Arabian Journal of Chemistry (2017) 10, 1029–1037

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

A simple reversed phase high-performance liquid

chromatography (HPLC) method for determination
of in situ gelling curcumin-loaded liquid crystals in
in vitro performance tests
Bruno Fonseca-Santos, Maria Palmira Daflon Gremião *, Marlus Chorilli *

School of Pharmaceutical Sciences, UNESP – São Paulo State University, Campus Araraquara, Department of Drugs and Medicines,
Araraquara, SP, Brazil

Received 22 October 2015; accepted 31 January 2016

Available online 6 February 2016

KEYWORDS Abstract A simple, rapid, and sensitive analytical procedure has been developed and validated for
Curcumin; the in vitro measurement of curcumin in samples from mucosae retention studies. Curcumin was
Validation; analyzed by HPLC using a C18 column with UV detection at 425 nm. The mobile phase was ace-
High-performance liquid tonitrile and water (50:50 v/v) acidified with 2% acetic acid at a flow rate of 1.2 mL min
1. The
chromatography; curve range was linear for the receptor solution concentration range 0.5–75 lg mL
1. The specificity
Liquid crystals; showed no interference with the biological matrix and excipients of the acceptor media. Intra and
Performance tests inter-day accuracy, and precision values were lower than 5% and were not statically different
(P < 0.05). Recoveries ranged from 99.7% to 108%. The limits of detection and quantitation were
11.61 and 500 ng mL
1, respectively. The method is adequate to assay curcumin from esophageal
porcine samples, enabling the determination of penetration profiles for in situ gelling curcumin-
loaded liquid crystals by in vitro studies, and fulfilled the requirements for reliability and feasibility
for application to the quantitative analysis of curcumin in porcine esophageal mucosae.
Ó 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding authors at: School of Pharmaceutical Sciences,

1. Introduction
Department of Drugs and Medicines, São Paulo State University
(UNESP), Rodovia Araraquara-Jaú, km. 1, Araraquara, São Paulo
14801-902, Brazil. Tel.: +55 16 3301 6998 (M. Chorilli). Tel.: +55 16 Curcumin is a natural product isolated as a yellow pigment from the
3301 6975 (M.P.D. Gremião). rhizome of Curcuma longa L. (Sharma et al., 1990). This drug has
E-mail addresses: [email protected] (M.P.D. Gremião), shown potential for use in treating cancer (Anand et al., 2008; Goel
[email protected] (M. Chorilli). et al., 2008; Zlotogorski et al., 2013) because it inhibits the nuclear
Peer review under responsibility of King Saud University. factor kappa beta (NF-kB), especially in oral squamous cancer cells
(OSCC) (Aggarwal et al., 2004), that is directly involved in tumorige-
nesis, (Richmond, 2002) angiogenesis, invasion tumors, and metastasis
(Ben-Neriah and Karin, 2011). Curcumin also has poor water solubil-
ity and low bioavailability, thus representing an interesting drug model
Production and hosting by Elsevier

http://dx.doi.org/10.1016/j.arabjc.2016.01.014
1878-5352 Ó 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1030 B. Fonseca-Santos et al.

