Drug Delivery

ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: https://www.tandfonline.com/loi/idrd20

Enhanced oral bioavailability of piperine by self-

emulsifying drug delivery systems: in vitro, in vivo
and in situ intestinal permeability studies

Bing Shao, Chao Cui, Hongyu Ji, Jingling Tang, Zhiyong Wang, Hongmei Liu,
Mengnan Qin, Xin Li & Linhua Wu

To cite this article: Bing Shao, Chao Cui, Hongyu Ji, Jingling Tang, Zhiyong Wang, Hongmei
Liu, Mengnan Qin, Xin Li & Linhua Wu (2015) Enhanced oral bioavailability of piperine by self-
emulsifying drug delivery systems: in�vitro,�in�vivo and in�situ intestinal permeability studies, Drug
Delivery, 22:6, 740-747, DOI: 10.3109/10717544.2014.898109

To link to this article: https://doi.org/10.3109/10717544.2014.898109

Published online: 27 Mar 2014.

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ISSN: 1071-7544 (print), 1521-0464 (electronic)

Drug Deliv, 2015; 22(6): 740–747

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.898109

RESEARCH ARTICLE

Enhanced oral bioavailability of piperine by self-emulsifying drug

delivery systems: in vitro, in vivo and in situ intestinal permeability
studies
Bing Shao1, Chao Cui1,2, Hongyu Ji1, Jingling Tang2, Zhiyong Wang1, Hongmei Liu1, Mengnan Qin1, Xin Li1, and
Linhua Wu1
1
Department of Pharmacy, The Second Affiliated Hospital, Harbin Medical University, Key Laboratory of medications research, College of
Heilongjiang Province, Harbin, P. R. China and 2Department of Pharmaceutics, School of Pharmacy, Harbin Medical University, Harbin, P. R. China

Abstract Keywords
The main purpose of this work was to develop and evaluate a self-emulsifying drug delivery Bioavailability, dissolution, piperine,
system (SEDDS) of piperine to enhance its solubility and bioavailability. The formulation was self-emulsifying drug delivery system,
optimized by solubility test and ternary phase diagrams. Then physiochemical properties and single-pass intestinal perfusion
in vitro release of SEDDS were characterized. In vivo pharmacokinetics study and in situ single-
pass intestinal perfusion were performed to investigate the effects of SEDDS on the History
bioavailability and intestinal absorption of piperine. The optimized formulation was composed
of ethyl oleate, Tween 80 and Transcutol P (3:5.5:1.5, w/w), with the level of the piperine Received 13 January 2014
reached 2.5% (w/w). The in vitro dissolution rates of piperine SEDDS were significantly higher Revised 21 February 2014
than the self-prepared capsules. In vivo pharmacokinetic study showed Cmax1, Cmax2 and area Accepted 21 February 2014
under the curve of piperine after oral administration of SEDDS in rats were 3.8-, 7.2- and 5.2-fold
higher than the self-prepared capsules, respectively, and the relative bioavailability of SEDDS
was 625.74%. The in situ intestinal absorption study revealed that the effective permeability and
the effective absorption rate values of piperine for SEDDS were significantly improved
comparing to solutions (p50.01). So SEDDS formulation could improve the oral bioavailability
and intestinal absorption of piperine effectively.

Introduction extent of intestinal absorption, then enhance the serum

concentration and bioavailability of it in rats and men with
Piperine is a major component alkaloid/amide present in the
no adverse effects (Shoba et al., 1998; Suresh & Srinivasan,
fruit of black pepper (Piper nigrum) and long pepper (Piper
2007, 2010). Meanwhile, piperine can inhibit the P-glycopro-
longum). The piperine is used not only for the dietaries as
tein (P-gp) which is abundantly expressed in cancer cell that
the spices, but also as a useful drug with great potential
impact anti-tumor drugs therapeutic outcome and leads to the
for medicine. The piperine is one of the most well-known
multidrug resistance (Han et al., 2008; Yoncheva et al., 2012;
components because of its high medicinal properties
Alzoubi et al., 2013). More recently, Singh (Singh et al.,
(Bhardwaj et al., 2002; Gao & Morozowich, 2006). In
2013) provided the direct proof that piperine could affect P-gp
Indian medicine, the piperine and its derivatives were always
function and expression in cell cultures in a manner.
used for treating epilepsy, headache and so on (Pattanaik
Therefore, it is important for piperine as an agent to overcome
et al., 2006). As increasingly popular modern-day uses of
multidrug resistance. The piperine was researched more about
piperine, it has been reported to inhibit oxidative stress, anti-
by the intravenous administration, such as the oil-in-water
fungal, anti-inflammatory activity and anti-tumor (Sunila &
emulsion of piperine, piperine lipid nanoparticle, positively
Kuttan, 2004; Bezerra et al., 2008; Ee et al., 2010). A new
charged stearylamine lipid nanosphere and pegylated lipid
study showed that the piperine could enhance the bioavail-
nanosphere (Veerareddy et al., 2004; Veerareddy &
ability of a number of therapeutic drugs recently (Srinivasan,
Vobalaboina, 2008). Pharmacokinetics of piperine in lipid
2007). Previous studies have indicated that piperine can
nanospheres showed a biexponential decline with signifi-
increase the absorption of curcumin not only by inhibiting of
cantly high AUC than piperine solution, and it meant that
hepatic and intestinal glucuronidation, but also enhancing the
some new formulations could change the bioavailability of
piperine in vivo. However, piperine has low solubility in water
Address for correspondence: Prof. Linhua Wu, Department of Pharmacy, and high lipid/water partition coefficient of 179. 33
the Second Affiliated Hospital, Harbin Medical University, Key (lgp ¼ 2.25) (Wu et al., 2012). The low solubility in water
Laboratory of medications research, College of Heilongjiang Province,
157 Baojian Road, Harbin, 150086, P. R. China. Tel: +86 45186605581. and poor dissolution may be the rate-determining step in the
Fax: +86 45186665559. E-mail: yaoji636@hotmail.com absorption processes for piperine. So, a great deal of
DOI: 10.3109/10717544.2014.898109 Studies on oral bioavailability of piperine 741

