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Journal of Thermal Analysis and Calorimetry

, Volume 133, Issue 1, pp 641–648 | Cite as

Investigation of the thermal behavior of inclusion complexes with antifungal activity

  • Fernando José de Lima Ramos Júnior
  • Karla Monik Alves da Silva
  • Deysiane Oliveira Brandão
  • José Venâncio Chaves Junior
  • Jonh Anderson Borges dos Santos
  • Fabrício Havy Dantas de Andrade
  • Rayanne Sales de Araújo Batista
  • Taynara Batista Lins
  • Damião Pergentino de Sousa
  • Ana Cláudia Dantas Medeiros
  • Marta Maria Conceição
  • Rui Oliveira Macêdo
  • Fábio Santos de Souza
Article
  • 136 Downloads

Abstract

The indiscriminate use of antifungal agents has led to the advancement of microorganisms tolerant to the various drugs known in the market. Therefore, the search for new compounds and new technologies capable of giving more stable formulations and better pharmacological activities is of paramount importance as an alternative for the development of new therapeutics. However, its effectiveness is directly related to the knowledge of its characteristics in the solid state. Thus, the objective of this work was evaluating the thermal behavior, physicochemical aspects and microbiological of the complexes of inclusion of β-cyclodextrin (BCD) and biphenyl-4-methyl carboxylate (B4CMET). Therefore, differential scanning calorimetry, thermogravimetry, Fourier-transformed infrared spectroscopy, X-ray diffraction and microbiological assay were used to generate robust fingerprint of the inclusion complexes. The results showed the formation of inclusion complexes of B4CMET with βCD, thereby stressing its greater stability and potential use as an antifungal agent against Candida spp. Thus, with thermoanalytical techniques it was possible to observe the increased thermal stability, with FTIR changes of characteristic bands were verified, with XRD the disappearance of diffraction peaks of the B4CMET was verified, and with the microbiological assay it was possible to visualize increased antifungal activity.

Keywords

β-Cyclodextrin Thermoanalytical techniques Antifungal agent Biphenyl-4-methyl carboxylate 

Introduction

The increase in the incidence diseases caused by genus Candida, the high toxicity of the commercial antifungals and the high degree of fungal resistance have contributed to the research of new drugs, in which it becomes alternatives to recent therapeutics. However, its effectiveness is directly related to the knowledge of its characteristics in the solid state [1, 2, 3].

In this way, the control and the knowledge of the characteristics of active pharmaceutical ingredient (API) as products formulated or pure substances is an important step in the development of safe and effective new medicines; thus, aspects as the bioavailability, expiration date of a formulation and its industrial processing are modified by the drug properties [3, 4, 5, 6, 7, 8].

In the pharmaceutical sector, the use of cyclodextrin as a good and promising tool in order to control the API characteristics has been widely employed because it shows complexing properties that result in higher solubility, dissolution and bioavailability of poor soluble drugs and also improves stability and organoleptic properties, reduces adverse effects and prevents interactions between some drugs and/or between drug and excipients [9].

On the other hand, for knowledge of characteristics of solid pharmaceuticals, methods such as differential scanning calorimetry (DSC), thermogravimetry (TG), Fourier-transformed infrared (FTIR) spectroscopy and X-ray diffraction can be employed [3, 4, 5, 6, 7, 8].

The differential scanning calorimetry (DSC) and thermogravimetry (TG) contribute to obtaining important information that makes it possible to evaluate the thermal stability, polymorphisms, incompatibility and degradation kinetics of the pharmaceutical products, and the Fourier-transformed infrared spectroscopy provides information about the identification of compounds or the investigation of a sample, since the chemical bonds of the substances have specific vibrational frequencies [10, 11, 12, 13, 14, 15, 16, 17, 18].

Thus, the objective of this work was evaluating the thermal behavior, physicochemical aspects and microbiological of the complexes of inclusion of β-cyclodextrin and biphenyl-4-methyl carboxylate.

Materials and methods

Materials

β-Cyclodextrin (βCD) was purchased from SIGMA ALDRICH®, and three batches of the biphenyl-4-methyl carboxylate (B4CMET) were provided by the Medicinal Chemical Laboratory/DCF/CCS/UFPB.

Preparation of the inclusion complexes (IC)

Equimolar amounts of the three batches of B4CMET and βCD were weighed and solubilized separately under mechanical stirring. B4CMET was solubilized in ethyl alcohol and βCD in purified water. After complete solubilization of the compounds, the B4CMET solution was poured into the βCD solution and kept under stirring for 24 h before being subjected to evaporation under reduced pressure (− 800 ± 20 mbar) at 45 ± 5 °C in a rotary evaporator. After evaporation of the solvents, the products were placed in a drying oven at 40 °C for 24 h.

