter, polydispersity and standard deviation of
ten measurements were calculated.
2.4. Drug entrapment efficiency
A Waters 2695 Alliance HPLC system (Milford, MA, USA) was
used for curcumin quantitation. The chromatographic analysis
was
performed in isocratic mode using a reverse phase C18 column
(VertiSep GES,Vertical Chromatography Co) with a 5 �m
particle
size, 4.6 mm internal diameter and 250 mm length. The mobile
phase consisted of a mixture of ethanol, acetonitrile and water
(80:10:10), pumped at a flow rate of 0.8 mL/min. The sample
injec-
tion volume was 20 �L, and the fluorescence detector was
operated
at an excitation wavelength of 365 nm and an emission
wavelength
of 512 nm.
The amount of curcumin incorporated into the nanoparticles
was determined directly after complete dissolution of nanoparti-

cles in acetonitrile. The solutions were centrifuged and
supernatant
was collected. After the appropriate dilutions in ethanol, 20 �L
of
the sample was injected into the HPLC system, and the drug
concen-
tration was obtained by comparison with a previously
constructed
analytical curve. Before injection, all of the solutions were
filtered
through a PVDF membrane filter (0.22-�m pore size,
Millipore). The
entrapment efficiency (%) was estimated by comparing the
amount
of curcumin extracted from nanoparticles with the initial
amount
used for the nanoparticles preparation.
2.5. In vitro release profile
The release of curcumin from nanoparticles was conducted by
suspending the nanoparticles (containing 1.5 mg of curcumin)
in
12 mL of phosphate saline buffer (PBS, 0.01 M, pH 7.4), and
the sus-
pension was divided in eight Eppendorf tubes. The experiments
were performed in triplicate and under sink conditions. The
tubes
were kept in a shaker at 37 ◦C at 150 rpm. At predetermined
time
intervals, the suspension was centrifuged at 19,975 × g for 15
min
to separate the released curcumin from the nanoparticles
[33,34].
The resulting precipitate in each tube was dispersed in 1.5 mL
of

phosphate saline buffer and incubated until the next sampling.
The
released curcumin present in the supernatant (1.5 mL in each
tube)
was diluted in ethanol, and 20 �L of this solution was injected
into
the HPLC to determine the amount of curcumin released at
different
time intervals.
2.6. Chromatograph system and conditions for curcumin
quantitation in plasma
The LC–MS/MS analysis was conducted in positive ion ESI
mode on a Quattro Micro API–Waters hexapole mass spectrom-
eter connected to a liquid chromatograph (Waters Alliance). The
analysis was conducted on a Phenomenex Luna C18(2) 100A
col-
umn (250 mm × 4.6 mm, 5 �m). The mobile phase consisted
of
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N.M. Khalil et al. / Colloids and Surfa
ethanol and 0.05% acetic acid solution (80:20, v/v) at a flow
rate
f 1.0 mL/min. The sheath gas and auxiliary gas were tuned to
give
n optimum response as necessary. The needle voltage was 4.5
kV.
albutamol was used as internal standard (IS) [35]. Argon was
used
s the collision gas at collision energy of 15 eV (curcumin) and
18 eV
salbutamol). The collision energy was individually tuned for
each
nalyte to obtain an optimum value. The analytes were quantified
sing selected ion reaction monitoring (SRM). The ion
transitions
/z 369.3→285.0 and m/z 240.0→147.7 were used for the
determi-
ation of curcumin and salbutamol, respectively. The

autosampler
ooler was maintained at 4 ◦C.
.7. Preparation of curcumin standards and quality control
A concentrated stock standard of curcumin and salbutamol (IS)
ere prepared by dissolving 4 mg of each compound in 20 mL
f methanol, generating a 200 �g/mL stock solution. Eight point
alibration curves were prepared by serial dilution of the cur-
umin stock solution (200 �g/mL in methanol) in the range of
.5–500 ng/mL. The calibration curve was prepared daily using
.45 mL of blank plasma with 50 �L of the appropriate working
olution, resulting in concentrations of 0.5, 10, 25, 50, 100, 200,
350
nd 500 ng/mL. Three quality controls (QC) were prepared at 1.5
low concentration), 225 (medium concentration) and 450 ng/mL
high concentration).
.8. Plasma sample preparation
To 100 �L of rat plasma sample (or a calibration standard or a
QC
ample) were added 100 �L of salbutamol (IS) and 100 �L of
0.5 M
odium hydroxide (to assist in the extraction of curcumin). The
mix-
ure was vortexed for 1 min. After the curcumin was extracted
with
300 �L of ethyl acetate (liquid–liquid extraction), followed by
agi-
ation in a shaker (10 min), it was centrifuged at 10,000 rpm at 4
◦C
or 10 min. The supernatant was evaporated using nitrogen gas in
a
ample concentrator. The obtained residue was reconstituted with

he mobile phase and vortexed for 20 s. The samples were
subjected
o LC–MS/MS analysis.
.9. Bioanalytical method validation
The specificity of the method was investigated by comparing
the
hromatogram of blank plasma with the blank plasma spiked with
tandard solutions and with the samples collected from rats after
urcumin administration.
The linearity of the bioanalytical assay was evaluated with a
otal of eight calibration standards over the concentration range
of
.5–500 ng/mL. Calibration curves were constructed by linear
least-
quares regression analysis by plotting the peak-area ratios
versus
he drug concentrations.
The limit of quantitation (LOQ) was defined as the lowest con-
entration of the analyte in the calibration curve that could be
etected with a variation of less than 15%.The intra-day preci-
ion and accuracy were determined within one day by analyzing
en replicates of the QC samples at concentrations of 1.5, 225
and
50 ng/mL of curcumin. The inter-day precision and accuracy
were
etermined on two separate occasions using replicates (n = 10) of
ach concentration used. The intra- and inter-day precision was
efined as the relative standard deviation (R.S.D.). The accuracy
was
xpressed using the following equation (1):
measured concentration

