Introduction
Diferuloylmethane, commonly called curcumin, is a yellow pigment present in the rhizomes
of turmeric (Curcuma longa L., Zingiberaceae). Curcumin exhibits antioxidative, anti-inflammatory, anticarcinogenic,
and chemopreventive properties. In clinical studies, curcumin has been shown to prevent
or treat various cancers in humans [1 ]. Resveratrol (3,5,4′-trihydroxystilbene) is a major component of grapes, wine, peanuts,
and Polygonum cuspidatum Sieb. & Zucc. (Polygonaceae). It also has anticancer, antioxidant, and anti-inflammatory
activities [2 ]. Recently, curcumin and resveratrol, two particularly important polyphenolic compounds,
have been found to exhibit a synergistic anticancer effect against various cancer
types, including colon cancer and hepatocellular carcinoma, and also in the treatment
of diseases associated with oxidative stress [3 ].
However, both curcumin and resveratrol have low aqueous solubility and are rapidly
metabolized. These problems result in poor oral bioavailability [4 ], which is an important restriction on their therapeutic usefulness. Many studies
have indicated the importance of using self-microemulsifying drug delivery systems
(SMEDDS) to improve solubility, absorption, and to increase the oral bioavailability
of poorly water-soluble drugs [5 ]. SMEDDS are defined as isotropic mixtures of oils, surfactants, and co-solvents/co-surfactants
that emulsify under conditions of gentle agitation, similar to those encountered in
the gastrointestinal tract [6 ]. The spontaneous formation of an emulsion presents the drug in a dissolved form,
and the resultant small droplet sizes provide a large interfacial surface area that
enhances the rate and extent of oral absorption [7 ].
In order to enhance the solubility and oral absorption of both compounds, a new self-microemulsifying
formulation containing curcumin in combination with resveratrol (CR-SME) was developed.
The synergistic antioxidant activity and cytotoxicity of the new formulation against
HT-29 cells were also evaluated. It is expected to be a promising approach to improve
the performance of medicines and functional foods used to prevent and treat some important
diseases in the future.
Results and Discussion
Suitable excipients in a self-microemulsifying system should have good solubilizing
properties for the drug combination and form a monophasic liquid at an ambient temperature.
The solubility in various vehicles of curcumin and resveratrol in combination is presented
in [Table 1 ]. The combination had a higher solubility in Cremophor EL than in other vehicles
with 85.98 ± 0.67 and 110.84 ± 0.54 mg/mL of curcumin and resveratrol, respectively,
therefore, Cremophor EL was selected as a surfactant. The result might be due to the
ability of the two polyphenols to form hydrogen bonds with the polyethylene oxide
(PEO) groups [8 ]. In the same manner, Labrasol as a co-surfactant, composed of PEO groups, also exhibited
a high solubilization capacity for the curcumin/resveratrol combination. Polyethylene
glycol (PEG) 400 had a high solubilizing capacity for both compounds. These probably
involved hydrophobic interactions between the ethylene units of PEG and the aromatic
rings of the compounds [9 ], but the formulation could not form a translucent microemulsion upon dilution. In
the case of Transcutol HP, it was not chosen to be a co-surfactant due to the color
change of the produced solution. Among the oily phase compounds, Capryol 90 provided
the highest solubility for both curcumin and resveratrol at 6.71 ± 0.07 mg/mL and
17.35 ± 0.02 mg/mL, respectively.
Table 1 The solubility of curcumin in combination with resveratrol in various vehicles.
