Journal of Thermal Analysis and Calorimetry

https://doi.org/10.1007/s10973-022-11504-3

Biological evaluation and compatibility study of oral mucoadhesive

hydrogel formulations using Schinopsis brasiliensis Engler. extract
and excipients
Thaíse Pereira Dantas Sampaio1 · Joao Augusto Oshiro Junior2   · Naara Felipe da Fonsêca2 ·
Ana Cláudia Dantas de Medeiros1,2

Received: 20 January 2022 / Accepted: 10 July 2022

© Akadémiai Kiadó, Budapest, Hungary 2022

Abstract
Native to Brazil, Schinopsis brasiliensis Engl. (Anacardiaceae) is the main representative species of the Schinopsis genus. It
brings anti-inflammatory, analgesic, hemostatic, antiseptic, antibacterial, and antifungal properties. The aims of this study
were to evaluate the antifungal potential of S. brasiliensis extract (API) against strains of Candida albicans, to perform a
compatibility study with mucoadhesive excipients, to evaluate antifungal activity, and characterize a mucoadhesive hydro-
gel for oral use. The antifungal activity was evaluated, and a pharmacological potency assay of different concentrations of
the hydroethanolic extract of the leaves of S. brasiliensis was performed using agar diffusion techniques, in a 5 × 1 cylinder
design method. The compatibility study between binary mixtures (BM) of extract and excipients was performed using dif-
ferential thermal analysis (DTA), thermogravimetry (TG), and Fourier transform infrared spectroscopy (FTIR). The hydrogels
formulated were characterized for pH, viscosity, and mucoadhesive strength and then evaluated for antifungal activity. C.
albicans was susceptible to the API at concentrations of 5, 10, 20, 40, and 80 mg ­mL−1. The pharmacological potency was
superior than the gallic acid chemical marker, with the API concentration for the hydrogel formulation at 50 mg ­mL−1. The
TG and DTA curves for the API—BM with carbopol (CAR) or pluronic hydrogel (PLU) maintained both the excipient and
API thermal characteristics, suggesting compatibility between them. The 30% PLU hydrogel/API association presented the
best performance and, considering the parameters of this study, should be further investigated as a potential oral antifungal
agent with activity against C. albicans.

Keywords  Schinopsis brasiliensis Engler. · Hydrogel · Excipients · Thermal analysis · Spectroscopy

Introduction

Schinopsis brasiliensis Engl. (Anacardiaceae), popularly

* Joao Augusto Oshiro Junior
joaooshiro@yahoo.com.br
* Ana Cláudia Dantas de Medeiros
anaclaudia@uepb.edu.br
1
Graduate Program in Dentistry, Department of Dentistry,
Center for Biological and Health Sciences, State University
of Paraíba, R. Baraúnas, 351, Cidade Universitária,
Campina Grande, Paraíba 58429‑500, Brazil
2
Drug Development and Testing Laboratory, Center
for Biological and Health Sciences, State University
of Paraíba, R. Baraúnas, 351, Cidade Universitária,
Campina Grande, Paraíba 58429‑500, Brazil
known as braúna and baraúna, is the principal representa-
tive species of the genus Schinopsis and native to Brazil. It
is distributed in the northeast, midwest, and southeastern
regions of the country and is common in the Caatinga
biome. The leaves, bark, stems, and fruits are used in tradi-
tional medicine as anti-inflammatories, analgesics, hemo-
statics, antiseptics, and antimicrobials [1], and we emphasize
its antifungal activity [2].
The antimicrobial activity of  S. brasiliensis  leaves
has been demonstrated against Escherichia coli [3], Pseu-
domonas aeruginosa, Staphylococcus aureus  [4,
5],  Streptococcus oralis  [5], and  Candida albicans  [6];
as well as its antimicrobial photosensitizing activity
in photodynamic therapy  against E. coli, S. aureus, P.