]
× 100 (1)
nominal spiked concentration
The freeze–thaw stability of the plasma samples was evaluated
y exposing QC samples at low and high concentrations to four
reeze–thaw (−20 ◦C to room temperature) cycles before sample
Biointerfaces 101 (2013) 353– 360 355
preparation. The stability of the samples in the autosampler was
evaluated by analyzing the extracted QC samples after being
placed
in the autosampler at 20 ◦C for 6 h, at which time the samples
were
analyzed. The long-term stability was verified by freezing (−20
◦C)
the QC samples for 250 days. Freshly processed standard
samples
were used to quantitate all of the QC samples. The analyses
were
performed in quintuplicate.
2.10. Pharmacokinetic study
Male adult Wistar rats with a mean body weight of 200–300 g
were fasted overnight prior to the experiments, with free access
to
water. The experimental protocol was approved by the
Institutional
Animal Ethics Committee of the Universidade Estadual de Ponta
Grossa, Brazil (Registration no. 06/2010). The rats were divided
randomly into three groups (n = 5). The formulations (curcumin
aqueous suspension, dispersion of the curcumin-loaded PLGA

nanoparticles and dispersion of the curcumin-loaded PLGA–
PEG
blend nanoparticles) were administered by oral gavage at a
single
dose of 50 mg/kg. The nanoparticles were dispersed in ultrapure
water.
Blood samples (500 �L) were withdrawn from the tail vein into
heparinized microtubes at the following times: 0.25, 0.5, 1, 1.5,
2,
4, 8, 12 and 24 h after dosing. The blood samples were
centrifuged
at 3020 × g for 10 min. The supernatant was collected,
transferred
to tightly sealed plastic tubes and stored at −20 ◦C until analysis
by LC–MS/MS. After each sampling the same volume removed
was
replaced with saline solution.
2.11. Data analysis and statistics
All of the in vitro results were expressed as the mean ± standard
deviation (S.D.) of three replicates. The in vivo results were
presented as the mean ± S.D. of five replicates. Pharmacokinetic
parameters were estimated using the model-independent method.
The terminal elimination rate constant (Ke) was estimated by
a linear regression analysis of the terminal portion of the log-
linear blood concentration–time profile of curcumin. The
terminal
elimination half-life (t1/2) was calculated from Ke using the
for-
mula t1/2 = 0.693/Ke. The maximum observed plasma
concentration
(Cmax) and the time taken to reach it (Tmax) were obtained
from
the curve plotting curcumin concentration vs. time. The area

under
each drug concentration time curve (AUC, ng/mL h) to the last
data
point was calculated by the linear trapezoidal rule and
extrapolated
to time infinity by the addition of CLast/Ke, where CLast is the
con-
centration of the last measured plasma sample. The apparent
body
clearance (Cl) was calculated using the equation Cl =
dose/AUC. The
apparent volume of distribution (Vd) was calculated by the
equation
Vd = dose/Ke AUC. Statistical analysis of the data was
performed via
one-way analysis of variance (ANOVA). The results were
considered
statistically significant if p < 0.05.
3. Results and discussion
3.1. Preparation of the curcumin-loaded PLGA and PLGA–PEG
blend nanoparticles
The nanoparticles containing curcumin were successfully
obtained by the single-emulsion solvent-evaporation method.
The
choice of a nanoencapsulation method is based on the drug
solubil-
ity, and because curcumin is hydrophobic, the method of
reducing
the size of the emulsion oil-in-water (O/W) is adequate for this
molecule. The ultrasonication was crucial to reduce the
emulsion
globules to nanometer size. Table 1 illustrates the size

characteris-
tics of the obtained nanoparticles. Both formulations, the PLGA
and
PLGA–PEG blend nanoparticles containing curcumin, presented
356 N.M. Khalil et al. / Colloids and Surfaces B: Biointerfaces
101 (2013) 353– 360
Table 1
Curcumin nanoparticle characteristics.
Polymer Particle size (nm)a Polydispersity indexa Size
distributiona Encapsulation efficiency (%)b
PLGA 161.93 ± 6.7 0.042 ± 0.01 146.2–200.7 nm (100%) 77.07
± 8.16
PLGA–PEG 152.37 ± 4.5 0.077 ± 0.01 109.9–185.1 nm (100%)
73.22 ± 9.77
Values reported as mean ± S.D.
m
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a
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F
t
a (n = 3).
b (n = 30).
onodisperse profiles and narrow size distributions. The presence
f PEG did not influence the mean particle size, but the polydis-
ersity index was superior (p < 0.05) than that obtained from
PLGA
anoparticles, while maintaining a monomodal profile.
The encapsulation efficiency was determined directly, and the
esults are presented in Table 1. The method used for
nanoencapsu-

ation resulted in significant enclosure of curcumin, and the
process
as found to be highly reproducible. The PEG did not influence
the
ncapsulation, as the values between batches were not
significantly
ifferent (p > 0.05). Indeed, the hydrophilic character of PEG
kept it
irected to aqueous phase, while the hydrophobic core of PLGA
can
ntrap the hydrophobic drugs.
.2. In vitro curcumin release profile
The in vitro release of curcumin from nanoparticles was evalu-
ted simulating physiological conditions (37 ◦C, PBS buffer pH
7.4).
he in vitro release profiles of curcumin were obtained by graph-
ng the cumulative percentage of the drug released with respect
o the amount of curcumin encapsulated as a function of the
time.
he experiment was performed over nine days. Fig. 1 illustrates
he curcumin release profiles from the PLGA and PLGA–PEG
blend
anoparticles and indicates that there was a pronounced time pro-
ongation of the drug release.
It is evident that the PEG influenced the curcumin release
ecause there was a great difference between the release profiles
f curcumin from nanoparticles of different compositions. A
bipha-
ic release pattern of curcumin was observed from the PLGA–
PEG
anoparticles, where the initial 24 h period released