Vehicles
Curcumin solubility (mg/mL)
Resveratrol solubility (mg/mL)
Oils
Corn oil
0.39 ± 0.01
0.11 ± 0.01
Capryol 90
6.71 ± 0.07
17.35 ± 0.02
Labrafac PG
0.90 ± 0.01
0.31 ± 0.17
Labrafac CC
0.78 ± 0.03
0.23 ± 0.15
Soyabean oil
0.23 ± 0.02
0.12 ± 0.11
Surfactants
Capryol PGMC
13.93 ± 0.07
16.83 ± 0.05
Cremophor EL
85.98 ± 0.67
110.84 ± 0.54
Cremophor RH 40
3.11 ± 0.04
2.46 ± 0.14
Labrafil M 2125 CS
0.60 ± 0.01
2.38 ± 0.07
Lauroglycol 90
4.24 ± 0.09
10.67 ± 0.02
Co-surfactants
Labrasol
62.99 ± 0.38
69.25 ± 0.17
Lauroglycol FCC
0.08 ± 0.01
1.20 ± 0.08
PEG 400
74.49 ± 0.20
66.68 ± 0.15
Propylene glycol
12.57 ± 0.53
18.25 ± 0.02
Transcutol HP
70.13 ± 0.36
76.50 ± 0.23
Ternary phase diagrams were constructed to study the proportion of components that
can produce the best microemulsion. The mixture of Cremophor EL, Capryol 90, and Labrasol
at the ratio 85 : 10 : 5 by weight was chosen as the best system for the curcumin/resveratrol
combination. This proportion can totally dissolve both compounds and there was no
sign of a phase separation into a cloudy emulsion upon dilution with water in different
ratios (1 : 10, 1 : 100, and 1 : 1000). The liquid CR-SME formulation of 900 mg (1
capsule) contained 25.5 mg of each curcumin and resveratrol.
Emulsion droplet size plays a vital role in the oral delivery of SME. A uniform and
small particle size has an influence on the transport of a drug for delivery to a
specific target [10 ]. The average droplet size of microemulsion from the formulation without compound
(blank-SME) and CR-SME after dilution with water was 13.10 ± 0.40 nm and 15.90 ± 0.10 nm,
respectively. In this study, deionized (DI) water, simulated gastric fluids (SGF),
and simulated intestinal fluids (SIF) were used as a medium for the dispersal of a
microemulsion from the CR-SME formulation. The average particle sizes in the DI water,
SGF, and SIF were 15.85 ± 0.07 nm, 18.29 ± 0.04 nm, and 20.15 ± 0.37 nm, respectively
([Fig. 1 ]). The polydispersity index (PDI) was in the range of 0.075–0.125. This result indicated
that the types of media had some influence on the particle size and PDI. Generally,
the small oil droplet size was observed in the presence of high amounts of surfactant
in the formulation. There is a possibility that Cremophor EL, a nonionic surfactant
was able to solubilize a hydrophobic drug (HLB = 12–14), promoted the small-sized
particles and augmented the entrapment property of the combination for delivery [11 ]. Transmission electron microscopy observations were performed with a volume ratio
of the CR-SME/water at 1/75. This demonstrated the spherical shape of the particles
with no signs of coalescence, even after 24 h of dilution. The image is shown in [Fig. 2 ] in which the average diameter of this formulation was less than 30 nm, which agreed
with the data obtained by the dynamic light scattering technique.
Fig. 1 The particle size and polydispersity index (PDI) of the CR-SME formulation diluted
by different dispersed media (n = 3). SGF: simulated gastric fluid; SIF: simulated
intestinal fluid.
Fig. 2 TEM micrographs of the CR-SME formulation (× 50 000). Bar = 200 nm. (Color figure
available online only.)
The in vitro release profiles of curcumin and resveratrol from the SME formulation in SGF, pH 1.2,
are shown in [Figs. 3 ] and [4 ]. This CR-SME formulation exhibited an immediate release of curcumin, with over 70 %
of curcumin released within 20 min, which was similar to that from the self-microemulsifying
formulation with individual curcumin (C-SME; [Fig. 3 ]). In contrast, only 5 % of the curcumin was released from the non-formulated combination.
A rapid release of resveratrol from the CR-SME formulation was also found with over
80 % within 20 min, and in a similar manner to that from the self-microemulsifying
formulation with individual resveratrol (R-SME; [Fig. 4 ]), whereas only 18 % of the dose was released from the non-formulated combination.