attentions should focus on the new formulations to improve
the solubility and dissolution of piperine.
Recently, self-emulsifying drug delivery system (SEDDS)
is a class of emulsion that receives particular attention as a
means of enhancing oral bioavailability of hydrophobic drugs
with poor aqueous solubility (Neslihan Gursoy & Benita,
2004). The self-emulsifying formulation is dispersed in the
gastrointestinal (GI) tract to form the spontaneous of
microemulsions with a droplet diameter usually within the
range of 10–100 nm, thus a large interfacial surface area is
provided for pancreatic lipase to promote the rapid release
of the drugs which is the limiting step in the absorption from
GI tract (Pouton, 1985). Many surfactants have been
reported to improve the lipophilic drugs oral bioavailability
by inhibiting human intestinal efflux transporters (Rege et al.,
2002; Tang et al., 2013). In brief, SEDDS is one formulation
technique which could be prepared as physically stable
formulation to enhance the bioavailability of the hydrophobic
drugs, especially for poorly water soluble traditional Chinese
medicines (Zhang et al., 2008). In our earlier studies,
Tang et al. (2008) and Shao et al. (2010) prepared the new
self-emulsifying formulation of Ginkgo Biloba extracts and
Wurenchun (alcohol extract from Fructus Schisandrae
Chinensis) for improving the bioavailability of the main
active components, respectively. However, it seems that
the relationship between the SEDDS formulations and the
mechanisms for enhancing the lipophilic drugs oral bioavail-
ability was not clear currently. Thus, the investigation of drug
absorption in the intestine is extremely important, and it will
increase the understanding of the mechanisms of drug across
the intestinal epithelium. In situ single pass intestinal
perfusion (SPIP) is usually used to investigate the intestinal
absorption behavior of drugs.
The main objective of the study was to develop an
optimized formulation of piperine SEDDS in order to improve
the solubility, the dissolution rate, the bioavailability after oral
administration compared with the self-prepared piperine
capsules. In the study, the SEDDS formulation of piperine
was developed by solubility tests and pseudo-ternary phase
diagram, meanwhile the particle size analysis and drug
release were used to characterize the optimized formulation.
Finally, the relative bioavailability of the piperine SEDDS
formulation was evaluated compared with the self-prepared
capsules in rats, and in situ SPIP was also conducted for
understanding the absorption mechanism.
Materials and methods
Materials
Piperine (purity 98%) was obtained from Shanxi Huike
Botanical development, Co, Ltd. (Xi’an, Shannxi, China).
Standard substance of piperine was supplied by the National
Institute for Food and Drug Control (Beijing, China). Ethyl
oleate and oleic acid were purchased from Sinopharm,
Shanghai Chemical Co. (Shanghai, China). Soybean oil
(food grade), Tween 80 and PEG 400 were obtained from
Tianjin Kemi’o Chemical Development Center (Tianjin,
China). 1, 2-propylene glycol and hydrochloric acid were
obtained from Tianjin Tianda Chemical Plant (Tianjin,
China). Cremophor (RH40, and ELP) was kindly provided
by BASF Co, Ltd. (Ludwigshafen, Germany). Transcutol
P was gifted by Gattefoss Corp (Saint-Priest, France). Sodium
dodecyl sulfate was purchased from Sigma Chemical Co.
(St. Louise, MO), heparin was purchased from Wanbang
Pharm. Co, Ltd. (Xuzhou, Jiangsu, China) and diazepam
was purchased from Jinyao Pharm. Co, Ltd. (Tianjin, China).
All other chemicals and solvents were reagent grade and used
without further purification.
Solubility studies
Solubility studies were conducted by adding an excess amount
of piperine (approximately 10 mg) to closed glass vials
containing 1 ml of the vehicle (Table 1). Then, the samples
were vortexed in a shaking water bath for 3 days at 25  C to
stimulate for equilibrium. After all, the resulting samples
were centrifuged at 5000 rpm for 10 minutes to separate the
undissolved piperine. After dilution of the supernatant using
methanol, the quantity of piperine presented in the solution
was determined by HPLC system (Waters Corp, Milford,
MA) consisting of Millenninum 32 software, 486 UV
spectrometer (Waters Corp) and 510 pump. Column was
DiamnsilÔ C18 column (200 mm  4.6 mm, 5 mm; Dikma,
Beijing, China). Mobile phase, a mixture of methanol and
water (77:23, v/v) was eluted at a flow rate of 1.0 ml/min.
The mobile phase effluents were monitored at 343 nm.