approximately
1% of drug, followed by a sustained release to a total of 56.9%
over
ine days of observation. This initial burst release may be due to
rug desorption from the particle surface, and the sustained
release
an be characterized by the drug diffusion through the polymeric
atrix and subsequent diffusion/erosion of the polymeric matrix.
ig. 1. In vitro release profile of curcumin from PLGA and
PLGA–PEG blend nanopar-
icles in PBS (0.01 M, pH 7.4) at 37 ◦ C. Values reported as the
mean ± S.D. (n = 3).
Curcumin release from the PLGA nanoparticles was slower than
from the PLGA–PEG blend nanoparticles (p < 0.05), and the
release
was progressive because it did not have a biphasic profile. After
24 h, only 5.8% of the drug had been released, and in nine days,
37%
of the curcumin had been released.
In general, it can be affirmed that the drug release depends upon
the solubility, diffusion and biodegradation of the matrix mate-
rials. Thus, the drug release mechanisms can be modified by the
choice of polymer matrices. Drug release also depends upon the
loading efficiency of the drug and the size of the nanoparticles
[24].
In our case, because the particle size and curcumin loading are
sim-
ilar for the PLGA and PLGA–PEG nanoparticles, we can attest
that
the difference between the amount of drug released from the
two
nanoparticles is due to the presence of PEG, as it has a

hydrophilic
character and can enhance the water permeation and drug diffu-
sion through the polymeric matrix [36]. It is possible that
curcumin
strongly interacts with the PLGA matrix, thus retarding the
release
capability, and that the PEG can increase the wettability of the
polymeric surface and matrix, contributing to the increase in
drug
release. The results show that the PLA-PEG nanoparticles
released
more curcumin than the PLGA nanoparticles (by approximately
1.5-fold; p < 0.05) during the period analyzed.
3.3. Bioanalytical method development and validation
A LC–MS/MS method for the determination and quantitation of
curcumin in rat plasma has been developed and validated. Initial
runs were conducted with mobile phases composed of acetoni-
trile:0.2% formic acid solution (40:60, v/v), acetonitrile:1%
formic
acid solution (70:30, v/v), acetonitrile:0.1% acetic acid solution
(70:30, v/v) and acetonitrile:0.005% acetic acid solution (70:30,
v/v). In all of these combinations, the curcumin peak resulted in
tailing, and the signal was slow.
Testing several ratios of methanol and acetic acid, the combina-
tion that resulted in a sharp peak with a sufficient response area
was using methanol and 0.05% acetic acid solution (80:20, v/v)
as
the mobile phase at a flow rate of 1 mL/min. The retention
times
were approximately 1.08 min and 0.82 min for curcumin and
IS,
respectively, and the total run time was 2 min.

The specificity of the method was evaluated by comparing the
chromatograms of curcumin in plasma (standard and sample)
and
those of potentially interfering plasma components. Representa-
tive chromatograms are shown in Fig. 2, including a blank
plasma
sample (Fig. 2A), plasma containing curcumin and salbutamol
stan-
dard (Fig. 2B) and a plasma sample obtained 30 min after the
oral
administration of 50 mg/kg of curcumin-loaded PLGA
nanoparticles
(Fig. 2C). The resulting chromatograms show the assay
specificity,
as there were no endogenous plasma components eluted at the
retention time of curcumin or IS.
The method was validated over a wide concentration range, and
the results were directly obtained and extrapolated on the
calibra-
tion curve. The calibration lines were shown to be linear from
0.5
to 500 ng/mL (r2 = 0.9941). The method was sensitive, and the
LOQ
was low (0.5 ng/mL). Other LC–MS/MS methods described in
the
N.M. Khalil et al. / Colloids and Surfaces B: Biointerfaces 101
(2013) 353– 360 357
F n stan
c

l
1
a
r
ig. 2. Representative chromatograms of (A) a blank plasma
sample, (B) a curcumi
urcumin, while that the lower are indicative of the peak of the
internal standard.
iterature for curcumin determination in plasma showed a LOQ
of
0 ng/mL [16] and 2.5 ng/mL [37].
Table 2 shows a summary of intra- and inter-day precision and
ccuracy for curcumin detection in rat plasma. The intra-day
accu-
acy of curcumin for rat plasma samples was 101.72–110.54%
for
dard, and (C) a curcumin sample. The upper peak of the figure
is representative of
QC samples with a R.S.D. of less than 6.70%. The inter-day
accu-
racy of curcumin for rat plasma samples ranged from 96.34% to
107.94% for QC samples with an R.S.D. of less than 4.34%.
These
results were within the limits established by the FDA guidelines
for the validation of bioanalytical methods [38].

358 N.M. Khalil et al. / Colloids and Surfaces B: Biointerfaces
101 (2013) 353– 360
Table 2
Intra-day and inter-day precision and accuracy of curcumin in
rat plasma (n = 10).
Nominal
concentration
(ng/mL)
Measured
concentration (ng/mL)
R.S.D. (%) Accuracy (%)
Intra-daya
1.5 1.66 ± 0.04 2.33 110.54
225 224.66 ± 8.00 3.56 99.85
450 457.76 ± 30.66 6.70 101.72
Inter-dayb
1.5 1.62 ± 0.05 3.39 107.94
225 216.77 ± 9.41 4.34 96.34
450 436.59 ± 18.74 4.29 97.02
a
t
o
r
s
s
c

p
a
w
o
o
s
f
3
o
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1
A
w
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f
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i
T
S
The analyses were performed in the same day; R.S.D. = relative
standard devia-
ion.