to be improved by nanotechnology (Ghalandarlaki et al., 2014; Sun individual curcuminoids, so various liquid chromatographic methods
et al., 2012; Ahmad et al., 2015) for the non-invasive administration have been developed for this purpose. High-pressure liquid chromatog-
of drugs (Mathias and Hussain, 2010), especially via the buccal route raphy with UV detection (HPLC–UV) is the most common method for
(Calixto et al., 2014; Shojaei, 1998; Campisi et al., 2010). the determination of curcuminoids and curcumin in turmeric samples,
The buccal route is attractive for the administration of drugs in biological samples, or dosage forms (Jadhav et al., 2007; Li et al., 2009;
delivery systems, such as loaded-liquid crystalline systems (Calixto Thorat and Jangle, 2013; Nascimento et al., 2012; Wichitnithad et al.,
et al., 2014; Salmazi et al., 2015), prolonging the residence time of 2009; Jayaprakasha et al., 2002; Syed et al., 2015; Koop et al., 2013).
the dosage in the oral mucosa (Shojaei, 1998; Grabovac et al., 2005) Due to the very labile characteristics of curcuminoids, C18 columns
and enabling local treatment of malignant lesions (Nielsen et al., are preferred for HPLC analysis (Khurana and Ho, 1988). These meth-
1998; Bhardwaj and Kumar, 2012; Hearnden et al., 2012). Liquid crys- ods reported by HPLC–UV for curcuminoids, especially those in older
tals (LCs) are matter in a state that has properties between those of literature, have several disadvantages, including unsatisfactory separa-
conventional liquids and solid crystals (Malmsten, 2002). In other tion times, poor resolution, complicated solvent mixtures with gradient
words, LCs have the structural behavior and rigidity of a solid, com- elution, and long analysis times.
bined with the mobility, disorder, and fluidity of an isotropic liquid The aim of this study was to develop and validate an analytical
(Kato, 2008). By increasing the concentration of solvents, such as method for evaluating the performance test of in situ gelling
water, in these systems, lamellar, hexagonal, and cubic liquid- curcumin-loaded liquid crystals.
crystalline forms can be generated (Malmsten, 2007; Mezzenga, 2012).
LCs can be used as drug delivery systems because of the high ability
of poorly water-soluble drug solubilization (Malmsten, 2002), like cur- 2. Experimental
cumin, besides showing interesting mucoadhesive properties being very
interesting to buccal administration to increasing the time retention of 2.1. Chemicals
LCs formulations.
Self-assembly systems display phase transformations and notable
in situ thickening after administration to body cavities, such as the Polyoxypropylene-(5)-polyoxyethylene-(20)-cetyl alcohol
mouth. When in contact with the oral environment, LCs have the (PPG-5-Ceteth-20) was purchased from Croda (Campinas,
ability to incorporate saliva, becoming a more viscous liquid- Brazil). Oleic acid and sodium dodecyl sulfate were purchased
crystalline mesophase (Malmsten, 2007) due to its transitional from from Synth (Diadema, Brazil). Curcumin (77% purity), low
a lamellar phase structure to a hexagonal or cubic mesophase (Lee molecular weight chitosan (Ch), and poloxamer 407 (Po) were
et al., 2001; Boyd et al., 2006; Bruschi et al., 2008; Carvalho et al., purchased from Sigma Aldrich (St Louis, USA). HPLC grade
2013) that can promote controlled release and, consequently, result acetonitrile and methanol were purchased from J. T. Baker
in the greatest mucoadhesion pharmaceutical form in the oral envi- (Mexico City, Mexico). Glacial acetic acid, sodium dihydrogen
ronment (Malmsten, 2002, 2007; Bruschi and de Freitas, 2005;