13
Vol.:(0123456789)

aeruginosa, and Enterococcus faecalis [7]. Bark extracts
from S. brasiliensis present antimicrobial activity against
strains and clinical isolates of E. faecalis [8], and Strepto-
coccus spp. [9]. Studies with animal models and human cells
with S. brasiliensis ethanolic bark extract have revealed little
toxicity [4, 9].
In the oral cavity, C. albicans is the species most often
isolated. It is part of the healthy microbiota in humans. Yet
in cases of immunosuppression, C. albicans can manifest
with clinical signs and symptoms in distinct forms of oral
candidiasis [10]. C. albicans is capable of undergoing phe-
notypic growth phase modifications (from yeast to hypha),
and when associated with immunosuppressive factors, can
invade tissue and form biofilm, reducing the effectiveness
of commonly used antifungal agents [11, 12]. Further, In
oral candidiasis, prolonged use of these agents can cause
resistance [13].
To fight microbial resistance, medicines derived from
medicinal plants are a promising alternative to traditional
antifungals, Considering the number of species with proven
antifungal potential, defined mechanisms of action, and
synergism between different classes and azole derivatives
[14], they have become essential raw materials for the
development of new drugs. Yet such medicines still require
physical–chemical studies to determine potential bioactive
compound/excipient incompatibility [15]. Increasing study
of products derived from medicinal plants has given rise
to pharmaceutical industry interest in plant-derived active
pharmaceutical ingredients (API) and improved quality con-
trol [16].
Pre-formulation thermoanalytic and spectroscopic meth-
ods help to identify and characterize potential incompat-
ibilities between the API and the pharmaceutical excipient,
revealing the most suitable excipients for formulation [17,
18]. In dry S. brasiliensis extracts, thermoanalytic and X-ray
diffraction techniques were essential to identify compatible
excipients for solid formulations [19]. Near-infrared spec-
troscopy has helped define compatibility between nanopar-
ticle excipients carrying pharmaceutical ingredients and S.
brasiliensis  extract  (with activity against multiresistant
microorganisms) [4]. Thermogravimetry and differential
thermal analysis associated with near-infrared spectros-
copy have proved to be important in the characterization of
medicinal plants [20].
With the emergence of new molecules in drug develop-
ment, bypassing first-pass metabolism and choosing local-
ized API delivery may become an alternative for overcoming
therapeutic challenges. Mucoadhesive systems, character-
ized by the union between two surfaces, one of which is
biological, are formed by several adhesion polymers, and
have been the choice for studies of new formulations [21].
Mucoadhesive polymers are hydrophilic macromolecules
(activated in the presence of water). Mucoadhesive polymers
13
T. P. D. Sampaio et al.
and gelling agents increase viscosity and promote gelation
and include: Carbopol, chitosan, sodium carboxymethyl-
cellulose, hydroxypropylmethylcellulose, hydroxyethylcel-
lulose, and pluronic F127 [22].
Hydrogels are systems formed by weakly cross-linked
three-dimensional polymeric networks, capable of retaining
water and indissolubility, important for biomedical appli-
cations, for example, as carriers of APIs [23]. Hydrogels
present promising results in carrying essential oils with
antifungal activity [24], demonstrate stability, safety, and
controlled release of APIs associated with flavonoids in C.
albicans biofilm control [25]. Hydrogels also present effi-
cacy in the treatment of skin infections caused by bacteria
and fungi by carrying active plant fractions of extracts with
antimicrobial activity [26].
In this context, our study aimed to evaluate the antifun-
gal potential of S. brasiliensis extract against strains of C.
albicans, to perform a compatibility study with mucoadhe-
sive excipients, and to develop and characterize the resulting
mucoadhesive hydrogel for oral antifungal use.
Materials and methods
Preparation of plant material and extract
S. brasiliensis  leaves  were collected  in a rural area
(7°13´50´´S, 35º52´52´´W) in the municipality of Pocin-
hos, semiarid region of the State of Paraíba. Exsiccates were
identified and deposited in the Professor Jayme Coelho de
Morais Herbarium, located at the Federal University of Par-
aíba (EAN 14,049). Registration for the use of this plant
was performed in the National System for the Management
of Genetic Heritage and Associated Traditional Knowledge
(SisGen) under the number AD768FA.
Hydroethanolic extract (HE) was prepared in an ethanol/
water solvent system at a concentration of 90% (90:10 v/v),
extractive solution of 20% (w/v) of plant drug, with ultra-
sound-assisted extraction at 40 °C for 1 h and subsequent
concentration on a rotary evaporator (40 °C, 55 rpm). The
remaining extract was submitted to a temperature of 40 °C,
in a forced air circulation oven. The dry extract was mac-
erated with grade and pistil and reduced to a fine powder
called active pharmaceutical ingredient (API) (vegetable)
used in all experiments.
Biological activity
Inoculum preparation
Strains of  C. albicans  from the  American Type Culture
Collection (ATCC 10,231) were reactivated in Sabouraud
Dextrose Broth and cultivated in Sabouraud Dextrose Agar
Biological evaluation and compatibility study of oral mucoadhesive hydrogel formulations…
(35 °C, 48 h). Colonies were suspended in 10 mL of ster-
ile 0.9% saline and standardized in a spectrophotometer
(530 nm) to contain the equivalent of ­106 CFU ­mL−1 [27].
Evaluation of the API antifungal activity
Agar diffusion technique [28] in cylinder methodology
using a 5 × 1 design was performed [5, 29]. Concentrations
2.5 (A0), 5 (A1), 10 (A2), 20 (A3), 40 (A4), and 80 (A5)
mg ml-1 diluted in 10% dimethyl sulfoxide (DMSO) were
evaluated. Nystatin (standard/positive control) was used at
concentrations of 0.125 (P1); 0.25 (P2); 0.5 (P3); 1 (P4); and
2 (P5) mg ­mL−1. The intermediate concentration (P3) was
determined according to Naeini [30]. DMSO was used as a
negative control. The assay was performed in triplicate and
in two independent experiments.
Petri dishes were prepared with a base layer (Mueller
Hinton agar) and a surface layer (Mueller Hinton agar inoc-
ulated with yeast suspension). On each plate, six stainless
steel cylinders with an external diameter of 8 mm, an inter-
nal diameter of 6 mm, and length of 10 mm were spaced at
equal distances. 100µL of the samples or the standard was
dispensed into each cylinder and the plates placed for 2 h in
a laminar flow chamber. These were then taken to an oven
at 35 °C for a period of 48 h and the diameters of the inhibi-
tion halos were read in millimeters (mm) with the aid of a
caliper. The results were expressed as the mean of the halos
in mm and by standard deviation (SD).
Pharmacological potency assay
The pharmacological potency of the API was determined
using agar diffusion technique, cylinder method, with a 5 × 1
design [29, 31]. The standard used was gallic acid at con-
centrations of 54, 60, 66, 73, and 80 mg ­mL−1. The API
concentration was 66 mg ­mL−1.
Once the pharmacological potency was determined, a
calibration curve for the API was produced, contemplating
the same concentrations of the standard gallic acid (54, 60,
66, 73, and 80 mg ­mL−1). According to Pinto, Kaneko, Pinto
[31], for the pharmacological potency test and determination
of the response at lower dose (L) and higher dose (H), it is
essential that the nominal dose level is equivalent between
sample and standard and that it contemplates five equidistant
points.
Correction of the standard curve points, production
of calibration curves, and determination of the L and H
responses was performed. The L and H response calcula-
tions determined the concentration of the API used in the
formulation. The assay was performed in triplicate in three
independent experiments. The results were expressed as the
mean of the halos in mm and SD.
API/Excipients Compatibility Study
The excipients used were: carbopol (CAR), chitosan (QUI),
carboxymethyl cellulose (CMC), hydroxypropyl methyl cel-
lulose (HPMC), hydroxyethyl cellulose (HIDRO), and plu-
ronic F127 (PLU). Binary mixtures (BM) of the pharmaceu-
tical excipients with the API were prepared in a porcelain
mortar and pestle, in proportions of 1:1, 1:2, and 2:1 (mg.
­mg−1). The samples were placed in closed plastic tubes at
room temperature. The excipients and BM were analyzed
using the techniques described below.
Thermal Analysis of the Samples
Differential thermal analysis (DTA) was performed with a
DTG 60 thermal analyzer (Shimadzu ®), under a nitrogen
atmosphere at a flow of 50 mL ­min−1, the temperature pro-
grammed from 25 to 400 ºC, with a heating rate of 10 ºC
­min−1. The mass used was 2 ± 0.5 mg, packed in an alu-
minum crucible.
Thermogravimetric (TG) curves were obtained using a
DTG 60 Thermal Analyzer (Shimadzu ®), under a nitrogen
atmosphere with a constant flow of 50 mL ­min−1, tempera-
ture programmed up to 900 ºC, with a heating rate of 10 ºC
­min−1. The mass used was 6 ± 0.5 mg, packed in an alumina
crucible.
Data were analyzed using  Shimadzu  ® software  and
graphs plotted using Origin ® Pro 8 software.
Fourier Transform Infrared Spectroscopy (FTIR)
FTIR analysis was performed on samples with signs of
incompatibility verified in the DTA and TG curves. The tests
were performed on a S ­ himadzu@ spectrophotometer, model
IR Prestige-21, with the following analysis conditions:
region 4000 – 400 ­cm−1; resolution: 4  ­cm−1; number of
accumulations: 20; mode: transmittance; sample preparation
dispersed in KBr (spectroscopic purity degree) in a propor-
tion of 1:100 mg for making 1.2 cm diameter pellets.
Hydrogel development and characterization
Once excipients compatible with the API were determined,
the hydrogels were formulated according to the technical
specifications proposed by Ferreira [22]. Each formulated
hydrogel was composed of a compatible gelling agent (car-
bopol or pluronic F127), preservatives (0.05% propylparaben
and 0.15% methylparaben), and API 50 mg ­mL−1 (defined
by the pharmacological potency assay).
For the formulation of the carbopol hydrogels, the
excipients were homogenized in previously heated distilled
water, and taken to ultrasound for, approximately five min-
utes (intermittently), for undissolved particles during the
13