b The analyses were performed in two different days within one
month.
Table 3 lists the data from the stability tests. No significant loss
f curcumin (≤1.4%) was observed after storage of the plasma at
oom temperature on the bench top for at least 6 h. The plasma
amples were stable over at least four freeze/thaw cycles and
were
table at −20 ◦C for at least 250 days, with no significant loss of
urcumin (≤1.8%). These results suggested that the plasma sam-
les could be stored at −20 ◦C for long periods, could be thawed
nd refrozen and could be maintained at room temperature for 6
h
ithout compromising the integrity and accuracy of the samples.
The sensitivity of this LC–MS/MS method offered advantages
ver other LC–MS/MS methods and conventional HPLC–UV
meth-
ds applied for curcumin pharmacokinetics. Also, the excellent
pecificity and short run time analysis make this method efficient
or curcumin pharmacokinetic applications.
.4. Pharmacokinetics study
The mean curcumin plasma concentration–time profiles after
ral administrations of 50 mg/kg of curcumin in different for-
ulations, curcumin aqueous suspension, curcumin-loaded PLGA
anoparticles and curcumin-loaded PLGA–PEG blend nanoparti-
les, are expressed in Fig. 3. The Table 4 summarizes the
relevant
harmacokinetic parameters.
After the oral administration of a curcumin aqueous suspen-
ion, the drug was absorbed quickly, and a maximum plasma

oncentration (Cmax) of approximately 4.066 ± 0.564 ng/mL was
eached in 30 min. Thereafter, the curcumin plasma concentra-
ion decreased abruptly, as the drug was distributed and rapidly
etabolized, resulting in a high Ke and short t1/2, approximately
.1 h. The curcumin was detected up to 8 h after administration.
sustained release of curcumin over 24 h was observed when it
as carried by the two nanoparticles formulations. Thirty minutes
fter oral administration of the curcumin-loaded PLGA
nanoparti-
les, the mean plasma concentration was 4.57 ± 0.35 ng/mL, and
or the curcumin-loaded PLGA–PEG nanoparticles, the plasma
oncentration was 9.1 ± 0.95 ng/mL. There was a significant
ncrease (p < 0.01) in curcumin absorption from the PLGA–PEG
able 3
tability of curcumin in rat plasma.
Sample condition Curcumin nominal concentration
1.5 ng/mL
Concentration measured
(ng/mL)a
R.S.D. (%)b Acc
6 h at room temperature 1.62 ± 0.1 1.42 108
Freeze–thaw four cycles 1.63 ± 0.04 0.7 109
250 days at −20 ◦ C 1.6 ± 0.05 1.1 107
a Values reported as mean ± S.D. (n = 5).
b Relative standard deviation, calculated comparing with CQ

freshly prepared.
Fig. 3. Comparison of in vivo plasma concentration vs. time
profiles of the different
curcumin formulations. All values reported are the mean ± S.D.
(n = 5).
nanoparticles in the first 30 min compared to free curcumin and
curcumin from the PLGA nanoparticles. The curcumin
concentra-
tion increased to 11.783 ± 0.454 ng/mL, Cmax, after 2 h
(Tmax), and
to 29.778 ± 4.632 ng/mL, Cmax, after 3 h (Tmax), with the
PLGA and
PLGA–PEG nanoparticles, respectively. Compared to free
curcumin,
the Cmax of curcumin from PLGA nanoparticles and PLGA–
PEG
nanoparticles was increased 2.9- and 7.4-fold, respectively.
The
increase in Cmax indicates that the nanoparticles were effective
in increasing drug absorption, and the delayed Tmax demon-
strates an obvious sustained release of curcumin. The
distribution
and metabolism of curcumin were decreased when it was car-
ried by nanoparticles (p < 0.01). The clearance of curcumin
from
the PLGA and PLGA–PEG nanoparticles was 16.3- and 61.6-
fold
lower than that of free curcumin, respectively. The PLGA–PEG
nanoparticles and PLGA nanoparticles decreased the curcumin
volume of distribution by 11.6- and 4.6-fold compared to free
curcumin. Thus, the t1/2 of curcumin from the PLGA increased
to 4 h, and that from the PLGA–PEG nanoparticles was
increased
to 6 h, while for free curcumin the t1/2 was 1 h. There was
a significant difference in the AUC0–inf between the curcumin

aqueous suspension, the curcumin–PLGA nanoparticles and the
curcumin–PLGA–PEG nanoparticles (p < 0.01). Between the
two
nanoparticle formulations, the curcumin from PLGA–PEG pre-
sented a relative bioavailability 3.5-fold superior to that of the
curcumin from PLGA nanoparticles. Compared to the curcumin
aqueous suspension, the PLGA and PLGA–PEG blend
nanoparticles
increased the curcumin bioavailability 15.6- and 55.4-fold,
respec-
tively.
Recently, Shaikh et al. [19] demonstrated that the PLGA
nanoparticles were able to increase the curcumin bioavailability
at least 9-fold when compared to curcumin administered with an
absorption enhancer. Tsai et al. [20] developed curcumin-loaded
450 ng/mL
uracy (%) Concentration measured
(ng/mL)a
R.S.D. (%)b Accuracy (%)
.31 474.50 ± 13.36 0.47 105.44
.00 481.07 ± 18.52 0.56 106.9
.25 483.99 ± 5.6 1.8 107.55
N.M. Khalil et al. / Colloids and Surfaces B: Biointerfaces 101
(2013) 353– 360 359
Table 4
Pharmacokinetic parameters of curcumin following single oral

administration of curcumin aqueous solution, curcumin-loaded
PLGA nanoparticles and curcumin-loaded
PLGA–PEG nanoparticles, in rats (n = 5).
Pharmacokinetic parameters Formulations
Curcumin aqueous suspension Curcumin PLGA nanoparticles
Curcumin PLGA–PEG nanoparticles
Dose (mg/kg) 50 50 50
AUC0–t (h ng/mL) 8.695 ± 1.872 134.251 ± 3.446* , # 447.80 ±
64.028*
AUC0–inf (h ng/mL) 8.762 ± 1.862 137.162 ± 3.694* , #
485.941 ± 54.663*
Cmax (ng/mL) 4.066 ± 0.564 11.783 ± 0.454* , # 29.778 ±
4.632*
Tmax (h) 0.5 2* , # 3*
Ke (1/h) 0.631 ± 0.072 0.178 ± 0.021* , # 0.119 ± 0.021*
t1/2 (h) 1.109 ± 0.124 3.929 ± 0.451* , # 5.979 ± 1.126*
Vd (L/kg) 9432.536 ± 2511.617 2073.664 ± 352.612* , #
900.544 ± 225.772*
Cl (L/h/kg) 5859.700 ± 1399.927 365.191 ± 37.351* , # 103.679
± 10.903*
Values reported as mean ± S.D. (n = 5). AUC: area under the
plasma concentration–time curve; Cmax : peak concentration;
Tmax : time to reach peak concentration; Ke : constant
of elimination; t1/2 : mean half-life; Vd : apparent volume of
distribution; Cl: clearance.
P
a
m
u
a