Table 1. Blank formulations compatibility test (vehicle compositions for screening SEDDS formulations,
with the proportion of oil 30%, surfactant 50%, cosurfactant 20%).

Oil Surfactant Cosurfactant Homogeneous Delamination (after 24 h)

1 Oleic acid RH40 Transcutol P Y Y
2 Oleic acid RH40 1, 2-Propylene glycol N Y
3 Oleic acid RH40 PEG400 Y Y
4 Oleic acid Tween80 Transcutol P Y N
5 Oleic acid Tween80 1, 2-Propylene glycol Y Y
6 Oleic acid Tween80 PEG400 Y Y
7 Ethyl oleate RH40 Transcutol P Y Y
8 Ethyl oleate RH40 1, 2-Propylene glycol Y Y
9 Ethyl oleate RH40 PEG400 Y Y
10 Ethyl oleate Tween80 Transcutol P Y N
11 Ethyl oleate Tween80 1, 2-Propylene glycol N Y
12 Ethyl oleate Tween80 PEG400 Y Y
13 Soybean oil RH40 Transcutol P N Y
14 Soybean oil Tween80 Transcutol P Y Y
742 B. Shao et al.
The inter- and intra-day variance of the method was within
the acceptable range of less than 2%. The curve was found
to be linear in the concentration range of 0.5–50.0 mg/ml
(r ¼ 0.9999). All solubility studies were repeated thrice.
Construction of pseudo-ternary phase diagram
The formulations were prepared in the mixture of excipients in
an isothermal water bath. A series of self-emulsifying systems
were prepared with different concentrations of oil, surfactant
and cosurfactant. All components ratio were regarded as being
100%. The mixture (0.2 ml) was added into 100 mL of water in
a glass beaker at 37  C under gentle agitation (Shao et al.,
2010). The system with clear appearance or slightly opaque
was considered to be a fine emulsion (Kim et al., 2011).
A pseudo-ternary phase diagram was built to identify the good
self-emulsifying region. All studies were repeated thrice.
Finally the effect of piperine on the self-emulsifying perform-
ance of SEDDS should be investigated, and 2.5% piperine
(w/v) was added to the boundary formulations of the self-
emulsifying domain of the ternary phase diagrams to observe
the emulsifying performance. The formulation with varying
concentrations of ethyl oleate (from 25% to 70%), Tween
80 (from 25% to 70%), Transcutol P (from 5% to 25%) and
piperine (25 mg) at 30  C in an glass beaker to obtain a fine
blend of mixture at a liquid state.
Preparation of self-emulsifying formulation and
particle size determination
The piperine dosage in self-emulsifying formulation was
determined according to the earlier report (Wang et al, 2010).
The oil, surfactant and cosurfactant were weighted by a glass
beaker and mixed by vortexing. A certain amount of piperine
was added to the mixed liquor and heated at 40  C with a low
speed to make it homogeneous. In the optimal prescription,
the fill volume of a size 0 capsule containing 12.5 mg piperine
was used for the next experiments at ambient temperature.
The particle size and the distribution of the emulsion were
determined by the Zetasizer Nano ZS90 (Malvern Instruments
Inc, Westborough, MA). SEDDS formulation (0.2 ml) was
diluted into 100 ml purified water with a gentle stirring for the
particle size analyzer.
In vitro dissolution study
Dissolution profiles of the SEDDS formulation and the
control piperine formulation were obtained using a VK 7000
dissolution testing station (Hanson Research Corporation,
Chatsworth, CA) according to the second method of Chinese
Pharmacopoeia dissolution procedure (paddle method) and
operating at a rotation speed of 100 rpm. The control piperine
capsules were self-prepared using the same piperine material
(25 mg). SEDDS capsules or the self-prepared capsules were
placed into the 900 mL hydrochloric acid at 37 ± 0.5  C.
In order to satisfy the sink conditions, 0.1% sodium dodecyl
sulfate was added in the medium. Each sample (5 ml) was
withdrawn at 5, 15, 30, 45 and 60 min with replacement by an
equal volume of fresh medium media to maintain a constant
volume of the dissolution medium. Samples were filtered
using a 0.45 mm microfiber filter and then 10 ml sample was
injected into the HPLC system. The quantification method of
Drug Deliv, 2015; 22(6): 740–747
piperine by HPLC was described above. Calibration curves
were linear over the range of 0.5–50.0 mg/ml for the piperine
(r ¼ 0.9999).
In vivo oral absorption study
Experimental protocols were approved by the Animal Care
Committee of Harbin Medical University. Twelve male
Sprague-Dawley rats weighting 180–220 g were obtained
from Experimental Animal Center of the Second Affiliated
Hospital of Harbin Medical University (Harbin, Heilongjiang,
China). The rats were randomly divided into two groups. All
animals were fasted for 24 h but allowed free access to water
before drug administration. Piperine SEDDS or self-prepared
capsules were given to six rats by intragastric administration
at a dose of two capsules containing 50 mg piperine. After
dosing, blood samples (0.3 ml) were collected into the
heparinized tubes at predetermined time intervals from rats
in each group. And then the plasma samples were immedi-
ately separated by centrifugation at 10 000 rpm for 10 min,