manipulation process, and the API was then added. Sub-
sequently, the alkalizing agent AMP 2000 was added drop
by drop, vigorously stirring until the formula acquired the
appearance and consistency of a gel.
For the formulation of pluronic F127 hydrogels, the
preservatives were dissolved in boiling water, and after
cooling they were kept under refrigeration until both water
and preservatives were cold. Pluronic F127 was dispensed
into this mixture which was refrigerated for 24 h, such that
the pluronic 127 was completely dispersed. API was added
by solubilizing in an ice bath.
The characterization assays evaluated hydrogen poten-
tial (pH), viscosity, and mucoadhesive strength of the
hydrogels alone or associated with API.
pH assessment
The pH of the hydrogels was visually assessed by meas-
urement using pH measuring strips with a colorimetric
scale (Macherey–Nagel ®), which classifies the sample
in a range of pH 0 (acid) to 14 (alkaline). Each hydrogel
sample was placed in a glass beaker and the pH indica-
tor paper strip was then immersed in the sample for five
seconds. The pH record was performed by means of a col-
orimetric comparison between the color scale presented
by the test and the color resulting from the reading. The
procedure was performed in duplicate.
Viscosity test
The viscosity reading of the hydrogels was performed with
a One R viscometer, spindle R7 standard at 100 rpm. The
samples were deposited in glass goblets at an approximate
volume of 60 ml. The measuring loop of the equipment
was introduced into the sample so that the largest possible
volume of the hydrogel was reached.
The PLU hydrogel samples were evaluated under two
conditions: under an ice bath, to avoid the rapid transition
from the sol phase (liquid state) to the gel phase, and under
increasing temperature, so that the viscosity was evalu-
ated during the transition process of the sol phase to gel
phase. In this condition, the cups with the samples were
placed in a water bath on a hot plate, while being read in
the viscometer. The temperature was measured with a digi-
tal thermometer. Analysis started at a temperature of 14 °C
and ended when the equipment presented result stability.
Each sample was analyzed in triplicate and the results
expressed in viscosity percentage (%) through the mean
of the values ​​and SD.
13
T. P. D. Sampaio et al.
In vitro mucoadhesion assay
Assessment of the in vitro mucoadhesive strength of the
hydrogels was performed according to Oshiro [32], with
modifications. Esophageal mucosa was obtained from pigs
from the Olhos d´Água slaughterhouse, located in Ipuã
(SP). The mucous membranes were transported and stored
in a medium with artificial saliva and in an ice bath, and
processing allowed for up to 6 h after the animals' death. The
esophageal mucosa was separated from the underlying tis-
sues using a scalpel and anatomical scissors, keeping the
slice thickness at approximately 1 mm. Mucin discs prepared
by mucosal compression (250 mg) were used.
Fifty microliters of artificial saliva was applied to the
surface of the mucin disc before the experiment, and the
mucosa was immersed for 30 s in the same saliva to simu-
late the oral environment. The force required to detach the
mucosal gels or mucin disc was assessed using the TA-XT
Plus texture analyzer (Stable Micro Systems, Surrey Eng-
land), in adhesion test mode.
The mucosa or mucin disc was fixed to the lower end
of the cylindrical probe of the equipment (10 mm in diam-
eter). The hydrogel was packaged in small plastic contain-
ers under the probe at 37 °C. The test was started with the
probe at a constant speed (1 mm/s) until the mucosa or disk
came into contact with the hydrogel. The mucosa or disc
remained in contact with the hydrogel for 60 s, and no force
was applied during this time. The probe was then raised
at constant speed (0.5 mm/s) until detachment between the
mucosa and the hydrogel resulted. During the experiment,
a force–time curve was recorded using the Expert Texture
Exponent 32 ® software, and the area under the force–dis-
tance curve during the withdrawal phase; adhesion peak was
calculated as adhesion strength. This process was performed
in triplicate. Results were expressed as mean values and
​​ SD.
Evaluation of hydrogel antifungal activity
The antifungal activity of the hydrogels was evaluated using
the diffusion in agar, cylinder method [29].
Each hydrogel sample was evaluated under two condi-
tions: undiluted hydrogel (H) and diluted hydrogel (Hd). Hd
was obtained by diluting 1 mg of sample in 1 ml of 10%
DMSO. The density of the sample was considered equal to
1 (d = 1).
In Petri dishes with half agar base and half agar inocu-
lated surface, six stainless steel cylinders were placed and
100µL of sample was dispensed in each, with three cylinders
of sample H and three cylinders of Hd. The plates remained
for 2 h in a laminar flow chamber and were then taken to an
oven at a temperature of 35 °C for 48 h. The diameters of
Biological evaluation and compatibility study of oral mucoadhesive hydrogel formulations…

the inhibition halos were read in millimeters (mm) with the Table 1   Evaluation of API and nystatin antifungal activity at differ-
aid of a caliper. The positive control was performed with ent concentrations
Nystatin 500 µg ­mL−1. Three independent experiments were Sample (mg mL-1) Inhibi-
performed. The results were expressed as the mean of the tion zones
halos in mm and SD. (mm ± SD)

P1 27.66 ± 0.81
Statistical analysis P2 28.33 ± 1.36
P3 29.56 ± 0.64
The results of the experiments were expressed as mean val- P4 30.66 ± 1.03
ues and
​​ standard deviation (SD), or median and interquartile P5 33.33 ± 0.81
range (IQR). A0 9.66 ± 0.51
For the viscosity, mucoadhesion and antimicrobial activ- A1 11.33 ± 0.51
ity tests of the hydrogels presenting data with normal distri- A2 14.33 ± 0.51
butions, parametric statistics were used through analysis of A3 17.66 ± 1.03
variance (ANOVA—one way), followed by Tukey's post hoc A4 19.00 ± 1.09
tests. For data with non-normal distributions, nonparametric A5 24.33 ± 0.51
statistics were used following the Kruskal–Wallis test. For DMSO 10% 0.00
data with non-normal distributions for related samples, the Legend: P1 (Nystatin=0.125 mg mL-1);  P2 (Nistatin=0.25 mg
Wilcoxon test was used. Data normality was tested using mL-1);  P3 (Nistatin=0.5 mg mL-1);  P4 (Nystatin=1 mg mL-1);  P5
the Shapiro–Wilk test. The significance level was set at (Nystatin=2mg mL-1);  A0 (API= 2.5 mg mL-1);  A1 (API= 5
mg mL-1);  A2 (API=10 mg mL-1);  A3 (API=20 mg mL-1);  A4
5% ( p = 0.05). The Statistical Package for the Social Sci-
(API=40 mg mL-1);  A5 (API=80 mg mL-1);  DMSO (Dimethylsul-
ences (SPSS, version 20.0) and Microsoft Excel (2016 ver- foxide)
sion) programs were used.
Table 2    Antifungal activity of gallic acid and API against strains
of C. albicans 
Results and discussion Concentrations Inhibition zones (mm ± SD)
(mg mL−1) Gallic acid API
Biological activity
54 9.33 ± 0.51 20.00 ± 0.57
API antifungal activity 60 9.66 ± 0.51 21.00 ± 0.00
66 10.00 ± 0.00 22.33 ± 0.51
Table 1 presents the mean diameters of the inhibition zones 73 10.66 ± 0.51 23.33 ± 0.81
(mm) for API and nystatin. 80 11.00 ± 0.51 24.33 ± 0.51
The agar diffusion method is used in many studies to eval-
uate the antimicrobial potential of therapeutic agents [14,
33] and plant species [28, 30, 34, 35] with the aim of devel- evident in extracts with ethanolic solvents in concentrations
oping new antimicrobial agents or comparing therapeutic greater than seventy percent.
agents with each other. Miranda-Cadena [36] has defined the Studies reveal that the antimicrobial activity of S. brasil-
susceptibility of clinical isolates of Candida species in rela- iensis leaves is expressive both in the ethanol extracts and
tion to the antifungal Nystatin. Clinical isolates were catego- in isolated molecules [37]. In addition, leaf extracts have
rized based on the diameter of the inhibition zones, using the also demonstrated potential bacterial resistance modulating
disk diffusion method. With inhibition halos of ≥ 15 mm, the activity and synergism with a synthetic antimicrobial, con-
isolate was considered susceptible; with 10–14 mm of halo, firming the S. brasiliensis as a potential natural antimicrobial
intermediate susceptibility; and with a halo of < 10 mm, the agent [38].
isolate was considered resistant to Nystatin.
Considering the results in Table 1, C. albicans was sus- Pharmacological potency of API
ceptible to samples A1, A2, A3, A4, and A5. Chaves [6]
demonstrated similar activity, corroborating the findings The antifungal activity results to determine the pharma-
of this study. However, Silva [5] did not find activity for cological potency of gallic acid and API are presented in
the same microorganism. This suggests that the antifungal Table 2.
activity of S. brasiliensis may be influenced by differences in Concentrations below 40 mg ­mL−1 of gallic acid pre-
extractive methods and solvent concentrations, being more sented no antifungal activity against  C. albicans.  Thus,

13
 T. P. D. Sampaio et al.

maintaining the ratio of 1.1 between concentrations of 80 25

and 40 mg ­mL−1, five equidistant points were obtained for
24
the pharmacological potency calculation, corresponding to