c
h
[
e
r
s
P
p
c
P
p
l
p
P
b
c
u
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t
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n
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[
p
T
r
C
t
d
t
t
i

n
n
r
w
m
t
[
[
* Significantly different of free curcumin (p < 0.01).
# Significantly different of curcumin from PLGA–PEG
nanoparticles (p < 0.01).
LGA nanoparticles. When these particles were intravenously
dministrated in rats, a significant amount of curcumin was
found
ainly in the spleen due to phagocytic cell uptake in the retic-
loendothelial system. Xie et al. [30] showed that after the oral
dministration of curcumin-loaded PLGA nanoparticles, the cur-
umin had a 5.6-fold higher relative bioavailability and had a
longer
alf-life than that of native curcumin. In a similar work, Anand
et al.
18] demonstrated that curcumin-loaded PLGA nanoparticles
have
nhanced cellular uptake, increased bioactivity in vitro and supe-
ior bioavailability in vivo relative to free curcumin. To date, no
tudy has compared the pharmacokinetics of curcumin loaded in
LGA and PLGA–PEG blend nanoparticles.
In our study, the significant difference in pharmacokinetic
arameters, mainly bioavailability and half-life, between the free
urcumin aqueous suspension and the curcumin-loaded PLGA
and

LGA–PEG nanoparticle dispersions is explained by the inherent
roperties of colloidal nanoparticles in biological media, which
pro-
ong drug release and its in vivo trajectory. The in vitro release
rofile demonstrated that curcumin is released more rapidly from
LGA–PEG nanoparticles than from PLGA nanoparticles and
could
e more quickly available in blood. It is well supported that
pharma-
okinetic parameters are altered depending upon the
nanoparticles
sed, and their surface composition plays an important role in
rug bioavailability [39,40]. PEG is frequently used for the sur-
ace modification of various polymeric nanoparticles because it
xhibits excellent biocompatibility and is able to improve the
long-
erm systemic circulation of the nanoparticles. The PEG coating
on
he surface of the polymer reduces the interactions between the
anoparticles and the enzymes of the digestive fluids and
increases
he uptake of the drug in the blood stream and lymphatic tissue
41]. This effect can explain the difference between the
curcumin
harmacokinetics from PLGA and PLGA–PEG blend
nanoparticles.
he ability of the PEG to make the coated nanoparticles invisible
to
ecognition by MPS cells gives the particles long circulation
time.
onsequently, the drug half-life and bioavailability are higher
than
hose of a drug carried in uncoated nanoparticles [27]. We
recently
emonstrated that the presence of PEG in PLA nanoparticles con-

aining the antiretroviral zidovudine was essential in promoting
he increase in drug bioavailability after intranasal
administration
n rats [42].
The increased curcumin bioavailability obtained with the
anoparticulate systems confirms the excellent abilities of the
anoparticles to modulate the physicochemical properties of
drugs,
esulting in improved pharmacokinetics profiles. Because the
poor
ater solubility and low oral bioavailability of curcumin are the
ajor drawbacks in its medicinal application, the studied
nanopar-
icles represent an important initial step in the development of
[
[
[
a medicine containing curcumin, using the nanotechnology as
a tool.
4. Conclusions
In this study, nanoparticles coated or not with PEG were suc-
cessfully prepared by the emulsion solvent-evaporation method.
Also, an analytical method for determining curcumin in plasma
was
optimized. We demonstrated that all curcumin pharmacokinetic
parameters were improved by nanoparticles, especially PLGA–
PEG
nanoparticles. The curcumin Cmax, Tmax, t1/2 and AUC were

signifi-
cantly increased by nanoparticles, while distribution and
clearance
were decreased. The PLGA–PEG nanoparticles were able to
increase
the curcumin bioavailability in 3.5-fold compared to curcumin
from PLGA nanoparticles. Compared to curcumin aqueous
suspen-
sion, PLGA and PLGA–PEG nanoparticles increased the
curcumin
bioavailability in 15.6 and 55.4-fold, respectively. These results
demonstrate the great potential of PLGA and mainly PLGA–
PEG
blend nanoparticles as carriers for the oral delivery of
curcumin.
Acknowledgments
This study was supported by Conselho Nacional de Desenvolvi-
mento Científico Tecnológico (CNPq) (577183/2008-7), Fundaç
ão
Araucária (462/2010) and FINEP (01.08.0211.00).
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Pharmacokinetics of curcumin-loaded PLGA and PLGA–PEG
blend nanoparticles after oral administration in rats1
Introduction2 Materials and methods2.1 Materials2.2
Preparation of curcumin-loaded PLGA and PLGA–PEG blend
nanoparticles2.3 Particle size2.4 Drug entrapment efficiency2.5
In vitro release profile2.6 Chromatograph system and conditions
for curcumin quantitation in plasma2.7 Preparation of curcumin
standards and quality control2.8 Plasma sample preparation2.9
Bioanalytical method validation2.10 Pharmacokinetic study2.11
Data analysis and statistics3 Results and discussion3.1
Preparation of the curcumin-loaded PLGA and PLGA–PEG
blend nanoparticles3.2 In vitro curcumin release profile3.3
Bioanalytical method development and validation3.4
Pharmacokinetics study4
ConclusionsAcknowledgmentsReferences
Abstract: The medicinal properties of Curcumin obtained
from Curcuma longa L. cannot be utilised because of poor
bioavailability due to its rapid metabolism in the lover and
intestinal wall. In this study, the effect of combining
piperine, a known inhibitor of hepatic and intestinal
glucuronidation, was evaluated on the bioavailability of
Curcumin in rats and healthy human volunteers. When
Curcumin was given alone, the dose 2g/kg to rats,
moderate serum concentrations were achieved over a
period of 4 h. Concomitant administration of piperine
20mg/kg increased (P < 0.02), and he bioavailability was
increased by 154%. On the other hand in humans after a
dose of 2g Curcumin alone, serum levels were either