These results demonstrated that the release profile of each polyphenol, either curcumin
or resveratrol, was not affected by the presence of the other ingredients in the formulation.
It should be noted, however, that resveratrol exhibited a greater percentage release
than curcumin, because of its greater number of hydroxyl groups that led to an increased
association of interactions with water. The hydrogen bonding of both the polyphenol
compounds seemed to be a vital variable during the solubilization process [12 ]. The stability of the CR-SME formulation was evaluated for its physicochemical properties
in the intermediate condition (30 ± 2 °C/65 ± 5 % RH) compared to the accelerated
stress condition of 45 °C/75 % RH for 0, 1, and 3 months. A brown glass or container
with light protection is recommended for storage of the CR-SME due to the resveratrol
undergoing photodegradation, and curcumin is also sensitive when exposed to light
[13 ]. From the results of [Table 2 ], the CR-SME formulation showed no significant change in appearance after 3 months
of storage in both conditions. The emulsion droplet size was in the range of 19.4–21.5 nm.
The liquid formulation was found to be stable, and there was no change in the content
of curcumin (99–101 %) and resveratrol (101–102 %).
Fig. 3 The cumulative release of curcumin from a curcumin/resveratrol combination in the
SME formulation (CR-SME), individual curcumin in SME (C-SME), and an unformulated
curcumin/resveratrol combination, in simulated gastric fluid (n = 6).
Fig. 4 The cumulative release of resveratrol from a curcumin/resveratrol combination in
the SME formulation (CR-SME), individual resveratrol in SME (R-SME), and an unformulated
curcumin/resveratrol combination, in simulated gastric fluid (n = 6).
Table 2 Stability data of the CR-SME in intermediate (30 ± 2 °C/65 ± 5 % RH) and accelerated
conditions (45 ± 2 °C/75 ± 5 % RH), mean ± S. D. (n = 3).
Sampling time
Appearance
Visual grading
Particle size (nm)
PDI
% Drug content
Curcumin
Resveratrol
Clear yellow liquid
A
18.29 ± 0.04
0.02 ± 0.01
101.12 ± 2.73
99.83 ± 4.11
A) 30 °C/65 % RH
Clear yellow liquid
A
19.40 ± 0.01
0.04 ± 0.02
99.45 ± 3.57
102.36 ± 3.13
Clear yellow liquid
A
20.22 ± 0.03
0.07 ± 0.01
101.35 ± 4.22
101.45 ± 5.44
B) 45 °C/75 % RH
Clear yellow liquid
A
20.45 ± 0.05
0.06 ± 0.01
99.43 ± 3.09
98.83 ± 4.85
Clear yellow liquid
A
21.51 ± 0.04
0.05 ± 0.00
96.12 ± 2.67
97.72 ± 5.14
Among the several types of cells, HT-29 (human colon adenocarcinoma cell lines) is
frequently used as an in vitro cancer model. In order to test the cytotoxicity of each compound and the CR-SME formulations
against HT-29 cells, MTT assays were carried out. The results in [Fig. 5 ] clearly showed that the CR-SME (the combination ratio for curcumin/resveratrol was
1 : 1) had a lower IC50 than each separate polyphenol used in the SME (18.25 µM for CR-SME, 25.4 µM for R-SME,
and 30.1 µM for C-SME). However, the blank SME had an IC50 value of 100 µg/mL. As Cremophor EL was the main component in this formulation, it
might further increase the therapeutic effect of some anticancer agents to produce
oxidative stress [14 ]. In a similar previous study from Majumdar et al. [3 ], they reported that curcumin in combination with resveratrol was a more effective
chemopreventive agent than each polyphenol alone. In this study, it has been demonstrated
that the co-delivery treatment of polyphenols prepared in the SME formulation caused
greater inhibition of colon cancer cells than using each compound in the same system.
Fig. 5 Cytotoxicity of HT-29 cells treated with different concentrations of CR-SME (each
polyphenol at 5 : 5, 10 : 10, 15 : 15, 30 : 30, and 50 : 50 µM) compared to the C-SME,
R-SME (individual compound at 10, 20, 30, 60, and 100 µM), and blank SME; (n = 8),
duplications.