Inhibition halos/mm
the concentrations shown in Table 2, in which the values 23
y = 0.1686x + 10.972
were plotted from the inhibition halos corresponding to gal- R 2 = 0.9929
lic acid (Table 2), and resulting in the straight line equation 22

y = 0.0669x + 5.6748 ­(R2 = 0.9886), shown in Fig. 1. 21

Figure  2 presents the calibration curve of the API
against strains of C. albicans. 20
Thus, according to the calculation of the L, H responses
19
and the straight line equation (Fig. 2), the dose range of 50 55 60 65 70 75 80 85
the API was established (Table  3).  A concentration of –1
Concentration/mg ml
50 mg ­mL−1 was determined for the hydrogel formulation.
Of the other compounds found in S. brasiliensis, gallic
Fig. 2   API—C. albicans calibration curve
acid is the most expressive, being determined as its chemi-
cal marker [39–41]. It was determined the main secondary
metabolite identified in S. brasiliensis leaves, followed by Considering the pharmacological potency of API com-
quercetin derivatives [39], and defined as a chemical marker pared to gallic acid in this study, it is suggested that syner-
in bark samples [41]. Pyrogallic tannin, phenols, flavonoids, gism between different phytocompounds may be responsible
resin, and alkaloids were identified in the phytochemical for the optimization of the potency of API compared to gal-
prospection of S. brasiliensis leaves [6], and there is evi- lic acid.
dence that the phenolic compounds present in the Anac- For the calculation of the L and H responses, a calibration
ardiaceae family are responsible for the antifungal activity curve was prepared for the API in the same concentrations
demonstrated in many of the species, among them S. brasil- as the standard.
iensis [40]. Among the compounds with proven antifungal
activity, we find gallic acid [42]. API‑Excipients compatibility study

API characterization by thermal analysis

12 The API, TG, and DTA curves are shown in Fig. 3.

y = 0.0669x + 5.6748 The API DTA curve presented three endothermic
R 2 = 0.9886
peaks. The first peak occurred at a temperature of 47.17ºC
Inhibition halos/mm

11
(Δ H −124.14 J ­g−1), the second peak occurred at a tem-
perature of 223.94 ºC (Δ H -31.30 J ­g −1), and the third
10
peak endothermic occurred at a temperature of 267.99ºC
(Δ H −64.30 J ­g−1). The first endothermic peak may be
related to loss of water and/or ethanol in the sample com-
9 position. In the thermogravimetric curve, the event caused
50 55 60 65 70 75 80 85 a mass loss of 5.56%. The second and third endothermic
Concentration/mg ml–1 peaks may be related to thermal decomposition events of
the sample.
Fig. 1   Calibration curve of gallic acid against C. albicans  In the TG curve, the second thermal event occurred from
221.87−294.46 °C, with a mass loss of 61.3%. According to
Fernandes [15], thermal decomposition is related to further
endothermic events. After the second thermogravimetric
Table 3    Response values L, and H in equivalent API—Gallic acid
concentrations
event, the sample continuously loses mass, entering into
decomposition.
Response Inhibition halos (y) Corresponding During new drug pre-formulation stages, the study of
(mm) concentration
(x) physicochemical interactions between active compounds
(mg mL-1) and excipients is essential to obtain a stable pharmaceuti-
cal form that ensures bioavailability and acceptable shelf
Lower (L) 17.53 38.90
life. Thermoanalytic techniques contribute to prediction of
Higher (H) 21.93 64.93

13
Biological evaluation and compatibility study of oral mucoadhesive hydrogel formulations…
100
80
60
Mass/%
40
20
0 100 200 300 400 500 600 700
Temperature/°C
Fig. 3   API (IFAV), TG, and DTA curves
potential incompatibilities, avoiding the waste of time and
supplies [17].
Figure 4 presents the TG curves for the API-BM excipi-
ent associations for CAR, CMC, HIDRO, HPMC, PLU, and
QUI.
Table 4 presents the results of thermal events in TG anal-
ysis of the API and BM.
Figure 5 presents the DTA curves for API associated in
BM with different excipients.
The CAR TG curve (Table 4) presents three stages of
thermal decomposition. The first takes place in the tem-
perature range between 45.09 and 60.94 °C. with a mass
loss of 5.08%; the second occurs in the temperature range
from 292.78 to 303.68 °C, with a mass loss of 12.02%; the
third takes place in the temperature range from 382.88 to
458.48 °C, with a mass loss of 51.88%. The BM with CAR
maintained the three thermal decomposition peaks, without
anticipating the first decomposition peak, compared to the
isolated excipient.
The DTA parameters of BM with CAR (Table 5) pre-
sented no anticipation of peaks, compared to API. The BM
CAR/API (2:1) was more stable, maintaining the third endo-
thermic peak (suppressed in the other BM with this excipi-
ent). These characteristics suggest compatibility between
CAR and API. A similar study identified carbopol as the
excipient with the best thermal behavior when associated
with S. brasiliensis in a sunscreen formulation [15].
In the CMC TG curve, three stages of thermal decompo-
sition were observed, between 46.73 and 60.73 °C, with a
mass loss of 22.54%; between 269.86 and 287.99 °C, with
a mass loss of 44.98%, and between 77.97 and 851.53 °C,
with a mass loss of 28.6%. The BM with CMC presented two
stages of thermal decomposition, suppressing third-stage
2
Temperature difference/mV;Exo up
IFAV
IFAV
0
–2
–4
–6
800 900 1000 50 100 150 200 250 300 350 400
Temperature/°C
decomposition found in the isolated excipient. The absence
of the third decomposition step in the BM with CMC may
have been influenced by the thermal decomposition range
of the API, which is lower than the last range of the excipi-
ent. The BM (1:1) presented anticipation in the decomposi-
tion process.
The DTA data (Table 5) show that when compared to the
API, the BM/CMC did not anticipate the endothermic peak.
However, the two subsequent endothermic peaks present in
the thermal analysis of API alone were suppressed. In the
BM, the two exothermic peaks characteristic of the excipi-
ent were maintained; however, anticipation of peaks and an
important reduction in enthalpy, compared to the excipient,
were observed. These characteristics suggest physical and
chemical incompatibility between CMC and the API.
The HIDRO TG curve presented two stages of thermal
decomposition. The first stage took place between 43.85
and 56.79 °C, with a mass loss of 10.16%; the second stage
occurred between 320.55 and346.61 °C, with a mass loss of
71.14%. When compared to the excipient, the thermogravi-
metric curves of the BM did not present anticipation of the
first decomposition step. Comparing the BM with the API,
there was an anticipation in the first stage of decomposi-
tion of the mixtures HYDRO: API (1:1) and HYDRO: API
(1:2). The BM (2:1) proved to be more thermally stable, and
anticipated the two decomposition steps. The thermogravi-
metric curves of the BM present similar characteristics to
the excipient, suggesting a potential interaction between the
excipient and the API.
The DTA curves of BM with HYDRO present suppres-
sion of two endothermic peaks and the presence of an exo-
thermic peak, (as compared to API). The suppression and
peaking characteristics suggest chemical incompatibility. It
13
 T. P. D. Sampaio et al.

(a) (b) 100

100
IFAV IFAV
1:1 1:1
1:2 1:2
80 2:1 80 2:1
CAR CMC

Mass/%
Mass/%

60
60

40
40

20

20

0 100 200 300 400 500 600 700 800 900 1000 0 100 200 300 400 500 600 700 800 900 1000
Temperature/°C Temperature/°C

(c) 100 (d) 100

IFAV IFAV
1:1 1:1
80 1:2 1:2
80
2:1 2:1
HIDRO HPMC
60
60
Mass/%

Mass/%

40 40

20 20

0 100 200 300 400 500 600 700 800 900 1000 0 100 200 300 400 500 600 700 800 900 1000
Temperature/°C Temperature/°C

(e) 100 (f) 100

IFAV
1:1 IFAV
1:2 1:1
80 1:2
2:1 80
PLU 2:1
OUT
60
Mass/%

60
Mass/%

40
40

20
20

0 100 200 300 400 500 600 700 800 900 1000 0 100 200 300 400 500 600 700 800 900 1000
Temperature/°C Temperature/°C

Fig. 4   TG curves of API (IFAV) BM associations with: A CAR and BM; B CMC and BM; C HYDRO and BM; D HPMC and BM; E PLU and
BM; F QUI and BM

was observed that the BM presented a predominance of HPMC presented a thermogravimetric curve with a ther-
excipient characteristics, suggesting interaction between mal decomposition step between 341.34 and 377.62 °C, with
excipient and API.