phosphate, and sodium hydrogen phosphate were purchased
Gilhotra et al., 2014). Due to their high viscosity, hexagonal and
cubic phases have been suggested as mucoadhesives (Carvalho
from Qhemis Hexis (Indaiatuba, Brazil). Ultrapure water was
et al., 2013; Fonseca-Santos et al., 2015; Oyafuso et al., 2015), as well obtained from a Milli-QÒ Direct Water Purification System.
as the incorporation of (muco)adhesive polymers such as chitosan
(Dhawan et al., 2004; Pendekal and Tegginamat, 2012) that can also 2.2. Chromatographic system and instrumentation
interact locally with oral tumor cells (He et al., 2010; Wu et al., 2012;
Yu et al., 2012).
The vertical diffusion cell, also known as the Franz cell, is widely A VarianÒ ProStar HPLC system was used for analyses, con-
used for the drug release-rate testing of semisolids and other topical sisting of two pumps (Solvent Delivery Module, model 210), a
formulations (Hanson and Heaney, 2013; Shah, 2005; Ng et al., ProStar 330 UV–vis PDA spectrophotometric detector (set at
2010). The dosage forms are placed in a donor area that then perme- 425 nm), a ProStar 410 auto sampler (sample injection volume,
ates and/or diffuses through a membrane into the receptor solution, 20 lL) and Timerline 101 column oven (set at 33 °C). A
from which samples are withdrawn periodically and analyzed for reverse-phase C18 column LunaÒ (250 mm  4.6 mm i.d.
drug concentrations (Hanson and Heaney, 2013). The experimental 5 lm particle size) was used. The mobile phase was a 50:50
conditions for drug release testing, such as acceptor media, types of (v/v) mixture of acetonitrile and 2% acetic acid in water at a
membrane, and usage of different animal tissue as models, depend
flow rate of 1.2 mL min
1.
on the purpose of the experiments, i.e., whether the aim is quality
control (Ng et al., 2010), biopharmaceutical characterization
(Siewert et al., 2003), or bioavailability testing in order to decrease 2.3. Standard and working solutions
or eliminate animal tests (Shah, 2005, 2001; Ng et al., 2010; Shah
et al., 2002). Curcumin was dissolved in methanol in a 20.0 mL volumetric
Analytical methods, employed for the quantitative determination flask to a final concentration of 1.0 mg mL
1. The solution was
of drugs in biological samples and pharmaceutical dosage forms sonicated for 5 min in an ultrasonic bath and completed to the
(Silva et al., 2014; Mattos et al., 2013; Chorilli et al., 2011a,b; Jain
final volume. Working solutions (0.5–75.0 lg mL
1) were pre-
et al., 2015; Dewani et al., 2015; Chiva Carvalho et al., 2013;
Carvalho et al., 2009), can influence the evaluation and interpretation
pared by dilution of aliquots of the standard solution with
of bioavailability, bioequivalence, and pharmacokinetic data. It is, methanol (1.0 mg mL
1) in a 5.0 mL volumetric flask.
therefore, essential to employ well-characterized and fully validated
analytical methods to give reliable results that can be interpreted with 2.4. Validation methods
satisfaction (Shah et al., 1992).
A variety of analysis techniques for the quantification of total and 2.4.1. Linearity
isolated curcuminoids in different matrices have been reported, espe-
To determine linearity, curcumin standard solution was
cially spectrophotometric methods for the determination of total cur-
cuminoids (Kadam et al., 2013; Silva-Buzanello et al., 2015; Ahmed diluted in triplicate at concentrations ranging from 0.5 to
et al., 2012). However, this approach cannot be used to quantify 75.0 lg mL
1 (working solutions). Linearity was evaluated
Determination of in situ gelling curcumin-loaded liquid crystals 1031