Thermal behavior

Differential scanning calorimetry

The DSC curves were obtained in a calorimeter equipment (Shimadzu, DSC-50), using a hermetically sealed aluminum crucible, with approximately 2.0 mg of the sample. The analysis was performed in the temperature range of 25–450 °C, at heating rates of 10 °C min−1 (B4CMET and βCD) and 2, 5, 10 and 15 °C min−1 (IC), under nitrogen atmosphere (flow rate of 50 mL min−1). DSC data were analyzed by the software Tasys® (Shimadzu).

Thermogravimetry

Dynamic thermogravimetric curves were obtained using a Shimadzu thermobalance, 50H TGA under an atmosphere of synthetic air at a flow rate of 20 mL min−1. The samples were placed in an alumina crucible, heated at a heating rate of 10 °C min−1 (B4CMET and βCD) and heating rate of 10, 20 and 40 °C min−1 (IC) up to 900 °C. The mass was 5.0 mg (± 0.003).

Physicochemical aspects

Fourier-transformed infrared spectroscopy

FTIR spectra were obtained by using a Shimadzu IR Prestige-21. The samples were previously prepared with potassium bromide in a ratio of about 2:100 (samples/KBr). The KBr disks were prepared by powder compression in a hydraulic press, at a pressure of 9 tons, and scanned against the KBr white in wave numbers in the range of 400–4000 cm−1.

X-ray diffraction

XRD spectra were recorded with a Shimadzu XRD 600 diffractometer, with Cu Kα radiation source. The measurement conditions were as follows: geometry, 2θ; voltage, 40 kV; current, 30 mA; θi, 5; θf, 30; step size, 0.02; and speed, 20° min−1.

Microbiological aspects

Determination of the antifungal activity

In order to evaluate the antifungal activity, solutions of the three batches of B4CMET and of the three batches of the inclusion complexes (IC) at the concentration of 2000 µg mL−1 in 50% ethanol were obtained and standard strains American Type Culture Collection (ATCC) Candida albicans (ATCC 18804), Candida tropicalis (ATCC 13803) and Candida parapsilosis (ATCC 22009) were used, which were made available by the Oswaldo Cruz Foundation (Fiocruz, RJ).

The microorganisms were maintained in test tubes containing Sabouraud agar, and then, they were cultured in plates containing the same medium, at 25 °C for 48 h. The determination of the minimum inhibitory concentration (MIC) was performed by the broth microdilution method, using 96-well microplates according to the procedures of the Clinical and Laboratory Standards Institute (CLSI) [19]. The inoculum was standardized into tubes containing 5 mL of sterile saline solution at 0.9%. The suspension was adjusted by spectrophotometer at 580 nm in a concentration equivalent to 103 UFC. Hundred microliters of each solution to 2000 µg mL−1 was subjected to serial dilution on Sabouraud broth in the microplates, obtaining concentrations of 1000 to 7.81 μg mL−1. Subsequently, 10 μL of each microorganism culture was added to each well. Fifty percent ethanol was used as a negative control, and Nystatin oral suspension 100.000 U I mL−1 as positive control. Fungal growth was indicated by the addition of 20 μL of aqueous resazurin (Sigma-Aldrich®) to 0.01%, with an additional incubation at 25 °C ± 1 °C for 2 h. The viable microorganisms modify the dye color from blue to pink. Thus, the MIC was defined as the lowest concentration in which no change of the color was noted in the dye.

Results and discussion

Thermal behavior

Differential scanning calorimetry

It was observed in the DSC curves of the inclusion complexes and their isolated components (Fig. 1) three events: one endothermic event to the βCD in the range of 99.11–127.25 °C (Tpeak = 115.03 °C),this finding can be related to dehydration of the molecule; another in the range of de 218.69–224.21 °C (Tpeak = 221.08 °C), related to the glass transition, this fact is already reported by Serafini [20] and Menezes [21], and another in the range of 311.16–336.78 °C (Tpeak = 323.09 °C) in which it was related to the βCD melting followed by its thermodecomposition. In the DSC curves of the B4CMET, only one endothermic event was observed that was related to the molecule melting. This event was found in the range of 117.65–123.91 °C (Tpeak = 120.30 °C) for the batch 1, in the range of 118.88–125.66 °C (Tpeak = 121.78 °C) for the batch 2 and in the range of 119.33–125.82 °C (Tpeak = 121.67 °C) for the batch 3. Additionally, in the inclusion complexes, the drug melting was not viewed, evidencing the encapsulation of this molecule in the cavity of the βCD with the formation of the complexes and not of a physical mixture.
Fig. 1

DSC curves of the biphenyl-4-methyl carboxylate, β-cyclodextrin and inclusion complexes at different heating rates

The DSC technique is employed as an important analytical tool in the characterization of drug and βCD interactions. Moreover, it is known that the molecules when are incorporation into the βCD cavity cease to interact with each other in the solid state and events such as melting point, boiling and sublimation are modified or simply disappear [22, 23, 24].