13
Biological evaluation and compatibility study of oral mucoadhesive hydrogel formulations…

Table 4   Thermogravimetric parameters for API, the pharmaceutical (2:1) samples, maintaining the characteristics of the excipi-
excipients, and their binary mixtures (BM) in proportions of 1: 1, 1: 2 ent curves. The PLU: API (1:2) sample maintained the three
and 2: 1 (Excipient: API)
endothermic peaks observed in the API. No peak antici-
Samples Initial temperature/°C Mass loss/% pation event occurred in the BM with this excipient com-
pared to API, characterizing chemical compatibility. The
API 71.68 5.56
permanence of the thermal characteristics of the API and
CAR​ 45.09 5.08
the excipient in the binary mixtures suggests the absence of
CAR + API (1:1) 38.40 6.72
interaction between them.
CAR + API (1:2) 48.07 5.34
The thermogravimetric curve of QUI presented two
CAR + API (2:1) 51.70 3.76
stages of thermal decomposition. The first stage took place
CMC 46.73 22.54
between 41.99 and 54.24 °C, with a mass loss of 14.6% and
CMC + API (1:1) 45.60 12.88
the second stage occurred between 274.81 and 305.36 °C,
CMC + API (1:2) 95.89 7.5
with a mass loss of 44.2%.  The thermogravimetric BM
CMC + API (2:1) 207.73 10.78
curves presented one stage of thermal decomposition and
HYDRO 43.85 10.16
did not show anticipation in the degradation process.
HYDRO + API (1:1) 53.40 10.52
The thermal stability of chitosan in this study was similar
HYDRO + API (1:2) 54.78 6.12
to Wang [43], with two degradation steps, the first associ-
HYDRO + API (2:1) 220.20 21.8
ated with water loss and the second associated with chitosan
HPMC 341.34 73.82
deacetylation. According to Sette-de-Souza [44], the degree
HPMC + API (1:1) 234.09 63.24
of chitosan deacetylation influences protection of the API
HPMC + API (1:2) 225.83 59.76
against degradation. This corroborates the present study,
HPMC + API (2:1) 266.08 72.86
where only one thermal event related to the mass loss API
PLUS 50.05 4.36
was associated with chitosan in the respective BMs.
PLU + API (1:1) 224.54 33.62
In the DTA curves of CHI-BM, suppression of the second
PLU + API (1:2) 220.72 37.30
and third endothermic peaks was observed in all samples, as
PLU + API (2:1) 221.91 27.90
compared to the API. The exothermic peak, characteristic
THU 41.99 14.60
CHI + API (1:1) 222.83 52.22
of the isolated excipient, was maintained; however the peak
CHI + API (1:2) 224.74 51.00
was anticipated. The BM curves were similar to the excipi-
ent curve. However, comparing the BM with the excipient,
CHI + API (2:1) 250.73 53.62
enthalpy reduction was observed in all thermal events. These
characteristics suggest chemical and physical incompatibil-
ity between this excipient and API.
a mass loss of 73.82%. Compared to the excipient, the BM The excipient and API thermal stability studies presented
presented anticipation in the degradation process. better compatibilities for CAR and PLU. The samples that
In the DTA curves for the BM with HPMC, suppres- presented signs of incompatibility (verified in the thermal
sion of an endothermic peak was observed in each sample, analyses) were submitted to FTIR analysis, in order to con-
compared to the API, maintaining the characteristics of the firm and elucidate these interactions.
excipient. In the BM (1:2), anticipation of the first endother-
mic peak is observed. Peak suppression and anticipation are
characteristics of chemical incompatibility between HPMC FTIR Analysis
and the API. The maintenance of the excipient characteris-
tics in detriment to of those of the API suggests interactions Spectroscopy is the science that studies the interactions
between them. of radiation with matter, elucidating molecular structures
In the PLU TG curve, two stages of thermal decomposi- and helping to determine organic and inorganic compounds
tion were observed. Between 50.05 and 57.52 °C, the first [45]. Infrared spectroscopy is used as an analytical instru-
stage occurred with a mass loss of 4.36%; and between ment to observe the presence of functional groups and plant
303.36 and 401.34 °C the second stage of decomposition metabolites in active pharmaceutical ingredients [46].
occurred with a mass loss of 92%. The BM did not present Figure  6 presents the FTIR spectra of the  S.
any anticipation event in the degradation process, maintain- brasiliensis API.
ing the decomposition steps for the excipient and the API. According to Fig. 6, the presence of a broad absorption
The DTA curves in the BM with PLU presented sup- peak in the range of 3354 ­cm−1, corresponding to the axial
pression of an endothermic peak in the PLU: API (1:1) and deformation of hydrogen, bound to oxygen or nitrogen was

13
 T. P. D. Sampaio et al.

(a) (b)
IFAV IFAV

Temperature difference/µV;Exo up
0 1:1 1:1
Temperature difference/µV;Exo up

1:2 1:2
2:1 2:1
CMC
CAR

–9

– 20
– 18

0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
Temperature/°C Temperature/°C

(c) IFAV
1:1 (d) IFAV
Temperature difference/µV;Exo up

1:2 1:1

Temperature difference/µV;Exo up
1:2
0 2:1
2:1
HIDRO HPMC

– 10

– 10
– 20

– 20
– 30

– 30 – 40
50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
Temperature/°C Temperature/°C

(e) IFAV (f)

1:1
0 IFAV
0
Temperature difference/µV;Exo up
Temperature difference/µV;Exo up

1:2 1:1
2:1 1:2
PLU 2:1
OUT

– 10 – 10

– 20 – 20

– 30 – 30

– 40
– 40
50 100 150 200 250 300 350 400
50 100 150 200 250 300 350 400
Temperature/°C
Temperature/°C

Fig. 5   DTA curves for API (IFAV) associated in BM: A CAR and BM; B CMC and BM; C HYDRO and BM; D HPMC and BM; E PLU and
BM; F QUI and BM

observed. Another absorption peak was observed in the bonds and were identified in samples of S. brasiliensis, as
range of 2922 ­cm−1, corresponding to axial deformation of well as a broad spectral band at 3334 ­cm−1, which refers to
hydrogen bonded to primary and secondary carbons. O–H bond stretching, characteristic of an extract abundant in
The absorption peaks in the 1714 to 1606 ­cm−1 range secondary metabolites such as polyphenols, rich in hydroxyl
correspond, respectively, to deformation of C = O and C = C groups [3]. Fernandes [16], in an extract of S. brasiliensis

13
Biological evaluation and compatibility study of oral mucoadhesive hydrogel formulations…

Table 5  DTA parameters for the API, the pharmaceutical excipients, and their binary mixtures in proportions of 1: 1, 1: 2 and 2: 1 (Excipient:
API)
Samples Stage 1 Stage 2 Stage 3
−1 −1
T onset—T T peak/DTA /°C ∆ H /Jg  T onset—T T peak/DTA /°C ∆ H /Jg  T onset—T T peak/DTA /°C ∆ H /Jg −1
final /°C final /°C final /°C