by linear regression analysis, which was calculated by least- 2.5.1. Method applicability
squares regression analysis and ANOVA test (a = 0.05). The proposed analytical method was used to evaluate the con-
tent of curcumin retained in the porcine mucosa when
2.4.2. Specificity and selectivity curcumin-loaded LCs were applied to the mucosa. A Hanson
The specificity of the method was determined by curcumin, the MicroettePlusÒ Franz diffusion cell (Chatsworth, USA) was
acceptor media (buffer phosphate, pH 6.8 containing 0.5% filled with a media volume of 7.0 mL (phosphate buffer, pH
sodium dodecyl sulfate, SDS), and methanol extract mucosae 6.8 containing 0.5% SDS) and stirred at 300 rpm at a temper-
esophageal porcine (biological membrane used in permeation ature of 37 ± 0.5 °C.
and retention studies) to evaluate interference in the quantita- Porcine mucosa esophageal tissue was treated with buffer
tion of the drug. phosphate for 5 min, then fixed to the diffusion cell. Formula-
tions were placed (600 mg) above the membrane. The experi-
2.4.3. Accuracy ment was carried out with curcumin-loaded LC1
To evaluate the accuracy of the method, a recovery experiment (5.0 mg g
1) and a formulation diluted to 10% with artificial
was performed. Curcumin concentrations of 1.0, 50.0 and saliva (LC1d), and the results were compared to the release
70 lg mL
1 were added to the matrix samples: phosphate buf- profile of curcumin dissolved in oleic acid as a control. During
fer (pH 6.8 containing SDS; acceptor media) and mucosae eso- the test, media aliquots of 1.0 mL were withdrawn at intervals
phageal methanolic extract. These results were expressed as (0.5, 1, 2, 4, 8 and 12 h) using a Hanson AutoPlusTM MultifillTM
recovery data and values should be within the interval 80– autosampler (Chatsworth, USA). The aliquots were filtered
120% (Resolução RE, 2003). through 0.45 lm PTFE disk filters and then injected into
HPLC.
2.4.4. Precision The extraction of curcumin from the retained pig esopha-
geal mucosa was carried out according to an adapted method
Intra and inter-day precision was analyzed by injecting cur-
(Mazzarino et al., 2015). After 12 h of ex vivo permeation, the
cumin working solutions in triplicate (n = 3) at three concen-
mucosa was removed from the Franz cell and the area of tis-
trations (1.0, 50.0 and 70.0 lg mL
1) over an interval of two
sue exposed to permeation was cleaned with cotton dipped in
days between repeatability and intermediate precision tests
methanol, and then the tissue was cut and perforated. The
(Validation of Analytical Procedures, 2005). The results were
fragments obtained were triturated with 4 mL of methanol
expressed as percentage of RSD and inter-day averages were
in a tissue homogenizer at 10,000 rpm for 2 min to achieve
evaluated statically by Student’s t-test (tailed test, P < 0.05).
total dispersion of the mucosa. The resulting suspension
was sonicated in an ultrasonic bath for 50 min to break the
2.4.5. Limits of detection and quantitation
cells.
Limits of detection (LOD) and quantitation (LOQ) were deter- The homogenate was centrifuged at 4000 rpm for 10 min.
mined using a calibration curve method according to ICH rec- The obtained supernatant solution was filtered into a 5 mL
ommendations (Validation of Analytical Procedures, 2005). volumetric flask and the volume completed with methanol.
The response standard deviation (RSD) was taken as the SD The solution was filtered through 0.45 lm PTFE disk filters
of y-intercepts of the regression lines of the three calibration and injected into HPLC for analysis. Extractions were per-
curves. The LOD were calculated as follows: formed from the mucosa of each diffusion cell (n = 6) and
LOD ¼ 10  r=S ð1Þ are expressed as percentages. All average values of retention
were tested by ANOVA following Tukey’s post hoc test
where r is the standard deviation of the response, and S is the (a = 0.05).
slope of the calibration curve. Limit of quantitation (LOQ)
was determined as the first concentration of calibration curves.
3. Results and discussion
2.5. Liquid crystal preparation and characterization
3.1. Validation studies
LCs were prepared at room temperature by mixing oleic acid as
the oily phase (O), PPG-5-Ceteth-20 as the surfactant (S), and 3.1.1. Linearity
water (W) containing 0.5% Ch and Po as the aqueous phase. Statistical analysis, using least-squares regression analysis of
The samples contained the following O/S/W proportions: curcumin assay results in the range 0.5–75 lg mL
1, gave a
(LC1) 3:4:3, (LC2) 1:2:2 and (LC3) 1:4:5. Sample LC1 was linear regression equation for the three calibration curves
diluted with artificial saliva (10:3) to generate the LC1d formu- for curcumin of: y = 0.002027x
0.000228, r = 0.999859
lation. These formulations were analyzed by polarized light (Fig. 1). ANOVA of regression showed an F-critical value
microscopy (PLM) to confirm liquid crystal mesophases at of 6.0993  10
36 and an F-tabulated value of 80768.6, which
20 magnification. The artificial saliva is composed of sodium indicated a significant regression (because F-critical
phosphate dibasic anhydrous (802.9 mg), sodium phosphate value < F-tabulated value). According to the validation crite-
monobasic anhydrous (362.6 mg), 70% sorbitol solution ria adopted, this method was linear over the whole tested
(42.7 g), potassium chloride (625.0 mg), sodium chloride concentration range. The calibration curves are shown in
(865.0 mg), magnesium chloride hexahydrate (125.0 mg), cal- Fig. 1 and ANOVA is shown in Table 1. The validity of
cium chloride dehydrate (72.0 mg), methylparaben (1.8 g), the assay was verified by means of ANOVA, which showed
propylene glycol (10 mL) and purified water to 1000 mL that there is linear regression with no deviation from linearity
(Nakamoto, 1979). (P < 0.05).
1032 B. Fonseca-Santos et al.