and the supernatant was transferred to the labeled tubes and
stored at 80  C until analysis. The plasma drug concentra-
tion was assayed with the HPLC method.
For 100 ml of plasma samples, 10 ml of internal standard
(diazepam, 150 mg/ml in methanol) was added and vortex
mixed. Then the plasma was mixed with 0.3 ml absolute
methanol in the 2 ml centrifuge tube for assay and centrifuged
at 10 000 rpm for 2 min to precipitate the proteins. Finally, an
aliquot of 10 ml the supernatant was injected into the HPLC
system. The concentration of piperine in rat plasma was
determined by HPLC as described above, except using the
methanol and water (68:32, v/v) mobile phase at a flow rate of
1.0 ml/min and detection wavelength was set a 320 nm. The
calibration curve was linear over the range 0.25–20 mg/ml in
plasma (r ¼ 0.9997) with the lower limit of quantification
(LLOQ) of 0.1 mg/ml. Meanwhile the method showed well
reproducibility with intra-day and inter-day precision, as well
as accuracy for piperine. The extraction recovery was 90.15%
for piperine and 73.40% for internal standard, respectively.
Pharmacokinetic data analysis
The maximum plasma concentration (Cmax), and the time to
reach maximum plasma concentration (Tmax) were compiled
from the blood data. The elimination rate constant (Ke) was
counted by means of the least-squares regression technique
according to the data for the last three or four points of the
plasma concentration-time curve and the half-life (t1/2) of the
drug was got by 0.693/Ke. The area under the drug
concentration time curve was calculated from 0 to 60 h
(AUC0!60 h) using the liner trapezoidal method. The relative
bioavailability (BA) of SEDDS (test formulation) to the self-
prepared capsules (reference formulation) was figured up with
the following equation:
AUCtest Dosereference
Relative BAð%Þ ¼ 
AUCreference Dosetest
Statistical analyses used an unpaired Student’s t-test. The
data were presented as mean with standard error (SE) for
the each group. The p50.05 and p50.01 were considered as
marginal significant and dramatic significant, respectively.
DOI: 10.3109/10717544.2014.898109
In situ intestinal absorption studies
The SPIP technique was applied as previously reported
(Luo et al., 2013; Samiei et al., 2013). The male Sprague-
Dawley rats were fasted for 24 h but allowed free access to
water. According to the method of Huo et al, three intestine
segments of duodenum, jejunum and ileum were tested for
the intestinal absorption studies (Huo et al., 2011). Firstly, the
rats were anaesthetized using an intraperitoneal injection of
sodium pentobarbital to get each of the small intestinal
segments about 10 cm and cannulated at both ends with
plastic tubing. During the experiment a heating lamp was
used and the exposed intestines were covered with gauze
moistened by frequent applications of warm saline to
maintain body temperature. The intestinal segment was
washed with phosphate buffer saline maintained at 37  C for
approximately 30 min until the outlet solution was visually
clear. Then the intestinal segment was infused by the
perfusion fluids which removed by idling the DHL-A
peristaltic pump (Shanghai huixin Precision Pump,
Shanghai, China) at a rate of 0.25 ml/min for 30 min for
balance. Phosphate buffered solution (PBS) was used in the
SPIP and it comprised 7.78 mM D-glucose. Each perfusion
experiment lasted for 60 min, and samples were collected at
an interval of 10 min in pre-weighed glass tubes. The samples
were centrifuged at 10 000 rpm for 5 min, the upper layers
were filtered through a membrane filter and 10 ml samples
were injected into the column for the HPLC assay. The
quantification method of piperine by HPLC was also
described as before. Calibration curves were linear over the
range of 0.10–10.00 mg/ml for the piperine (r ¼ 0.9997). At
the end of experiment, the length and perimeter of the
perfused intestinal segments were accurately measured.
In the SPIP studies, in order to study whether the
absorption of piperine has dose-dependent. The low, middle
and high concentration (1, 5 and 10 mg/ml) of piperine in
SEDDS solution and control piperine perfusion solution were
pumped in the best absorption of intestine segment respect-
ively. All the perfusion solution was maintained at 37  C.
For the absorption segment test, the concentration of 5 mg/ml
for SEDDS or control piperine solution were pumped the
intestine segment which was taken about 10 cm from duode-
num, jejunum and ileum, respectively. For further study,
Transcutol P and Tween 80 were selected to investigate the
effect of excipients on absorption process. The concentration
of piperine solution on was 5 mg/ml, and the quantity of the
accessories was added by its proportion in the SEDDS
formulation.
Analysis of perfusion date
Intestinal permeability coefficient (Peff) of the drug and the
effective absorption rate (Ka) were determined according the
following equations:
CoutðcorrÞ ¼ Cout Qout =Qin