API 38.47−57.60 47.17  − 124.14 202.15−228.69 223.94   −  31.30 248.28−274.11 267.99   −  64.30
CAR​ 54.70−108.73 54.51   −  303.80 201.35−297.17 266.42   −  1.18 309.21−397.46 343.74   −  682.65
CAR + API (1:1) 66.35−110.50 77.38   −  67.15 230.78−268.51 231.23   −  44.26 –  –  – 
CAR + API (1:2) 57.98−113.84 60.47   −  176.96 211.31−278.89 272.90   −  287.69 – – –
CAR + API (2:1) 84.36−124.87 84.37   −  98.33 230.54−285.51 234.90   −  254.50 333.78−360.68 353.28   −  17.16
CMC 61.59−118.98 96.69   −  827.54 277.13−305.45 293.74 434.17 313.63−327.03 316.86 14.04
CMC + API (1:1) 78.48−118.63 114.39   −  143.49 268.13−303.81 282.69 159.04 312.26−329.29 312.38 9.04
CMC + API (1:2) 61.15−112.95 58.74   −  149.87 181.53−212.89 179.91 64.31 269.30−299.72 263.07 70.09
CMC + API (2:1) 58.09−132.69 67.53   −  236.36 279.56−307.03 291.49 298.07 313.52−326.28 312.74 17.58
HYDRO 66.30−105.17 87.34   −  240.20 323.27−354.46 343.59 450.32 –  –  – 
HYDRO + API 50.08−106.33 113.40 – 124.53 367.69−390.60 361.60 39.25 –  –  – 
(1:1)
HYDRO + API 51.07−90.03 51.44   −  129.64 153.94−217.75 187.84 19.65 –  –  – 
(1:2)
HYDRO + API 55.21−104.03 56.33   −  143.44 286.31−310.68 302.16   − 11.76 - –  – 
(2:1)
HPMC 47.29−91.56 56.60 -62.29 340.68−385.20 360.89   −  541.84 –  –  – 
HPMC + API (1:1) 55.14−82.50 57.52 -90.63 261.93−290.94 262.50 -51.91 –  –  – 
HPMC + API (1:2) 38.64−98.23 42.98   − 34.54 242.88−271.33 271.06   − 61.44 –  –  – 
HPMC + API (2:1) 64.27−69.87 67.10 -19.47 298.89−363.97 303.40 -156.93 –  –  – 
PLUS 54.96−65.70 58.87   − 332.14 359.66−399.33 371.12   − 760.02 –  –  – 
PLU + API (1:1) 51.43−61.92 56.56   − 132.89 225.28−245.74 245.52   −  29.45  −   −   − 
PLU + API (1:2) 49.39−61.20 55.30   −  129.98 238.05−265.60 261.51   −  199.31 365.10−394.02 386.70   −  142.40
PLU + API (2:1) 52.27−64.58 57.75   −  313.54 381.00−400.69 394.07   −  75.60 – –  – 
THU 65.96−128.33 99.64   −  522.65 282.77−319.36 300.33 548.45 – – –
CHI + API (1:1) 73.95−113.20 112.61 -83.86 282.39−319.13 297.90 125.09 – – –
CHI + API (1:2) 56.15−98.85 56.69   −  153.24 271.26−312.11 271.26 44.57 – – –
CHI + API (2:1) 67.55−130.27 67.71   −  361.63 282.47−321.91 300.70 283.75 – – –

bark, identified sharp peaks in the 1307–1025 ­cm−1 region
related to carbon–oxygen (CO) bonds. This indicates the
presence of tannins, flavonoids, anthraquinones, and other
metabolites.
The absorption peaks in the range of 1444 and
1193  ­cm−1, respectively, correspond to angular deforma-
tions of CH 2 functional groups, and intense bands of axial
deformation of SO 2 functional groups.
Figure 7 presents the FTIR spectra of the API and binary
mixtures in proportions of 1: 1, 1: 2, and 2: 1 (Excipient:
API).
The FTIR graphs of the BM of the API associated with
CMC presented a reduction in the intensity of the absorption
bands, as seen in Fig. 7A, not resembling the FTIR spectra
of the API and CAR, suggesting an interaction between them
in the BM proportion of 1:1.
In Fig.  7B, the BM spectral bands (1:2) are similar
to the API absorption spectra, demonstrating potential
compatibility between excipient and API.  However, in
Fig. 7C, it is observed that the BM (2:1) presented no simi-
larity with the absorption spectra of the API and the excipi-
ent, suggesting incompatibility, probably chemical, between
CMC and API.
In Fig. 7D, a reduction in peak intensity was observed in
the absorption spectra of the HYDRO: API (1:1) BM. Fig-
ure 7E suggests potential compatibility between the excipi-
ents, however the higher proportion of API in this sample
may have influenced continuance of the same peak intensity
when compared to the spectra of API alone. Figure 7F pre-
sents a reduction in peaks in the BM absorption spectra,
compared to the API.
Figure 7G presents a reduction in peak intensity in the
BM absorption spectra (1:1), compared to the API, sug-
gesting a potential interaction between the API and this
excipient.

13

Transmitance/%
2922
1714
1606
1444
3354
1028
1193
4400 4000 3600 3200 2800 2400 2000 1600 1200
Wavenumber/cm–1
Fig. 6   API (IFAV) FTIR Spectra
Figure 7H presents the absence of the characteristic spec-
tral bands of the API and the excipient, demonstrating inter-
action between them. Considering that this BM presents a
higher proportion of API, it can be suggested that, regardless
of the spectral characteristics of the other binary mixtures,
API presents characteristics of incompatibility with this
excipient.
Figure 7I demonstrates a potential interaction between
API and HPMC, with reduced peaks in the BM absorp-
tion spectra, compared to the API. Figures 7J, 7L, and 7M
reveal similarities regarding the arrangement of peaks and
absorption spectra of the BM, compared to the API. In the
three BM analyzed, a reduction in the intensity of absorp-
tion peaks was observed in all spectral bands, suggesting
incompatibility between QUI and the API.
Considering the characteristics of the BM absorption
spectra between the API and the excipients CAR, HIDRO,
HPMC, and QUI, incompatibility between the API and the
analyzed excipients is suggested, corroborating the results
revealed in the thermal analysis studies (TG and DTA).
Considering the results of the compatibility study, CAR
and PLU were eligible as excipients for hydrogel formulation
associated with the API.
Hydrogel characterization
The characterization results for pH, viscosity, and mucoad-
hesion in pure carbopol hydrogel (HCAR), and pluronic
F127 hydrogel (HPLU), and associations with the API are
presented in Table 6.
Salivary pH oscillates between 5.8 and 7.6, which can
be influenced by local factors [22]. According to Sheshala
[47] pH 7 is physiologically ideal for application of hydrogel
13
T. P. D. Sampaio et al.
when treating oral disease. Thus, the closer to the physi-
IFAV
ological pH, the more suitable the formulation will be for
application in the oral cavity.
According to Table 6, it was found that when associating
the API with the formulations of hydrogels with CAR and
PLU, there was a reduction in pH in all samples.
This characteristic reduction of pH when associating
the API with hydrogels may be related to the presence of
gallic acid or other phenolic compounds in the S. brasil-
iensis samples [39, 40]. Phenolic compounds are impor-
tant antioxidants, reacting with free radicals and reactive
compounds with lower energy, favoring the abstraction of
hydrogen [48]. These isolated hydrogen molecules may have
800 400 contribute to pH reductions in the hydrogels associated with
the API, considering ionic interactions occurring between
excipients and the API polyphenols.
All samples of HCAR associated with the API reduced
the pH (pH = 4), this value being below the ideal in formula-
tions for oral use.
Of the HPLU samples associated with the API, the sam-
ples HPLU 30% + API and HPLU 40% + API presented the
lowest pH variation (pH = 5). Compared to the other hydro-
gel samples associated with the API, they presented values​​
closer to the physiological pH. To improve the hydrogen
potential of these samples, bringing them closer to physi-
ological pH, Ferreira [22] suggests the use of pH modifiers
as a convenient control for pH at the application site, and
better ensure absorption of the API.
Considering the results of Table 6, under the conditions
of this study, the samples HPLU 30% + API and HPLU
40% + API presented favorable pH for oral use formulation,
if adding pH modifiers.
Considering viscosity, it was observed that the higher
the excipient concentration, the higher the viscosity of the
hydrogels, see Table 6. This corroborates Sheshala [47],
who presented a direct proportional relationship between
viscosity and concentration in gel formulations based on
poloxamers.
As for the CAR hydrogels not associated with the API,
it was observed that there is a difference in the viscosity
values between the HCAR 1%, HCAR 1.5%, and HCAR
2% samples (p < 0.001).  Tukey's post hoc showed that
these samples differ significantly, comparing HCAR 1%
with HCAR 1.5% (p < 0.001), HCAR 1% with HCAR 2%
(p < 0.001), and HCAR 1.5% with 2% HCAR (p < 0.001).
When associated with the API, this significant dif-
ference remained only between the HCAR 1% + API,
and HCAR 2% + API samples (p = 0.02). Thus, HCAR
1.5% + API, and HCAR 2% + API, presented better per-
formance in terms of viscosity, for the CAR hydrogel
samples.
Of the PLU hydrogel samples not associated with the
API, HPLU 20% and HPLU 40% presented a statistically
Biological evaluation and compatibility study of oral mucoadhesive hydrogel formulations…