short retention times were very convenient for routine proce-

dures in quality control. The methodology was selective,
because no interfering signals were detected at the tR of accep-
tor media (phosphate buffer, pH 6.8) and mucosae methanolic
extract, and none interfered with curcumin detection (Fig. 2B).

3.1.3. Accuracy
Curcumin working solution and spiked samples (methanol,
phosphate buffer with 0.5 DSD and mucosae extract) were for-
tified at three concentrations (1.0, 50.0 and 70.0 lg mL
1) by
diluting aliquots of the standard solution (1.0 mg mL
1). Each
replicate was injected into the C18 column and peak areas were
obtained. These values were substituted into the regression line
equation to calculate the recovered concentrations of the sam-
ples. Accuracy is reported in Table 2 as a percentage recovery
relative to the known amount of curcumin in the samples. The
Figure 1 Calibration curves of curcumin at concentrations of recovery values were within the confidence interval of 80–
0.5–75 lg mL
1 in methanol. 120%.
3.1.2. Specificity and selectivity
3.1.4. Precision
The chromatograms in Fig. 2 show good peak resolutions,
The intra-day precision was analyzed by injecting curcumin
indicating the high specificity and selectivity of this method.
working solutions in triplicate at 1.0, 50.0 and 70.0 lg mL
1,
The resolutions (R) of peaks were 2.2 and 3.0 between peaks
corresponding to low, average, and high concentrations in
2/1 and 3/2, respectively, indicating a high degree of peak sep-
the calibration curve range, respectively. The inter-day preci-
aration (R > 2). The chromatogram showed three peaks and
sion was analyzed by injecting each concentration three times
was reported by Jayaprakasha et al. (2002), where the major
(n = 3) on two consecutive days. The standard deviation (SD)
peak was attributed to curcumin (Goel et al., 2008) and two
and RSD were calculated for each sample (Table 3). The low
minor peaks were identified as demethoxycurcumin (Anand
values of RSD (<2%) reflected the high precision of the
et al., 2008) and bisdemethoxycurcumin (Sharma et al., 1990)
method and the P-value (<0.05) showed no mean significance.
by co-injection of standards. These authors confirmed the
compounds associated with elution peaks by 1H and 13C
3.1.5. LOD and LOQ
NMR analysis.
The method developed by these authors was similar to that The LOQ value was taken as the lowest concentration of cur-
developed in this work; however, the chromatographic condi- cumin in the acceptor media that could be quantitatively mea-
tions were slightly different with regard to mobile phase pro- sured. The LOD was 11.61 ng mL
1 and LOQ was
portions (acetonitrile/water acidified with 2% acetic acid; 0.5 lg mL
1.
60:40 vs. 50:50 v/v in this work), the length of the C18 column
(150 mm vs. 300 mm in this work), and the solvent flow rate 3.2. Liquid crystal preparations and characterization
(2 mL min
1 vs. 1.2 mL min
1 in this work). The total chro-
matographic analysis time was 16 min per sample, with cur- Fig. 3A shows that it was possible to obtain an optically trans-
cumin, desmethoxycurcumin, and bisdesmethoxycurcumin parent liquid system (OPCLS) when the concentrations of S,
eluting at retention times of 13.6, 12.1 and 10.8 min, respec- O, and W were 40%, 30–70%, and above 50–70%, respec-
tively (Wichitnithad et al., 2009). However, chromatographic tively, and a minor region with 30%, 50–70% and 70%,
analysis with a C18 column of 200 mm length would generate respectively. The transparent semisolid system (OPCSS) was
an analysis time greater than 15 min. Therefore, our method formed when concentrations of S, O, and W were 25–40–
used the small changes mentioned above to achieve the best 30%, 5–25%, and 45–55%, respectively. Emulsion systems
peak resolution and a shorter analysis time. were obtained in regions where concentrations of S, O, and
The chromatogram of curcumin exhibited a characteristic W were below 5–20%, 5–70%, and 85-95%, respectively.
peak (Goel et al., 2008) at a retention time (tR) of 11.5 min, Phase separation occurred in a large proportion of the formu-
and two minor peaks at a tR of 9.5 and 10.5 min, reported in lations, in two distinct regions: the first region was formed
the literature as bisdesmethoxycurcumin and desmethoxycur- when the concentrations of S, O, and W were greater than
cumin, respectively (Wichitnithad et al., 2009) (Fig. 2A). The 55–100%, 5–40%, and below 5–40%, respectively; the second