undetectable or very low. Concomitant administration of
piperine 20mg produced much higher concentrations from
0.25 to 1h post drug (P < 0.01 at 0.25 and 0.5h; P < 0.001 at
1h), the increase in bioavailability was 2000%. The study
shows that in the dosages used, piperine enhances the
serum concentrations, extent of absorption and
bioavailability of curcumin in both rats and humans with no
adverse effects.
Key words: Curcumin, piperine, pharmacokinetics, Curcuma
longa, Zingiberaceae
Introduction
Curcumin in obtained from Curcuma longa L
(Zingiberaceae), a perennial herb widely cultivated in
tropical regions of Asian. Its rhizome is extensively used for
imparting colour and flavour to food. Current traditional
Indian medicine claims the use of its powder, turmeric,
against a wide variety of diseases (1). Extensive scientific
research (2) on curcumin had demonstrated a wide
spectrum of therapeutic effects which range from anti-
inflammatory, wound healing, antispasmodic,
anticoagulant, antitumor activities (3) and recently, with
potential utility in autoimmune deficiency syndrome (4).
Pharmacokinetic properties of curcumin indicate that
following oral administration, it is poorly absorbed (3) and
only traces of the compound appear in the blood, while
most of it is excreted in the faeces (5). The transformation
of curcumin into an unidentified compound during
absorption (6) and its glucuronidation in the liver (5, 7) are
probably responsible for its low concentration in the blood.

Planta Medica 64 (1998) 353 – 356
©Georg Thieme Veriag Stuttgart – New York
Black pepper (Piper nigrum L) and long pepper (Piper
longum L) have been in use as spices from ancient time’s
thoughout the world. A major component of the Piper
species id the alkaloid piperine (l-piperoylpiprtidine), which
had been reported to enhance the bioavailability of drugs
by inhibition of glucuronidation in the liver (8) and small
intestine (9).
In view of the potential therapeutic utility of curcumin it
appeared pertinent to examine the effect of piperine, a
known hepatic and intestinal metabolic inhibitor, on the
pharmacokinetic disposition of curcumin in animals and
man, to provide a scientific rationale of assigning it a
rightful place in the pharmacologist’s armamentarium.
Materials and Methods
Animal Studies
Albina Wistar rats (n=96) of both sexes (150-200g) were
chosen for the study. They were housed in well ventilated
cages, fed on commercial rat pellets supplied by Hind Lever,
Mumbai, with tap water as libitum. They were divided into
two sex and weight matched groups (n=6/group/time cut),
one group for administration of curcumin and the other for
concomitant curcumin and piperine. Curcumin and piperine
were supplied in pure powder for by Sami Chemicals and
Extracts, Bangalore, India. Both the compounds were
administered orally to fasted rats as an aqueous
suspension. The in group that received both drugs,
curcumin was administered first followed immediately by
piperine. Control rats received water only. Curcumin was
given in a dose of 2g/kg and piperine, 20mg/kg.

Under ether anaesthesia, pre and post drug jugular vein
blood samples were collected from both groups of rats into
centrifuge tubes at the time intervals – 0, 0.25, 0.50, 0.75,
1, 2, 3, 4, 5 and 6h. The blood was allowed to clot at room
temperature for about 1h and then centrifuged at 3000rpm
for 10min. The serum was separated out carefully using
Pasteur pipettes into storage tubes and frozen at -20°C
prior to analysis.
Influence of Piperine on the Pharmacokinetics of Curcumin in
Animals and Human Volunteers
Guido Shoba₁, David Joy₁, Thangam Joseph₁, M. Majeed₂. R.
Rajendran₂, and P.S.S.R. Srinivas₂
₁Department of Pharmacology, St John’s Medical College,
Bangalore, India
₂5AMI Chemicals & Extracts (P) Ltd., Banaglore, India
Received: August 1, 1997; Revision accepted: October 18, 1997

Human volunteer studies
Ten healthy male volunteers, 20 to 26 years, weighing 50 –
75kg (mean 60 ± 1.93) participated in a randomized cross
over trial, to determine the comparative bioavailability and
pharmacokinetic profile of curcumin when given alone and
with piperine. Complete physical examination and an
electrocardiogram were done. Laboratory tests comprising
complete blood counts and haemoglobin percentage, blood
biochemistry consisting of blood urea nitrogen (BUN) serum
creatinine, total and conjugated bilirubin, alkaline
phosphatase, aspartate transaminase (ASAT), alanine
transaminase (ALAT), urine albumin, and sugar were
preformed to confirm that the subjects included in the
study were normal. The study was formally approved by
the Institutional Ethical Committee and informed consent
was obtained for all subjects.
Subjects abstained from food since 10pm of the previous
evening and reported to the laboratory at 7am.
Venepuncture was done using a 20g scalp vein set with
heparin lock and left in situ. Blood samples (5ml) were
collected (without anticoagulant) at 0, 0.25, 0.50, 0.75, 1, 2,
3, 4, 5 and 6h post drug. Blood was allowed to clot at room
temperature for 1h. Serum separation and storage until
analysis was as explained earlier. Following basal blood
sample collection 2g of pure curcumin powder (4 capsules
of 500mg curcumin each) or 2g of pure curcumin powder
combined with 20mg of pure piperine powder (4 capsules
of 500mg curcumin + 5mg piperine each; identical capsules
prepared by Sami Chemicals and Extracts, Bangalore, India)
was given with 150ml of water. Blood sampling after
curcumin per se and curcumin with piperine was done two
occasions, separated by a two week wash out period on the
same volunteers. The following precautions were taken