Q ln CoutðcorrÞ =Cin
Peff ¼
 2rL  PEG 400
Q 1  CoutðcorrÞ =Cin
Ka ¼
r 2 L
Studies on oral bioavailability of piperine 743
where Qin is the measured flow rate (ml/min) of entering
intestinal perfusate; Qout is the measured flow (ml/min) of
exiting intestinal perfusate for the specified time interval
calculated from the actual intestinal perfusate density (g/ml),
which is determined by weighing the contents of a known
volume of perfusate; Q is perfusion flow rate (0.25 ml/min);
Cin and Cout were the concentration of drug in the effluent
perfusate through the inlet and outlet tube (mg/ml), respect-
ively. Meanwhile the 2rl is the surface area of the intestinal
segment concerned (cm2)
Statistical analyses used an unpaired Student’s t-test. The
data were presented as mean with standard deviation (SD) for
the each group. The p50.05 and p50.01 were considered as
marginal significant and dramatic significant, respectively.
Results and discussions
Solubility study
Self-emulsifying formulations which consisted of the oil,
surfactant, cosurfactant and drug should be clear and
monophasic liquid at ambient temperature. Meanwhile it
usually provides the advantage of increased capacity for
loading more poorly water-soluble drugs when compared with
lipid solutions. The surfactants which are used in the SEDDS
improve the bioavailability by various mechanisms
(Kommuru et al., 2001). So it is important to select the
appropriate vehicle for the efficiency of self-emulsification.
As the piperine could be clearly seen in the solution, the
solubility of the piperine in the vehicles could be estimated by
visual observations, and the data of solubility in the good
vehicles should be determined. And then excipients with high
solubility were selected to carry out the blank formulation
compatibility experiment (Table 1). Among the tested that
No.4 (ethyl oleate, Transcutol P, Tween 80) and No.10 (oleic
acid, Transcutol P, Tween 80) blank formulations were not
layered and formed uniform liquid. The solubility results of
piperine in various vehicles were presented in Table 2. The
piperine appeared to have higher solubility in ethyl oleate
among three oils of soybean oils, oleic acid and ethyl oleate.
Among the various cosurfactants, the Transcutol P showed
highest drug solubility with 185.29 ± 0.16 mg/ml. It is
important to select the appropriate vehicle for the piperine
to improve the solubility. The Transcutol P had the best
solubilizing capacity for piperine, so Transcutol P was
extremely important for the formulation. The solubility of
piperine in Cremophor RH40 and Tween 80 were
30.77 ± 0.11 and 70.71 ± 0.08 mg/ml, respectively (Table 2).
Table 2. Solubility of piperine in various vehicles at 25  C (n ¼ 3,
mean ± SD; mg/ml).
Vehicle Solubility
Water 0.164 ± 0.002
Oleic acid 23.672 ± 0.095
ethyl oleate 37.812 ± 0.058
Soybean oil 18.023 ± 0.038
Transcutol P 185.296 ± 0.158
1, 2-propylene glycol 38.623 ± 0.029
38.702 ± 0.025
Cremophor RH40 30.774 ± 0.106
Tween 80 70.712 ± 0.084
744 B. Shao et al. Drug Deliv, 2015; 22(6): 740–747

