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2.5.7. Scanning electron microscopy (SEM)
The morphological characteristics of the samples were analyzed
in the electron microscope Shimadzu SSX-550 Superscan to visu-
alize the surfaces of Rip A, b-CD, PM and Rip-A/b-CD. The particles
were fixed in metallic support under vacuum with subsequent
metallization by a thin gold film. Microphotographs were acquired
in different magnifications by acceleration voltages from 8 to 15 kV.
2.6. Artemicidal cytotoxic assay
Larvae of Artemia salina Leach were treated with Rip A and Rip
A/b-CD complex according to Meyer et al. [20]. Briefly, A. salina eggs
were hatched in a synthetic saline (NaCl 36 g/L) at 25 ꢀC with
continuous aeration. Following 48 h of hatching, nauplii were
collected (n ¼ 10 nauplii/sample/concentration) and samples (Rip
A,
b-CD and Rip-A/b-CD complex) were added at the concentrations
of 50 50, 100, 250, 500 and 1000
mg/mL. Potassium dichromate
(K2Cr2O7) 0.1% was used as positive control. After 24 h exposure,
death nauplii were quantified to obtain CL50 (average lethal con-
centration) values. Experiments were performed in triplicate.
2.7. Statistical analysis
All results were presented as mean standard error of mean
(SEM). Data were evaluated by analysis of variance (ANOVA) fol-
lowed by Student-Neuman-Keuls. Differences were considered
significant when p < 0.05. CL50 values and their 95% confidence
intervals were obtained by nonlinear regression (GraphPad pro-
gram, Intuitive Software for Science, San Diego, CA).
3. Results and discussion
3.1. Phase solubility diagram
Fig. 2. UVeVis absorption spectrum of Riparin A (A) and its phase solubility diagram
(B) in the presence of -cyclodextrin.
Complexation is an interaction between a host molecule, such as
-CD, and its guest molecule, as Rip A. Recently, investigations have
b
b
been conducted to acquire inclusion complexes of active sub-
stances, such as neurotransmitters (dopamine), hormones
(epinephrine) and the neuroactive amino acid tyramine, and these
inclusions improved stability and optimized some pharmaceutical
parameters [21,22].
The method of Higuchi and Connors [15] is widely cited to carry
out phase solubility diagrams and analysis of inclusion complexes
and was used based on the increase of Rip A solubility as function of
A/
enhancement of 240.2% of Rip A solubility was found following the
addition of -CD. Solubility diagram allows the classification of
complexes in two profiles: A and B. Profile A consists of soluble
complexes and profile B represents limited solubility [24,25].
b-CD. The intrinsic solubility of Rip A (S0) 1.48 mM and an
b
Herein, inclusion complexes of Rip A/
AL.
b-CD were classified as type
increasing concentrations of b-CD. Fig. 2A details the UVeVis ab-
Type A diagrams are subdivided into i) AP: positive deviation
diagram with more effective solubilization for high concentrations
of CDs; ii) AL: linear diagram with drug solubility increasing linearly
with CDs' concentration and iii) AN: negative deviation diagram in
which CDs are proportionally less effective at higher concentrations
due to complex interactions between solute-solute and solute-
solvent. On the other hand, Type B diagrams can be of two sub-
types: i) BS: diagram with precipitation of insoluble complex with
increase of cyclodextrin after initial increase of guest substance
solubility and ii) BI: highly insoluble complexes without the pos-
sibility of initial increase in solubility of guest substances [26,27].
sorption spectrum of Rip A in a concentration-dependent way and
Rip A solubility diagram. Rip-A revealed absorption at approxi-
mately 225 nm in water:ethanol (1:9). In this wavelength, no
spectral reading of b-CD absorption was detected, indicating that
cyclodextrin does not interfere in the spectrophotometric analysis
of Rip A [21,23].
Considerations about the UVeVis scan of Rip A allowed to obtain
a calibration curve: y ¼ 0.1664x þ 0.0008 (r2 ¼ 0.9992) (Fig. 2B). A
linear increase between aqueous solubility of Rip A and
b-CD
concentrations was found, suggesting inclusion complexes' pro-
duction in the molar ratio of 1:1 (Rip A: -CD). This stoichiometric
ratio is present in most complexes obtained with -CD, in which the
b
b
guest molecule usually has a molecular weight between 100 and
400 Da, and Rip A, with 225.29 Da, has fit parameters. The stability
constant (Kst) was 373 L/mol [14,22].
3.2. Dissolution study
After 5 min of reaction, dissolution analysis showed free Rip A,
The constant Kst indicates the presence of significant interaction
PM and Rip A/b-CD complex as 18.4, 26.1 and 67.6% of the disso-
between Rip A and
b
-CD and generation of stable inclusion com-
lution, respectively (Fig. 3). Both free Rip A (61.7%) and PM (62.5%)
displayed time-dependent increasing for their percentages of dis-
solved Rip A until 60 min [18,28].
plexes, since low Kst values indicate poor interaction and big
amount of free host molecules. Ideal Kst values range from 100 to
1000 L/mol, and this was verified in the inclusion complexes of Rip
The establishment of a soluble complex was corroborated by