Table 1 ANOVA for simple linear regression.

Model d.f. Sum of squares Mean squares F-tabulated F-critical
Regression 1 0.060102483 0.060102 80768.6 6.1  10
36
Residue 19 1.41385  10
05 7.44  10
07
Total 20 0.060116621
Determination of in situ gelling curcumin-loaded liquid crystals 1033

Figure 2 Chromatograms of curcumin (50 lg mL
1), blank methanol, and buffer phosphate as acceptor media and mucosae methanolic
extract, fortified or unfortified.

Table 2 Accuracy of curcumin at different concentrations (1.0, 50.0 and 70.0 lg mL
1) in matrices (methanol, phosphate buffer with
0.5 DSD, and mucosae extract).
Matrix Theoretical concentration (lg mL
1) Experimental concentration (lg mL
1) RSD (%) Recovery (%)
Methanol 1.0 1.01 ± 0.02 1.98 101.0 ± 2.0
50.0 50.3 ± 0.05 0.10 100.7 ± 0.1
70.0 70.4 ± 0.7 0.30 100.0 ± 0.1
Phosphate buffer w/DSD 1.0 1.02 ± 0.03 2.94 102.0 ± 3.0
50.0 50.12 ± 0.11 0.22 100.2 ± 0.2
70.0 69.80 ± 0.09 0.13 99.7 ± 0.13
Mucosae extract 1.0 1.08 ± 0.03 2.78 108.0 ± 3.0
50.0 50.10 ± 0.59 1.18 100.4 ± 1.18
70.0 71.00 ± 0.9 1.41 101.4 ± 1.28
1034 B. Fonseca-Santos et al.

Table 3 Intra- and inter-day of curcumin at different concentrations (1.0, 50.0 and 70.0 lg mL
1).
Theoretical concentration (lg mL
1) Intra- and inter-day precision P-valuec
Experimental concentration (lg mL
1)a RSD (%)b Recovery (%)a
Intra-day Inter-day Intra-day Inter-day Intra-day Inter-day
1.0 1.01 ± 0.02 1.01 ± 0.01 1.98 1.25 101.0 ± 2.0 100.8 ± 1.3 0.5000
50.0 50.3 ± 0.05 50.7 ± 0.61 0.1 1.20 100.7 ± 0.1 101.4 ± 1.22 0.2576
70.0 70.4 ± 0.7 70.03 ± 0.208 0.3 0.99 100.0 ± 1.0 100.6 ± 0.3 0.2168
a
Mean and SD of samples.
b
Triplicate in two different days.
c
Student’s t-test with significance level of 95%.