Therefore, ethyl oleate, Transcutol P and Tween 80 were indicated that the mean droplet size will be declined when
selected as the oil phase, cosurfactant and surfactant for the emulsification ability of the system increased (Devani
constructing the pseudo-ternary phase diagram to optimize et al., 2004; Li et al., 2005). That is, the cosurfactant with
the formulation. the highest solubilization capacities of piperine is a good
candidate to fit the formulation. Therefore, a little Transcutol
Construction of pseudo-ternary phase diagram P was required which could not only dissolve more drugs but
also reduced the interfacial energy between the oil and water
Pseudo-ternary phase diagram were constructed to determine interface to readily deformed oil droplets. In the study, there
the isotropic clear self-microemulsifying regions and to were no significant differences in the self-emulsifying region
obtain optimum concentration of components in the SEDDS when the piperine was added into the boundary of blank
formulations (Pouton, 1997). A series of blank formulation prescription. Based on the in vitro experiment, the self-
were prepared and their self-emulsifying properties were emulsifying region had a fine emulsion that was clear and
observed through naked eyes. The phase diagram of the slightly opaque in appearance (Figure 2).
systems was observed that increasing the concentration of the Finally, the formulation containing ethyl oleate (30%),
surfactants in formulation could increase the spontaneity of Tween 80 (55%), Transcutol P (15%), and piperine (2.5%, w/w
the self-emulsifying region of the self-emulsification process of the vehicle) were selected as the optimized formulation for
and decrease the mean droplet size (Figure 1). The surfactant the next experiments according to the results of pseudo-
concentration in self-emulsifying formulations usually ranged ternary phase diagram.
from 30% to 70% which will form an emulsion state in the GI
tract. Meanwhile the GI tract may be irritated by majority Droplet size of self-emulsifying formulation
surfactants. Thus 55% surfactant was considered for the
formulation. Surfactants can reduce the energy from the The distribution of the particle size is one of the most
interfacial to form a layer around the emulsion droplets, important characteristics of emulsion for it influences dissol-
stabilize the oil-water interface and provide a mechanical ution in vitro and the time of distribution in vivo which will
barrier to coalescence (Craig et al., 1995). Therefore, there affect the bioavailability (Pouton, 1997, Yap & Yuen, 2004).
will be more self-emulsifying region in phase diagrams with To evaluate the property of the SEDDS formulation, the
more cosurfactant in the formulation. Previous study droplet size of the self-emulsifying system was determined by
the Zetasizer measurement. The mean droplet size of the
Tween 80 piperine emulsions was 89.82 ± 2.16 nm (Figure 2), which is a
0.0
1.0 typical characteristic of SEDDS, suggesting small nanosized
0.1
0.9 droplets were obtained.
0.2
0.8
0.3
0.7
In vitro dissolution study
0.4
0.6 As the piperine has low solubility and high membrane
0.5 permeable, dissolution rate is the primary limiting aspect to
0.5
0.6
0.4
absorption (Six et al., 2004). The drug release profiles of
0.7 piperine from self-emulsifying formulation and self-prepared
0.3
0.8 capsules were compared (Figure 3). For the SEDDDS system,
0.2
0.9
the release rates of piperine were remarkably higher than
0.1 those of the self-prepared capsules. The release percentages of
1.0
0.0 piperine at 60 min were 80.27% and 13.60% in the dissolution
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Transcutol P Ethyloleate medium from SEDDS formulation and the self-prepared
capsules, respectively. Within 15 min, the total release amount
Figure 1. Pseudo-ternary phase diagrams indicating the efficient self- of piperine from the SEDDS formulation rapidly reached
emulsification region with ethyl oleate as oil, Tween 80 as surfactant, more than 75%. The piperine from the SEDDS formulation
and Transcutol P as the cosurfactant, with a drug loading of 2.5%. The
solid outline represents the area explored for nano-emulsion region; and could be released rapidly due to the spontaneous formation of
the filled squares represent the compositions which were evaluated. emulsion and the resultant small droplet size, compared with