To test the combined antioxidant effect of curcumin and resveratrol in the SME, the
ferric reducing antioxidant power (FRAP) assay was performed. The CR-SME formulation
produced a higher absorbance at 590 nm than both agents alone in the formulation (C-SME
and R-SME) and the blank SME ([Fig. 6 ]). The number and position of the hydroxyl groups in phenolic acids play an important
role in their antioxidant activity. The antioxidant activity of CR-SME could be related
to the functional groups of resveratrol. It has three hydroxyl groups and a conjugation
between both the aromatic rings [15 ]. Similarly, the phenolic and the methoxy group on the phenyl ring and the 1,3-diketone
system are important for contributing to the antioxidant activity of curcumin. When
both compounds were present, there was a potential for synergy that one antioxidant
helped regenerate the other. In a previous study, the decay kinetics of a curcumin/resveratrol
combination was also found to be lower than each individual compound [16 ].
Fig. 6 Total antioxidant power of CR-SME, C-SME, and R-SME in comparison with ascorbic acid
(5–30 µg/ml) measured by the ferric reducing antioxidant power (FRAP) assay.
Finally, in order to evaluate whether the combination administration of curcumin and
resveratrol affected the oral absorption, studies were performed on CR-SME, C-SME
plus R-SME, and curcumin/resveratrol suspensions. The oral absorption of the combination
or individual compound in SME was much greater than that of the combination in suspension
form, as shown in [Figs. 7 ] and [8 ] for curcumin and resveratrol, respectively. The plasma concentration time profiles
were similar between the administration of CR-SME and C-SME following with R-SME.
The pharmacokinetic parameters in rabbits are summarized in [Table 3 ]. The total plasma concentrations of each compound from CR-SME were significantly
higher when compared with the curcumin/resveratrol suspension (p < 0.05). The AUC0–6 h of CR-SME increased by about 13.7-fold for resveratrol and 34.5-fold for curcumin
than that of the suspension. The CR-SME administration gave a significantly higher
AUC0–6 h of curcumin than did C-SME following with R-SME (p < 0.05). However, there was no
significant difference in the AUC0–6 h of resveratrol between these two administration methods. In addition, all of the
treatments have similar Tmax values at 60 min for curcumin and 90 min for resveratrol.
Fig. 7 Curcumin plasma concentration vs. time profiles after oral administration of curcumin
(50 mg/kg) in combination with resveratrol (50 mg/kg) in the SME formulation, curcumin
in combination with resveratrol in aqueous suspension, or curcumin (50 mg/kg) SME
formulation followed by a resveratrol (50 mg/kg) SME formulation. All values reported
are mean values ± SD (n = 3).
Fig. 8 Resveratrol plasma concentration vs. time profiles after oral administration of curcumin
(50 mg/kg) in combination with resveratrol (50 mg/kg) in the SME formulation, curcumin
in combination with resveratrol in aqueous suspension, or a curcumin (50 mg/kg) SME
formulation followed by a resveratrol (50 mg/kg) SME formulation. All values reported
are mean values ± SD (n = 3).
Table 3 Pharmacokinetics value of CR-SME after oral administration compared to C-SME plus
R-SME and a combined suspension (equivalent to 50 mg/kg of curcumin and resveratrol),
mean ± S. D. (n = 3).
Formulation
Cmax (µg/mL)
Tmax (min)
AUC0–6 h (µg h/mL)
Curcumin
Resveratrol
Curcumin
Resveratrol
Curcumin
Resveratrol
CR-SME
2.02 ± 0.09
3.22 ± 0.25
60
90
1593.75 ± 142.53
2688.75 ± 292.65
C-SME plus R-SME
1.86 ± 0.02
3.57 ± 0.19
60
90
1304.50 ± 118.51
2246.00 ± 284.45
Curcumin plus resveratrol suspension
0.19 ± 0.06
0.55 ± 0.01
60
90
46.25 ± 5.72
196.25 ± 39.29
Materials and Methods
Chemicals
Curcumin (purity ≥ 70 %) was from Sigma-Aldrich. Trans-resveratrol (purity ≥ 98 %)
(P. cuspidatum root extract resveratrol powder) was from Pioneer Herb. Capryol 90, Labrafac CC,
Labrasol, Lauroglycol FCC, Labrafil M 2125 CS, and Lauroglycol 90 were from Gattefosse.