Figure 3 (A) Ternary phase diagram of PPG-5-CETETH-20, oleic acid, and aqueous phase (0.5% chitosan and poloxamer 407); (B)
macroscopic appearance and MPL of LC1, LC2, and LC3 formulations; and (C) photomicrographs of LC1 diluted with artificial saliva to
10% (d10), 20% (d20), and 30% (LC1d).

region was formed when the concentrations of S, O, and W 0.5%), respectively. These formulations were not adequate
were below 40%, 50–100%, and 10–100%, respectively. for buccal administration due to their poor syringeability.
Based on the ternary phase diagram, formulation LC1, a Moreover, LC1 is located in a phase transition region.
liquid formulation composed of 40% (w/w) PPG-5- Thus, when diluted with artificial saliva, its viscosity increased
CETETH-20, 30% (w/w) oleic acid, and 30% (w/w) aqueous in situ. This can be observed in the dilution line in the diagram
phase (chitosan and PO 407 at 0.5%), was selected as the liq- in Fig. 3A.
uid crystal precursor system because its liquid phase facilitated Carvalho et al. (2013) previously developed a phase dia-
buccal administration (e.g., by syringe) (Bruschi et al., 2008). gram consisting of oleic acid as the oil phase, PPG-5-
Both LC2 and LC3 were semisolid formulations composed CETETH-20 as the surfactant, and water as the aqueous
of 40% PPG-5-CETETH-20, 20% and 10% oleic acid, and phase. This study also resulted in different regions, but
40% and 50% of aqueous phase (chitosan and PO 407 at reported a lower region of transparent viscous systems,
Determination of in situ gelling curcumin-loaded liquid crystals 1035