Figure 2. Size distribution of Piperine (Pip) Size Distribution by Intensity

15
SEDDS in water.

10
Intensity (%)

0
0.1 1 10 100 1000 10000
Size (r.nm)
DOI: 10.3109/10717544.2014.898109 Studies on oral bioavailability of piperine 745

that from the self-prepared capsules which was only arrived improvement about the area under the curve of piperine from
at 1.05%. The release profile of formulation indicated the SEDDS formulation compared with the self-prepared
that SEDDS could increase the dissolution of piperine in capsules. It suggested that the SEDDS can increase the
the dissolution medium which may probably affect the dissolution of piperine in the dissolution medium and the GI
bioavailability in rats. absorption after oral administration. For the SEDDS formu-
lation, it had a double-peaks phenomenon in the plasma
In vivo study concentration-time profiles (Figure 4). The first piperine peak
An in vivo pharmacokinetic study was undertaken in rats in occurred at about 8 h after dosing, and the second peak
order to evaluate the bioavailability of the piperine SEDDS occurred at about 26 h. Although the values of the Cmax1 and
formulation after oral administration compared with the self- Cmax2 for the SEDDS formulation were 5-fold greater than
prepared capsules group. The resultant plasma concentration those for the self-prepared capsules (p50.01), there were no
versus time profiles for the treatments of the oral SEDDS significant difference between Tmax1 and Tmax2 for both
formulation and the self-prepared capsules were shown in plasmas peaks (p40.05), which indicated that the Tmax was
Figure 4, likewise the corresponding pharmacokinetic par- not affected by formulation. In particular, the mean values of
ameters were presented in Table 3. There was a significant AUC0!60h estimated from the individual plasma drug
concentration profiles of the SEDDS formulation was
416.64 mg/ml h and the self-prepared capsules was 79.80 mg/
SEDDS ml h (p50.05). The analyses for the value of t1/2 did not show
100 Self-prepared capsule
apparent differences between of both formulations. As we all
know, the double-peak phenomenon is often happened to the
Piperine Dissolved(%)

80
drug with the hepato-enteric circulation caused by the bile
60 elimination. The earliest study also reported that the piperine
was not detectable in urine at any time interval, however,
40
piperine was excreted as such in the faeces over a period of
20
4 days orally administered to rats (Suresh & Srinivasan,
2010). It might signify that the most of piperine was excreted
0 by bile and probably had hepato-enteric circulation, which
0 20 40 60 could lead to part of drug was absorbed once again in the
Time (minutes) intestine. In present study, the SEDDS formulation had
the smaller emulsion droplet, the faster drug release and
Figure 3. Dissolution profiles of Pip from SEDDS and self-prepared the higher permeability in intestine, which could enhance the
capsule (n ¼ 6).
quantity of the absorption for piperine once again into the
intestine (Porter et al., 2007). Meanwhile when the emulsion
Table 3. Main pharmacokinetic parameters of piperine after a single oral was excreted by the bile, it will have a smaller droplet (Zhang
dose of SEDDS or self-prepared capsules in rats (n ¼ 6, mean ± SE,
**p50.01; *p50.05).
et al., 2013). In the SEDDS group, the initial peak was caused
by the absorption of piperine in the GI tract, and the later peak
Parameters SEDDS self-prepared capsule was caused by the hepato-enteric circulation of piperine in the
plasma concentration profile. The double-peak phenomenon
Cmax1 (mg/ml) 13.81 ± 1.69** 3.63 ± 0.85
Tmax1 (h) 8.33 ± 0.61 6.33 ± 0.95 of the self-prepared capsules was not conspicuous, maybe
Cmax2 (mg/ml) 12.90 ± 1.79** 1.79 ± 0.23 induced by the lower dissolution and absorption of piperine.
Tmax2 (h) 26.66 ± 0.84 26.00 ± 0.89 Hence, the SEDDS would be a favorable formulation in
ke (h1) 0.93 ± 0.66 0.56 ± 0.31
clinical point of view as it could enhance the bioavailability of
t1/2 (h) 9.30 ± 1.39 12.85 ± 2.05
AUC0!60 h (mg/mlh) 416.64 ± 68.34* 79.80 ± 14.27 poorly water-soluble piperine.
AUC0!1 (mg/ml h) 438.42 ± 69.73* 85.21 ± 14.64 As the piperine is widely used in the anti-tumor and
F (%) 625.74 ± 177.10 100 overcoming multidrug resistance, it is important to make sure
the time of first administration in order to affect the function

Figure 4. Plasma concentration-time profiles 16

SEDDS
Pip plasma concentration (µg/ml)

of Pip from SEDDS and self-prepared cap- 14

sule following oral administrations in rats. Self-prepared capsule
Each value is mean ± SE (n ¼ 6). 12
10
8
6
4
2
0
0 10 20 30 40 50 60
Time (hours)
746 B. Shao et al. Drug Deliv, 2015; 22(6): 740–747

of P-gp. For this property, it could probably reduce the (A) 3 # 2

Ka Peff
** # **
## **
administration time for anti-tumor and will provide some 2.5 1.6

Ka (× 10−1 min−1)

Peff (× 10−3 cm/s)

convenience for the patients. Thus the metabolism and 2
1.2
transformation of piperine need to be deeply researched for
1.5
widely using in clinic. 0.8
1