Cremophor EL and Cremophor RH 40 were from BASF. PEG 400 and propylene glycol (PG)
were from the PC Drug Center Co., Ltd. Corn oil was from the Thai Vegetable Oil Public
Company limited. Trichloroacetic acid, sodium phosphate dibasic, sodium phosphate
monobasic, acetonitrile, and methanol (HPLC grade) were from RCI Labscan. Potassium
ferricyanide was from Ajax Finechem Pty Ltd. Ferric chloride was from Sigma-Aldrich.
Ascorbic acid was from Chem-Supply Pty Ltd. Hard gelatin capsules (size 00) were from
Capsugel. All other chemicals used were of analytical grade.
Cell culture
Human colon adenocarcinoma cell lines (HT-29 cells; HTB-38) were from ATCC. McCoyʼs
5 a, FBS, and penicillin (100 IU/mL)-streptomycin (100 mg/mL) (pen-strep) were from
Gibco, Invitrogen. Trypsin-EDTA 0.25 % was from Gibco, Invitrogen. MTT was from Molecular
Probes, Invitrogen. PBS (pH 7.4), 2-(N-Morpholino) ethanesulfonic acid (MES) sodium
salt, was from Sigma. DMSO was from Amresco.
Solubility measurement
The shake flask method was utilized to study the equilibrium solubility of the curcumin/resveratrol
combinations in different oils (corn oil, Capryol 90, Labrafac PG, Labrafac CC, soyabean
oil), surfactants (Capryol PGMC, Cremophor EL, Cremophor RH40, Labrafil M 2125 CS,
Lauroglycol 90) and co-surfactants (Labrasol, Lauroglycol FCC, PEG 400, propylene
glycol, Transcutol HP). An excess amount of curcumin and resveratrol in the ratio
of 1 : 1 was added to each Eppendorf tube containing 1 g of the vehicle. The sample
was vortexed at a maximum speed for 10 min using a mixer (Vortex-gene 2, Becthai Bangkok
Equipment & Chemical) and allowed to equilibrate in a water bath shaker (Heto Lab,
Scientific Promotion) (37 °C at 100 rpm); the equilibration time was set at 48 h.
Solid-phase separation was achieved using centrifugation (30 min, 1000 rpm at 37 °C)
and filtration [0.2 µm polyvinylidenedifluoride (PVDF) filter]. The supernatants were
collected and diluted with the mobile phase [acetonitrile and 1 % citric acid (v/v)
(55 : 45)] for quantification of curcumin and resveratrol by the HPLC method. All
solubility experiments were performed in triplicate.
Ternary phase assay
The compositions of oil, surfactant, and co-surfactant from the solubility study were
selected to construct ternary phase diagrams. The ternary phase diagrams were plotted
to identify the self-microemulsifying regions and to find the optimal concentrations
of components. The mixture series of oil, surfactant, and co-surfactant were prepared.
The vehicles were weighed into glass test tubes and mixed using a vortex mixer. The
concentration range of each component was 10–50 % oil, 25–90 % surfactant, and 0–25 %
co-surfactant. One gram of each mixture was dispersed in 20 mL of distilled water.
The efficiency of the self-microemulsification was observed visually and scored according
to the grading system described by Singh et al. [17 ].