perhaps due to the absence of mucoadhesive polymers such as

chitosan and poloxamer, which are present in the diagram in
Fig. 3A. Such polymers may have thickening properties
(Chattopadhyay and Inamdar, 2010), changing the rheological
properties of these systems and leading to an increase in the
regions of viscous systems in the diagram.
Using the MPL technique, Malta crosses were observed in
the photomicrograph of LC1, suggesting that a lamellar liquid
crystalline mesophase had formed (Chorilli et al., 2011c).
Thus, the LC2 and LC3 photomicrographs showed striated
structures that may be more organized structures typical of a
hexagonal liquid crystalline mesophase (Carvalho et al.,
2013, 2010), as shown in Fig. 3B. When, LC1 was diluted with
artificial saliva in increasing proportions (10%, 20%, and
30%), resulting in LC1d10%, LC1d20% and LC1d, respec- Figure 4 Bar chart of curcumin retained (%) in mucosae
tively, the photomicrographs showed striated structures that esophageal tissue obtained from retention studies after 12 h in
might be the more organized structures typical of a hexagonal oleic acid, lamellar liquid crystal (LC1), and in situ gelling liquid
liquid crystalline mesophase (Salmazi et al., 2015). The results crystal (LC1d) *P > 0.05.
for these phase transitions are shown in Fig. 3C.
Therefore, these results demonstrated that LC1 behaved as
a precursor of a LC system and was able to form a strong LC could be related to the similarity of the lamellar liquid-
mesophase with the incorporation of artificial saliva. This tran- crystalline structure with the cell membrane leading to
sition could be attributed to an increase in the packing con- enhanced interactions between them and causing the release
straint in the hydrophilic core of PPG-5-CETETH-20, which of tissue target from this interaction bio-nanodevice (Nel
reduced the interfacial curvature of the aggregate. Moreover, et al., 2009; Liu and Wang, 2014). In addition, slow release
the subsequent hydration of the surfactant generated a large and high-capacity carrying of hydrophobic drugs, such as anti-
repulsive force between the head groups of the surfactant, tumor drugs, into liquid-crystal systems, is particularly inter-
increasing the distances between lamellae (Carvalho et al., esting for cancer therapy, where the cytotoxicity of
2010). This resulted in the formation of different liquid crys- anticancer drugs can be improved.
talline structures (Mezzenga, 2012).
4. Conclusions
3.2.1. Method applicability
The HPLC method presented here can be considered suitable for ana-
During pharmaceutical development, it is appropriate to lytical determination of curcumin concentrations for in vitro perfor-
employ in vitro release or permeation studies to select formula- mance tests. The method was capable of detecting low
tions that can provide adequate therapeutic activity. Drug concentrations of curcumin in the diffusion cell acceptor media, and
release studies provide valuable data on the structural charac- the calibration curve was linear over the concentration range. The val-
teristics of the vehicle and its ability to release the drug. idation process also showed high selectivity and specificity, since there
LC phases have received considerable attention due to their was no interference between the curcumin peak and media or mucosae
potential as drug delivery systems. Cubic and hexagonal components. The methodology was accurate and precise as observed
from the recovery and RSD values. The analytical procedure had a
phases provide a slow drug release matrix (Mezzenga et al.,
15 min chromatographic run time, which allowed the analysis of a
2005; Ubbink et al., 2008) when compared with lamellae
large number of samples in a short period of time. Retention studies
phase, which has a rapid drug release matrix (Farkas et al., showed higher concentrations on mucosae tissue to lamellar meso-
2000; Makai et al., 2003). phase than hexagonal mesophase.
Recently, Martiel et al. (2015) published work that Designed LCs showed in situ gelling properties caused by the addi-
explained controlled release from LCs, detailing the influence tion of artificial saliva. This artificial saliva caused the formation of a
of drug hydrophobicity and oil-surfactant type on this prop- lamellae mesophase, transforming into hexagonal liquid crystalline
erty. The authors summarized that partition coefficients of formulations with mucoadhesive properties (data not shown).
drugs and the ability of the drug to diffuse through the hydro- This paper proposes a HPLC method to quantify curcumin in per-
carbon tail region were important. This phenomenon was formance in vitro assays for LCs containing curcumin; however, this
methodology can be applied to other pharmaceutical dosage forms
influenced by the bulkiness of the drug, the oil-type, and by
intended for buccal release of curcumin.
either chain stiffening (Martiel et al., 2015). The optimal struc-
tural control was obtained by combining a drug with high
hydrophobicity with LCs (Martiel et al., 2015), which may Disclosure
not be true for curcumin due to its poor-water solubility.
Fig. 4 shows the cumulative percentage in the esophageal The authors report no conflict of interests in this work.
mucosal tissue after a 12 h ex vivo test. Statistical analysis
showed that there were differences between the mean Acknowledgments
(P > 0.05) percentages of retention for the formulations. The
retentions were approximately 3%, 15%, and 10% for the con- This work was financially supported by Fundação de Amparo
trol, LC1, and LC1d, respectively. The lamellar phase reten- à Pesquisa do Estado de São Paulo (FAPESP), grant
tion values were greater than hexagonal systems, which 2013/03746-3, Coordination for the Improvement of Higher
1036 B. Fonseca-Santos et al.

Education Personnel (CAPES) for research fellowships, and Chorilli, M., Bonfilio, R., Louvandini, C.R., Gonçalves, F.A.R.M.,
Programa de Apoio ao Desenvolvimento Cientı́fico (PADC- Salgado, H.R.N., 2011b. Development and Validation of an LC-
FCF-UNESP). MS/MS method for quantitative analysis of mirtazapine in human
plasma. Am. J. Anal. Chem. 02 (06), 650–657.
Chorilli, M., Prestes, P.S., Rigon, R.B., Leonardi, G.R., Chiavacci, L.
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