0.5 0.4
In situ intestinal absorption studies
0 0
There are two kinds of technique for the in situ intestinal
perfusion method, recirculating perfusion model and single-
pass perfusion model. The absorption in the recirculating (L: low concentration, M: middle concentration, H: high concentration)
perfusion model will be increased just because it has a long Ka Peff
(B) 2.5 **## 0.8
retention time for the solution in the intestine (Cao et al.,
2013). The single-pass perfusion is suitable for drugs that are 2
* ##

Ka (× 10−1 min−1)

Peff (× 10−3 cm/s)

* 0.6
rapidly absorbed, meanwhile it could have a comparatively # * ##
1.5
absorption just by its slowly single-pass rate (Luo et al., 0.4
2013). Thus it is widely used for the research of the intestinal 1
absorption. The intestinal permeability of the piperine in 0.2
0.5
different concentration of SEDDS or solution formulation was
studied as a function in duodenum using the in situ SPIP 0 0
technique. Three concentrations were selected for this part to
determine whether the permeability of piperine was depended
on concentration. And there was no significant difference (D: Duodenum, J: Jejunum, I: Ileum)
among those concentrations for the two perfusion solutions ka Peff
(C) **
(Figure 5A). Meanwhile Ka and Peff of piperine in the SEDDS 2.5 ## 0.8

Ka (× 10−1 min−1)

Peff (× 10−3 cm/s)

were significantly higher than the control group (p50.01,
p50.05) (Figure 5A). The intestinal permeability of piperine
was increased about 1.3-times for the SEDDS formulation,
compared to the control solution. As a result, the effect of
SEDDS was dependent on the solubilized state of piperine. As
the emulsion has a small droplet size that permits an easily
access epithelial cells and enhances the absorption in
intestine. This result was highly consistent with the dissol-
ution study, and those results revealed that the transport
mechanism may fit the active passive transport. The Peff
values of piperine SEDDS formulation (5 mg/mL) in duode-
num, jejunum and ileum were 0.64  103 ± 0.05  103 cm/
s, 0.45  103 ± 0.04  103 cm/s and 0.39  103 ± 0.07
 103 cm/s, respectively (Figure 5B), which was higher
than the piperine solution. The absorption in duodenum was
significantly better than other intestine segments (p50.01),
not only for the piperine solution but also for the SEDDS
formulation, and those results may caused as the duodenum
has great amount of blood vessels to transform the drug for
absorption. In addition, it indicated that the SEDDS formu-
lation did not change the absorption segment of piperine.
Numerous reports indicated that the surfactants could increase
drug absorption by reversible injury in biological cell
membranes (Neslihan Gursoy & Benita, 2004; Rahman
et al., 2013). So Transcutol P and Tween 80 were selected
for next study. The two excipients in the piperine solution had
increased the absorption which was not extremely significant
(p40.05) (Figure 5C). The SEDDS showed significant
difference compared to the control solution (p50.01).
As a result, enhancement of the effect of SEDDS was
depend on the forming small size of emulsions droplet which is
provided a large interfacial surface area for drug absorption not
only the alone role of complementary (Rahman et al., 2013).
In the dissolution study, the SEDDS formulation had
significant greater extent of the dissolution and it had
#
2
0.6
1.5
1
0.4
0.5 0.2
0 0
Figure 5. (A) Comparison of Peff and Ka of different concentration
perfusion solutions determined by SPIP study in rat duodenum;
(B) Effect of different intestine segments on compounds absorption;
(C) Effect of adjuvant on absorption parameters of Pip solution. (n ¼ 5,
mean ± SD, #p50.05, ##p50.01 represented for Peff, *p50.05,
**p50.01 represented for Ka).
increased contact surface with dissolution media. In the
intestinal absorption studies, the SEDDS formulation had
higher permeability than the control reference. The above
points may explain why the SEDDS formulation can improve
the bioavailability of piperine in vivo.
Conclusions
The piperine is widely used to inhibit oxidative stress,
anti-fungal, anti-inflammatory activity, anti-tumor and so on.
Recently, most hot topics of researches on piperine are
applied to enhance the bioavailability of many therapeutic
drugs, so it is used as an overcome multidrug resistance agent.
But it is not used widely in clinic just because it has the low
solubility and bioavailability. In the present study, the
piperine SEDDS formulation was successfully developed
with an increased dissolution rate, bioavailability and perme-
ability. The relative bioavailability of SEDDS formulation
was 625.7% after oral administration compared to the
DOI: 10.3109/10717544.2014.898109
self-prepared capsules. Therefore, the present study suggested
that the SEDDS formulation could be a potential system to
oral delivery of poorly water-soluble drugs, such as piperine.
Declarations of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
Financial support was provided by the Education Department
of Heilongjiang Province, No. 12541482.
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added to the Declarations of interest section in this version.
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