Preparation of curcumin/resveratrol in self-microemulsifying formulations
According to the ternary phase diagram studies, the SME formulation at the optimal
component ratios was selected for the incorporation of curcumin and resveratrol. Thirty
mg of each compound was added to 1 g of the self-microemulsifying mixture and the
dispersion was stirred continuously until a homogenous solution was formed. The formulations
were left for 48 h at room temperature. Hard gelatin capsules (size 00) were manually
filled with the CR-SME formulation and stored in a tightly sealed glass bottle at
room temperature until examined.
Emulsion droplet size and size distribution
One gram of each formulation was diluted with DI water, SGF, and SIF (20-fold dilution).
The content was gently stirred by a magnetic stirrer for 5 min. The droplet size and
the polydispersity index of the resultant microemulsion were determined by the dynamic
light scattering technique using ZetaPALS, Zeta potential, and a particle size analyzer
(Brookhaven Instruments Corporation). The light scattering was performed at a fixed
angle of 90 at a temperature of 25 °C. The measurement time was 1 min, and each run
comprised 10 subruns.
Morphological characterization
The morphology of microemulsion formed was observed by transmission electron microscopy
(TEM; JEOL). The CR-SME formulation was diluted with distilled water at a ratio of
1 : 75 and mixed by gentle shaking. A drop of the sample obtained after the dilution
was placed on copper grids. Any excess liquid was drawn off with filter paper. The
grid surface was then air-dried at room temperature.
Release of curcumin and resveratrol from the self-microemulsifying formulation
The release profiles from the capsules filled with CR-SME containing 30 mg of each
curcumin and resveratrol, C-SME containing 30 mg of curcumin, and R-SME containing
30 mg of resveratrol were compared to the unformulated combination. This study was
carried out using the USP 30 rotating paddle apparatus with 900 mL of simulated gastric
fluid (SGF, pH 1.2) at 37.0 ± 0.5 °C and 75 rpm. The prepared formulations were subjected
to the release studies for 2 h. Samples were withdrawn and replaced with the fresh
medium at 5, 10, 15, 30, 45, 60, 90, and 120 min. The concentrations of curcumin and
resveratrol were assayed by HPLC. The test was repeated six times, and the data were
reported as the mean ± SD. A plot of the cumulative % release of curcumin and resveratrol
against time was constructed to illustrate the drug release profiles.
HPLC analysis of curcumin in combination with resveratrol
The analysis of curcumin in combination with resveratrol in the drug release samples
was performed on an Agilent separation module with a photodiode array detector (HP
1100, Agilent). A C18 column (VertiSep™ UPS C18 column 4.6 × 250 mm, 5 µm, Ligand
Scientific) was used. The mobile phase consisted of a mixture of acetonitrile and
1 % citric acid (v/v) (55 : 45) [18 ] with an isocratic solvent system. The injection volume was 20 µL. The flow rate
of the mobile phase was 1 mL/min, and the detector wavelength was kept at 325 nm and
425 for curcumin and resveratrol detection, respectively.
Stability studies
The stability testing was carried out according to the ICH guidelines (2003) on the
topic of Q1 A (R2): stability testing of the new drug substances and products. The
optimized CR-SME formulation was subjected to stability studies in order to evaluate
its physical and chemical stability. Samples were kept in a stability chamber (Patron
AH-80) under intermediate conditions [30 ± 2 °C, 65 ± 5 % relative humidity (RH)],
and evaluated under accelerated conditions (45 ± 2 °C, 75 ± 5 % RH), with the humidity
and temperature control taken at 0, 1, and, 3 months for both conditions. Samples
were prepared for the assay in the mobile phase and injected directly onto the HPLC
column three separate times (n = 3).
Ferric reducing antioxidant power assay
The FRAP assay was used to evaluate the antioxidant capacity in order to determine
the ferric reducing activity of the polyphenol co-delivery in the SME formulation.
PBS 2.5 mL was mixed with the same amount of potassium ferricyanide and the sample
solution (CR-SME, R-SME, and C-SME), standard ascorbic acid, or blank formulation
was added into the tube. After incubation for 20 min at 50 °C, 2.5 mL of trichloroacetic
acid were added and centrifuged for 10 min at 3000 rpm to separate the layers. The
supernatant was transferred for mixing with 2.5 mL of DI water and 0.5 mL of ferric
chloride. Finally, the absorbance of all samples, standards, and blank were measured
at 590 nm.