during the trial; subjects refrained from smoking,
consuming alcohol, or beverages, and from taking drugs of
any kind 24h prior to and during the trial. Standard meals
were given to all the participants on the day of the test.
Analytical methods
Estimation of curcumin was done by reverse phase high
pressure liquid chromatography (HPLC) using modification
of the method described by Tannesen et al. (10). The
modification was done by Sami Chemicals and Extracts,
Bangalore, India, and is detailed below, the mobile phase
used was ethanol : methanol (60 : 40) instead of only
ethanol and the flow rate was changed from 1.2ml/min to
1ml/min. HPLC grade methanol and low actinic glassware
protected from light were used for the entire procedure.
Extraction and preparation of standard solution
Curcumin (25mg) was dissolved and diluted to 25ml with
methanol in a volumetric flask; 0.1ml (100 µm) of this was
transferred to a volumetric flask and diluted with methanol
up to 10ml making a 10ppm solution; 0.1ml of this 10ppm
solution was transferred to another 10ml volumetric flask
and the volume make up with methanol making a 0.1ppm
solution.
Extraction of curcumin from serum and preparation of
sample
Serum samples stored t -20°C were equilibrated to room
temperature before analysis. A portion of 1ml was
transferred into a 10ml volumetric flask and about 5ml of
methanol added. The mixture was shaken thoroughly and
heated at 80°C on a water bath for half an hour. After
cooling to room temperature, methanol was added to make

up the volume to 10ml and mixed well. The turbid solution
was transferred into a 15ml centrifuge tube and centrifuged
at 4000 RPM for 10 minutes. The supernatant was collected
by means of a 25ml syringe and 10cm needle (Luer lock)
and the clear solution filtered through a 0.45µm, 13mm
Millipore membrane filter, into a narrow end test tube.
20µl of the solution were injected into the chomatograph
for carry out the HPLC analysis.
Samples were read by UV absorbance of 254nM. The
recovery rate experiments were carried out by adding a
known amount of standard curcumin to the serum and the
added curcumin extracted as per procedure and quantified.
The recovery rate of curcumin from serum ranged from 87
– 89.9%. The minimum level of detection of curcumin was
0.001µg/ml.
Calculation
Content of curcumin in µg/ml in the test sample
Standard Reading x standard concentration
Standard Reading x standard concentration
Treatment of pharmacokinetic data
For calculation of pharmacokinetic parameters (PK), curve
fitting was carried out by a model independent method
with non-linear least-square regression analysis using a
computer designed programme “PHARMKIT”. This
programme uses an algorithm call “SIMPLEX” for calculating
non-linear least-squares. The various PK parameters
calculated were: absorption half-life (t½(a)), elimination half
live (t½el), volume of distribution (Vd); and clearance (CI).
Areas under the concentration time curve (AUCo-m) was

calculated using the trapezoidal method. Maximum
concentration (Cmax) and time to max (Tmax) are observed
values. Relative bioavailability (F) was calculated using
formula:
AUC Curcumin + piperine
AUC Curcumin
Statistical analysis
Serum concentration time curves and the PK parameter
from animal data were analysed using the Students ‘t’ test
while the paired ‘t’ test was used for comparing serum
concentration curves in humans. PK parameters of
curcumin when given alone in humans could not be
calculated as curcumin could not be detected in most of the
samples.
Planta Med. 64 (1998) Guido Shoba, David Joy, Thangam
Joseph, M. Majeed. R. Rajendran, and P.S.S.R. Srinivas
=
F = x 100
Results
Animal studies
Curcumin alone at 2g/kg or when combined with piperine,

20mg/kg, was well tolerated by the rats as they showed no
untoward effects for 48h. Yellow coloured faecal pellets
appeared at 30h post drug and continued up to 48h.
Perusal of Figure 1 indicates that when curcumin was given
alone, peak serum concentrations of 1.00 ≠ 0.26 µg/ml
were attained rapidly within 0.75h and plateaued till 1h.
Thereafter, the levels declined gradually reaching zero at
5h. The plasma concentration time curve of curcumin in
combination with piperine followed a similar pattern from 0
to 0.75h and 3 to 5h. However piperine produced higher
serum concentrations of curcumin at 1 and 2 h (1.55 ± 0.21
and 1.50 ± 0.25 µg/ml) respectively, being significantly
higher (P <0.02) at 2h. Thus piperine significantly enhanced
the serum concentration of curcumin, albeit for a limited
duration (although serum samples were collected up to 6h,
values are depicted till 5h only, since the 6h value was also
‘0’ in all animals).
Table 1 shows the values (mean ± SEM) of the
pharmacokinetic parameters if curcumin per se and when
combined with piperine. Cmax was increased from 1.35 ±
0.23 to 1.80 ± 0.16 µg/ml, but was not statistically
significant, while Tmax was significantly increased from 0.83
± 0.05 to 1.29 ± 0.23h (P <0.02). The t½(el) significantly
decreased from 1.70 ± 0.58 to 1.05 ± 0.18h (P<0.002).
Though t½(a) increased from 0.31 ± 0.07 to 0.47 ± 0.03h and
AUC increased from 2.36 ± 0.28 to 3.64 ± 0.31 µg/h/ml,
these increases were not statistically significant. CI
significantly decreased from 713.00 ± 12.00 to 495.00 ±
37.00:/h (P<0.02) but the decrease in the Vd from 1366.00
±248.70 to 782.60 ± 193.90L/kg was not significant. The
relative bioavailability of curcumin when combined with