(a) (b) (c) IFAV

IFAV 2:1
1:1 IFAV
CMC
CMC 1:2
CMC

Transmitance/%
Transmitance/%

Transmitance/%
4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500

Wavenumber/cm
–1 Wavenumber/cm–1 Wavenumber/cm–1

(d) IFAV (e) (f) IFAV

1:1 IFAV
2:1
HIDRO 1:2
HIDRO
HIDRO

Transmitance/%
Transmitance/%

Transmitance/%

4000 3500 3000 2500 2000 1500 1000 500

4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500
–1
–1 –1 Wavenumber/cm
Wavenumber/cm Wavenumber/cm

(g) (h) IFAV (i)

IFAV IFAV
1:2
1:1 2:1
HPMC
HPMC
HPMC
Transmitance/%

Transmitance/%

Transmitance/%

4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber/cm–1 Wavenumber/cm–1 Wavenumber/cm–1
(j) IFAV (k) (l)
IFAV IFAV
1:1
1:2 2:1
QUI
QUI QUI
Transmitance/%

Transmitance/%
Transmitance/%

4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500
–1 –1
Wavenumber/cm Wavenumber/cm
Wavenumber/cm–1

Fig. 7   API (IFAV) FTIR spectra associated with BM. CMC Fig.a, b, c), HYDRO (Fig. d, e, f), HPMC (Fig. g, h, i) and CHI (Fig. j, l,  m)

13
 T. P. D. Sampaio et al.

Table 6    Evaluation of pH, viscosity, and mucoadhesion in pure CAR, which when solubilized in water, presents increased
HCAR and HPLU hydrogel, and in associations with the API elasticity, influencing its mucoadhesive properties [49].
Samples pH Viscosity (%) Mucoadhesion (N) Considering HCAR samples associated with the API, the
(mean ± SD) (median / IQR) results continued to maintain the order of adhesion strength
between the samples, being HCAR 2% + API, followed by
HCAR 1% 11 7.96 ± 0.20 (*) 0.00680 /
HCAR 1.5% + API, and then HCAR 1% + API. However,
0.00022 (****)
no significant difference was observed between the samples
HCAR 1.5% 5 12.66 ± 0.15 (*) 0.00795 / 0.00172
(p = 0.63). Under the conditions of this study and consider-
HCAR 2% 5 39.86 ± 0.20 (*) 0.02340 /
0.02316 (****) ing the statistical significance of the experiment, it is sug-
HCAR 1% + API 4 12.76 ± 0.05 (**) 0.01320 / 0.00167 gested that mucoadhesion does not determine (clarify or
HCAR 1.5% + API 4 26.06 ± 0.05 0.01505 / 0.00470 direct) which of the HCAR samples associated with the API
HCAR 2% + API 4 34.96 ± 0.05 (**) 0.01665 / 0.00797 would be a better choice. Likewise, it was found that the API
HPLU 20%  5–6 0.13 ± 0.05 a (***) 0.01345 / benefited the HCAR mucoadhesive properties, such that the
0.00195 (****) significant difference that existed between HCAR 2% and
HPLU 30% 5–6 0.30 ± 0.00 a 0.03305 / HCAR 1% samples was not observed for samples HCAR
0.01452 (****) 2% + API and HCAR 1% + API. The hydrophilic potential
HPLU 40% 5–6 33.03 ± 2.40 a (***) 0.02260 / 0.01087 may be a characteristic of the APIs added into mucoadhesive
HPLU 20% + API 1 4 0.50 ± 0.00 a (***) 0.00810 / 0.00155 (*) gels, compromising the mobility of the polymer chain and
HPLU 30% + API 1 5 2.50 ± 0.17 a 0.01770 / 0.00430 the intimate contact between mucin and the gel surface [50],
HPLU 40% + API 1 5 67.13 ± 14.75 a (***) 0.01770 / 0.00592 in contrast to the result of the association of the API with
a Viscosity measured with samples in ice bath
HCAR in this study.
(*) ( p < 0.001)
The HPLU samples not associated with the API revealed
that the HPLU 30% sample possessed the highest adhe-
(**) ( p = 0.02)
sion strength, followed by HPLU 40%, and HPLU 20%
(***) ( p = 0.018)
(p = 0.001). Tukey's post hoc showed that the difference in
(****) ( p = 0.001)
the mucoadhesive strength of HPLU 30% was significantly
greater than HPLU 20% (p = 0.001).
As for the HPLU samples associated with the API, it was
significant difference (p = 0.018). This significant differ- observed that the results continue to maintain the adhesion
ence was maintained for the HPLU 20% + API, and HPLU strength order between samples, so that HPLU 30% + API
40% + API samples (p = 0.018). It is thus assumed that the presented the highest adhesion strength, followed by
association of the API with HPLU did not change the vis- HPLU 40% + API, and 20% HPLU + API (p = 0.001). Tuk-
cosity of the hydrogel, characterizing stability in the sys- ey's post hoc showed that there is a difference between
tem. Therefore, HPLU 30% + API and HPLU 40% + API HPLU 20% + API, and HPLU 30% + API (p < 0.001), and
presented better performance in terms of viscosity as HPLU 40% + API (  p < 0.001).  There was no difference
against the PLU hydrogel samples. between HPLU 30% + API and HPLU 40% + API. Under
No statistically significant difference was observed when these conditions, in terms of mucoadhesion strength, the
comparing the hydrogel samples presenting the best perfor- HPLU 30% + API and HPLU 40% + API samples are simi-
mance in terms of viscosity. lar.  Pagano [51] suggested that different concentrations
Evaluating mucoadhesion for the HCAR hydrogels not between pluronic hydrogels can increase viscosity with
associated with the API, it was observed that the HCAR consequent impairment of mucoadhesion, differing from
2% sample presented the highest adhesion strength, fol- the results of this study. However, the interaction between
lowed by HCAR 1.5%, and HCAR 1% ( p = 0.001). The pluronic and other mucoadhesive polymers may be a good
adjusted significance value after multiple comparisons strategy to improve the mucoadhesion of the API-carrying
using the Kruskal–Wallis test indicates that the difference is system [52].
between the 2% HCAR and 1% HCAR samples (p = 0.001). Due to its thermo-reversible characteristics, the viscosity
The difference is significantly greater for the 2% HCAR of the HPLU samples was also evaluated in function of tem-
( p = 0.001). Thus, mucoadhesion strength was similar in perature. Table 7 presents viscosity results for pure HPLU,
the HCAR 2% and HCAR 1.5% samples. and the API association in function of temperature.
Under these study conditions, the mean sample val- Pagano [51] investigated the sol–gel phase transition
ues revealed that concentration is directly proportional to process of thermogel-based formulations in the treatment
mucoadhesion strength in CAR hydrogels when not associ- of mucositis, determining a temperature range from 15° to
ated with the API. This may be related to the properties of 37 °C to observe this event, and considering that the oral