Cell culture studies
The HT-29 cells were grown in McCoyʼs 5 A (modified) medium supplemented with 10 %
v/v FBS, and 1 % v/v pen-strep. The cells were maintained at 37 ± 0.5 °C in an atmosphere
with 5 % CO2 and 90 % RH, and passaged every 2 days. When the cell monolayer reached 80–100 %
confluency, the cells were removed from the culture flask using a 0.25 % trypsin-EDTA
solution. Viable cell numbers were counted prior to use by a standard hemocytometer.
The cells were then used in cell cytotoxicity studies.
The cytotoxicity test
To evaluate the cytotoxicity of CR-SME at different concentrations, each polyphenol
in the formulation was mixed at ratios of 5 : 5, 10 : 10, 15 : 15, 30 : 30, and 50 : 50 µM
to get the final concentrations of the combination at 10, 20, 30, 60, and 100 µM,
respectively. Each sample was dispersed in DI water and then diluted with the complete
medium. The HT-29 cells were seeded in 96-well cell culture plates at a density of
8 × 103 cells/well and incubated for 24 h. After overnight incubation, the culture medium
was removed, and the cells were washed with 100 µL of PBS. One hundred microliters
of the samples at different concentrations were added to each well. Complete medium
and 1 % sodium lauryl sulfate were used as the negative control and positive control,
respectively. After 24 h treatment, the samples were removed and the cells were washed
with PBS. Fifty microliters of 0.5 mg/mL MTT solution were added to each well and
the cells were incubated for another 4 h. After removing the MTT solution carefully,
100 µL of DMSO were added to dissolve the formazan crystals formed by the living cells.
The absorbance of the samples was measured by a microplate reader (DTX 880 Multimode
Detector, Beckman Coulter Inc.) at a wavelength of 570 nm. Duplications were performed.
The percentage of cell viability was calculated relative to the measured absorbance
of the negative control that exhibited 100 % cell viability.
In vivo absorption studies
The male New Zealand white rabbits with a mean body weight of 2.5 ± 0.2 kg were supplied
by the Animal House, Faculty of Science, Prince of Songkla University. The animal
protocol was approved under the guidelines of the Animal Care and the Committee of
Prince of Songkla University (MOE 0521.11/061). The overnight fasted rabbits were
divided into three groups with three rabbits per each group. The compounds 1) CR-SME
formulation (curcumin 50 mg/kg and resveratrol 50 mg/kg) 2) curcumin/resveratrol in
aqueous suspension (curcumin 50 mg/kg and resveratrol 50 mg/kg), or 3) C-SME formulation
(50 mg/kg) followed by R-SME formulation (50 mg/kg) were orally administered as a
single dose. Blood samples (1 mL) were collected via the auricular artery [19 ] 0, 15, 30, 45, 60, 90, 120, 180, 210, 240, 300, and 360 min after oral administration
and were immediately transferred to a heparinized microcentrifuge tube and centrifuged
at 4000 g for 20 min at 4 °C. The plasma samples were separated. Acetonitrile was added to
each plasma sample (acetonitrile : plasma was 1 : 1 v/v), vortexed, sonicated, and
allowed to stand for 5 min for deproteinization. The protein precipitate was removed
by centrifugation. The supernatant was pipetted into a tube. Samples were then diluted
with methanol in a ratio of 1 : 0.5. The solution was filtered using a 0.2-µm membrane
filter and subjected to the validated HPLC method. The pharmacokinetic parameters
including the maximum concentration (Cmax ), time to reach maximum concentration (Tmax ), and the area under the concentration-time curve (AUC 0–6 h ) were determined.
Statistical analysis
All results are expressed as the mean ± SD. Differences between two related parameters
were assessed by Studentʼs t-test or one-way ANOVA. Differences were considered significant
at p < 0.05.