Piperine is 154%.
Human volunteer studies
Curcumin alone or when combined with piperine was well
tolerated by all the subjects and there were no adverse or
untoward reactions; 2 subjects dropped out of the study for
non-medical reasons. Therefore all calculations presented
here are based on the data obtained from 8 subjects. In
figure 2 is shown the serum concentration if curcumin per
se and when given with piperine. Although serum samples
were collected up to 6h, we have depicted values till 3h,
since the 4, 5 and 6h values were also ‘0’ in all subjects.
Serum levels’ of curcumin when given alone were either
very low or undetectable at mot time points in most
subjects, explaining he almost flat serum concentration
curve (Fig. 2). However, when piperine was added the
serum concentrations of curcumin were significantly
increased at the time points up to 0.75h; P <0.01
Fig. 1 Serum concentrations µg/ml (mean ± SEM) of
curcumin 20g/kg oral alone and with piperine 20mg/kg in
rats (n= 6/group/time cut). Significance as compared to
curcumin alone; *P <0.02.
Fig. 2 Serum concentrations µg/ml (mean ± SEM) of
curcumin 2g oral alone and with piperine 20mg in humans

(n= 8). Significance as compared to curcumin alone;
*P <0.01 **P <0.001.
at 0.25h and 0.5h; P <0.001 and at 0.75h. Subsequently
there was a rapid decline up to 1h and thereafter a gradual
decline to zero by 3h.
In Table 2 are depicted the PL parameters (Mean ± SEM) of
curcumin when given alone and with piperine. Cmax
(observed values) when curcumin was given alone was only
0.006 ± 0.005 µg/ml at 1h whereas when piperine was
added the Cmax (observed value) was increased to 0.18 ±
0.16 µg/ml and was attained earlier, i.e. at 0.75h. Vd and CI
could not be calculated with curcumin alone as serum
levels were not detected at most time points in most
subjects. The mean AUC(0-tn) however, was calculated using
the trapezoidal method and was found to be 0.004 µg/ml,
the relative bioavailability of curcumin when given with
piperine was therefore 2000%.
Discussion
The results obtained in the study demonstrate that piperine
enhances the oral bioavailability of curcumin in both rats
and humans at does that we devoid of adverse side effects.
However, certain differences between rats and human with
respect to curcumin were evident. Curcumin per se attained
overall moderate serum concentrations over a 4h period in
rats with peak levels occurring between 0.75h to 1h. On the
other hand, in humans when curcumin was given alone only
negligible serum concentrations of curcumin were
detectable the serum concentration-time curve being

almost flat. This difference may be due to high oral dose
employed in the rat (2g/kg), whereas the human does was
about 60 times less, approximately 33mg/kg. Curcumin
serum concentrations reached zero at 5h in rats and 3h in
humans. Further in rats with the addition of piperine,
curcumin achieved high concentrations than in humans
albeit for a short period, took s longer time to peak and
declined slowly. Whereas in humans Tmax was attained
earlier and then declined rapidly. This rapidity in decline is
more apparent probably because of the high levels of
curcumin achieved with piperine as compared to curcumin
alone. There was an increase in the AUC though not
significant as an increase in bioavailability of curcumin by
about one and a half times as compared to curcumin given
alone in both rats and humans. In rats when piperine was
added to curcumin both Vd and CI decreased which may
have also contributed to the higher concentration, such a
comparison was not possible in humans for season
explained earlier. Our findings concerning absorption f
curcumin in rats are in agreement with data obtained by
Wahlstrom and Belnnow (11), who sowed that when
Sprague Dawley rats were given curcumin 1g/kg p.o.,
measurement of blood plasma levels and biliary excretion
indicated some absorption from the gut with no apparent
toxic effect unto 5g/kg p.o. Likewise, Khanna et al. (12);
found that after curcumin, 100mg/kg p.o., 74% was
absorbed from the gastrointestinal tract within the first 5h,
while complete elimination occurred within 48h. Our
results are, however, in conflict with studies by
Ravindranath and Chandrasekhara (6), who could not
detect curcumin 400mg/kg p.o. They, however, did report
60% absorption of curcumin as determined by the amount
excreted in the faeces.
There is evidence that piperine is a potent inhibitor of drug
metabolism, and glucuronidation altering the disposition

and bioavailability of a large number of drugs (8). Further
piperine at 20mg in humans has also been shown to
produce earlier Tmax higher Cmax and AUC of drugs like
propranolol and theophylline (13). This property of piperine
suggests that it may be involved in inhibiting the
metabolism of curcumin and enhancing bioavailability.
In conclusion, the study shows that piperine enhances the
serum concentration and bioavailability of curcumin in rats
and man probably due to increased absorption and reduced
metabolism.
References
1. Kuhup, P.N.V. (1977) Handbook of Medicinal Plants, Vol. 1.
Central Council for Research in Indians Medicine and
Homeopathy (CCRIMH), New Delhi.
2. Srimal, R.C., Dhawan, R.N. (1985) in: Development of
Unuani
drugs from herbal sources and the role of elements in their
mechanism of action. Hamdard National Foundation
Monograph. New Delhi.
3. Ammon, H.R.T., Wahi, M.A. (1991) Planta Med. 57, 1-7.
4. James, J. S. (1993) AIDS Treatment News 176, 1-3.
5. Ravindranath, V., Chandrasekhara, N. (1980) Toxicology
(Ireland) 16. 259-260.
6. Ravindranath, V., Chandrasekhara, N. (1981) Toxicology 20.
251-257.
7. Holder, G.M., Plummer, J.L., Ryan, A.J. (1978) Xenobiotica
8.

761-768.
8. Atal, C.K., Dubey, R.K., Singh, J.J. (1985) Pharmacol. Exp.
Ther.
232. 258-262.
9. Singh, J., Dubey, R.K., Atal, C.K. (1985) J. Pharmacol. Exp.
Ther
236. 488-493.
10. Tonnesen, H.H., Karlsen, J. (1993) J. Chromatography 259.
367-
369.
11. Wahlstrom, B., Blennow, G. (1978) Acta Pharmacol.
Toxxicol.
43. 86-92.
12. Khanna, M., Singh, S., Sarin, J.P.S. (1981) Indian Drugs 19.
31.
13. Bano, G., Raina, R.K., Zutshi, U., Bedi, K.L., Johri, R.K.,
Sharma,
S.C. (1991) Eur. J. Clin. Pharmacol. 14. 615-617.
Dr Guido Shoba
Department of Pharmacology
St. John’s Medical College
Bangalore 560 034
India

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