13
Biological evaluation and compatibility study of oral mucoadhesive hydrogel formulations…

cavity has a lower temperature of around 32 °C, for being in 40% + API sample, the hydrogel formed at a temperature of
contact with the external environment. 14 °C was observed. Within the parameters of the study, the
When choosing a mucoadhesive polymer to develop for- samples did not present measurable viscosity percentages
mulations for treatment of antifungal or antimicrobial dis- due to the gel consistency of the samples not being measured
ease, poloxamer-based gel thermoreversion is a favorable by the equipment.
property. Poloxamer-based gels present promise in the treat- According to Giuliano [53], poloxamers retain a promis-
ment of periodontal disease, with satisfactory gel formation, ing thermoreversible property for releasing APIs. The sol
viscosity and bioavailability of APIs, positively interfering phase facilitates manipulation and administration of the
in the frequency of the therapeutic dose and improving hydrogel, and during the sol–gel transition combined with
patient compliance [47]. mucoadhesive polymers, it establishes a prolonged and sus-
In view of these considerations and according to the tained release of trapped APIs, decreasing the needed dos-
results of Table 7, analyzing the HPLU samples not asso- age, side effects, and number of administrations.
ciated with the API, it was observed that the HPLU 20% Considering that viscosity interferes with fluidity and
sample presented the lowest viscosity percentage (20.6%) permanence of the hydrogel at the treatment locale, it can
and the HPLU 40% sample presented the highest viscosity be inferred that depending on the temperature, both HPLU
percentage (98.8%); in function of temperature, and consid- 30% + API and HPLU 40% + API present good viscosities,
ering the maximum gelation percentage measured by the favorable for use in the oral cavity.
equipment as 100%.
When evaluating the concentrations of HPLU associated
with the API, it was observed that in the HPLU 20% + API Hydrogel antifungal activity
sample there was a reduction in viscosity percentage in func-
tion of temperature, compared to the HPLU 20% sample. Table 8 presents the antifungal activity of diluted and undi-
This was observed starting at 23ºC. A reduction in viscosity luted HCAR and HPLU associations with the API.
may cause greater sample flow and less contact permanence Initially, when evaluating the HCAR samples associ-
at the treatment locale. ated with the API, it was observed that all of the samples
In the HPLU 30% + API and HPLU 40% + API sam- presented inhibition halos greater than 10 mm, both for the
ples, the association of the API with this excipient did diluted and undiluted hydrogel samples. According to the
not reduce the hydrogel viscosity in function of tempera- categorization proposed Miranda-Cadena [36],  C. albi-
ture. In the HPLU 30% + API sample, the transition from cans was considered susceptible to all samples, except the
the sol phase to the gel phase was observed immediately undiluted 2% HCAR + API sample, which was considered
after removal from the refrigerator (14 °C), with the appear- as possessing intermediate susceptibility.
ance of a hydrogel at f 15 °C, without heating. In the HPLU Analyzing the undiluted HCAR samples, there was a sta-
tistically significant difference between all: HCAR 1% + API
and HCAR 1.5% + API (p = 0.03); 1% HCAR + API and 2%
Table 7   Viscosity of pure HPLU and when associated with the API, HCAR + API (p ˂0.001); and 1.5% HCAR + API and 2%
in function of temperature HCAR + API ( p = 0.03). Thus, undiluted 1% HCAR + API
Samples Temperature (°C) Viscosity (%) presented better antifungal activity than the other undiluted
HCAR samples.
HPLU 20% 14−22 0.1−0.4
Analyzing the diluted HCAR samples, a statistical dif-
23−26 1.7−4.9
ference was observed between the: 1% diluted HCAR + API
27−32 20.4−20.6
and 1.5% diluted HCAR + API (p = 0.03); and the 1% diluted
HPLU 30% 14−15 0.4−0.8
HCAR + API and 2% diluted HCAR + API (p ˂0.001) sam-
16 1.2−10.1
ples. Thus, the 1% diluted HCAR + API presented better
17 25.1−65.5
antifungal activity than the other diluted HCAR samples.
HPLU 40% 14 38.9−72.8
Comparing the HCAR samples with the best antifungal
15 74.9−87.3
activity, 1% diluted HCAR + API was the most effective,
16 93.2−98.8
suggesting that against C. albicans, and under the conditions
HPLU 20% + API 14−22 0.5−0.8
of this study, dilution may improve the antifungal activity of
23−26 0.8−1.2
carbopol hydrogels (p = 0.005).
27−32 2.6−17.1
Considering the HPLU samples, it was observed that C.
HPLU 30% + API 14−15 hydrogel formed at
albicans presented intermediate susceptibility against all
HPLU 40% + API  14 hydrogel formed at
samples, except for the 40% undiluted HPLU + API, for
a
  Viscosity was not measurable in the equipment reading parameters which C. albicans was considered resistant.

13

Table 8    Antifungal activity of diluted and undiluted HCAR and
HPLU-API associations
Samples Undiluted hydrogel Diluted hydrogel (1:1)
Halos Diameters (mm ± SD)
HCAR 1% + API 17.00 ± 0.00 19.22 ± 0.44
HCAR 1.5% + API 15.00 ± 0.00 16.77 ± 0.44
HCAR 2% + API 12.88 ± 0.33 15.22 ± 0.44
HPLU 20% + API  10.00 ± 0.00 12.00 ± 0.70
HPLU 30% + API  10.00 ± 0.00 11.00 ± 0.00
HPLU 40% + API 0.00 10.00 ± 0.00
Analyzing the undiluted HPLU samples, it was observed
that the HPLU 20% + API and HPLU 30% + API samples
presented similar antifungal activity while HPLU 40% + API
presented no antifungal activity. This result may be related
to the pluronic concentration in the hydrogel, affecting vis-
cosity and thus sample flow and permeability in the culture
medium.
Analyzing the diluted HPLU samples, a statistically sig-
nificant difference was observed between the HPLU 20%
diluted + API and HPLU 40% diluted + API (p < 0.001); 30%
diluted HPLU + API and 40% diluted HPLU + API
(p = 0.01) samples. Thus, the 20% diluted HPLU + API, and
30% diluted HPLU + API samples presented better antifun-
gal activity than the other diluted HPLU samples.
Comparing the HPLU samples with the best antifungal
activity, 20% diluted HPLU + API ( p < 0.001) and 30%
diluted HPLU + API ( p = 0.003) were the most effective,
suggesting that dilution may have improved antifungal
activity.
Pluronic hydrogels have been investigated as a promising
alternative for API transport, potentiating antifungal activity
through API diffusion control, and providing longer contact
time with C. albicans [54]. Pluronic hydrogels were found
to be active against bacteria and fungi associated with eye
infections; carrying bioactive compounds [55] and maintain-
ing desirable release profile characteristics the API against
periodontal disease [56]. This corroborates the results of
this study, bringing forth a promising carrier of API with
antifungal activity against C. albicans.
Conclusions
The API from S. brasiliensis leaves presented antifungal
potential against strains of C. albicans, a pharmacological
potency considerably superior than the gallic acid chemi-
cal marker. Synergism between phenolic compounds, char-
acteristic of this plant species, may have influenced this
result. Under our study conditions, Carbopol and Pluronic
13
T. P. D. Sampaio et al.
F127 were the most compatible excipients for the formu-
lation of mucoadhesive hydrogels with S. brasiliensis. The
combination of thermoanalytical and spectroscopic tech-
niques allowed identification of these compatibilities and
demonstrates the relevance of analytical instruments in
pre-formulation studies. The mucoadhesive hydrogel com-
posed of 30% pluronic F127, associated with the S. bra-
siliensis API, presented the best performance and should
be further investigated as a potential antifungal agent for
oral use. Still more studies should be performed in order to
elucidate potential API mechanisms of action (for S. brasil-
iensis leaves) on multispecies biofilms, bioactive compound
release profiles, and cytotoxicity in oral epithelial cells.
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