Thermogravimetry

In the TG curves (Fig. 2), it was possibly viewed to the βCD that in the range of 99.11–127.25 °C occurred a mass loss event of 12.34%, this loss was related to the release of water molecules from the βCD cavity, confirming the results obtained in the DSC curve (Fig. 1). Then, it was checked a thermal stability in the range of 128.00–311.16 °C. However, above 311.00 °C in the range of 311.16–361.85 °C, it was found a mass loss of 69.03%, in which it was related to the fast melting/decomposition of the βCD. Additionally, in the range of 518.00–900.00 °C it was observed a mass loss of 5.70%, related to the final decomposition.
Fig. 2

TG curves of the biphenyl-4-methyl carboxylate, β-cyclodextrin and inclusion complexes at a heating rate of 10 °C min−1

It was observed in Fig. 2 that the B4CMET showed a mass loss event related to the volatilization of the molecule in the range of 213.27–241.21 °C (Δm = 94.15%) to the batch 1, in the range of 220.79–254.13 °C (Δm = 94.11%) to the batch 2 and in the range of 220.20–254.00 °C (Δm = 94.10%) to the batch 3. This research finding indicated that three batches of B4CMET showed the same thermal behavior.

According to Fig. 2, in the all inclusion complexes two mass loss events can be observed in the temperature range of 44.11–178.41 °C, indicating the loss of water from the solvation of the complex cavity and the release of a small amount of B4CMET, respectively. In the range of 259.64–367.30 °C occurred a higher mass loss event probably with the release of B4CMET included. Finally, in the range of 486.06–900.00 °C an additional mass loss was detected, 11.17% for the batch 1, 4.4% for the batch 2 and 6.75% for the batch 3. This shows an increase in thermal stability for the encapsulated B4CMET, probably as a result of intermolecular interactions with the βCD cavity.

Kinetic study of thermal degradation

Table 1 shows the kinetic parameters for B4CMET and ICs. Comparison of the activation energy values for the three batches of pure B4CMET was found to be lower than the values obtained for this substance when associated with βCD (inclusion complexes). Thus, the energy barrier to the B4CMET volatilization reaction became relatively higher when that substance was associated with βCD, which confirms the increase in the thermal stability of B4CMET in the inclusion complexes, which was observed in the TG curves.
Table 1

Kinetic parameters of the B4CMET and IC by Ozawa method with decomposition fractions α0.1 and α0.9

 

Activation energy/kJ mol−1

Order reaction

Frequency factor/min

B4CMET

 Batch 1

78.64

0

4.50 × 107

 Batch 2

72.29

0

5.22 × 106

 Batch 3

75.21

0

1.07 × 107

IC

 Batch 1

126.98

0

2.394 × 107

 Batch 2

121.82

0

1.030 × 1010

 Batch 3

120.73

0

8.765 × 108

Physicochemical aspects

Fourier-transformed infrared spectroscopy

Infrared spectroscopy allows to determine the complexation of guest molecules with cyclodextrin by observing changes in their peaks in terms of shape, position and intensity [22].

In the FTIR spectra (Fig. 3), it was possible to visualize for βCD, symmetric and asymmetric stretch of the OH group at 3346 cm−1, stretch of the C–H bond at 2928 cm−1, deformation of the -OH group of water molecules complexed with βCD at approximately 1640 cm−1, C–OH absorption corresponding to the modes of OH deformation in the plane of βCD at 1200–1500 cm−1 and asymmetric stretching of COC at 800–1200 cm−1 and βCD ring vibrations at 400–800 cm−1.
Fig. 3

FTIR spectra of the biphenyl-4-methyl carboxylate, β-cyclodextrin and inclusion complexes

For the B4CMET (Fig. 3), in the three batches, were observed axial deformation vibrations of aromatic CH at approximately 3000 cm−1, axial deformation vibrations of the double bond of C=O at 1740–1750 cm−1, C=C vibrations of the aromatic nuclei at 1450–1600 cm−1, deformation of the C–O bond of the ester at 1050–1300 cm−1 and the angular deformation of adjacent 3H of metasubstituted aromatic rings 710, 750 and 810 cm−1.

All spectra of inclusion complexes (Fig. 3) showed a strong similarity to the free βCD spectrum, although the existence of bands characteristic of B4CMET at approximately 3000, 1745, 1460, 1292, 1273 and 750 cm−1 was also observable, with exhibited markedly less intensity than spectra of the isolated molecule. That result could relate to the insertion of the B4CMET molecule into the βCD cavity, which causes a conformational restriction that reduces its free movement and, in turn, the signal intensity. Along with that effect, the OH and C–H stretching bands of βCD were displaced at 3346–3340 and 2928–2920 cm−1, respectively. Such results reinforce data obtained in DSC and TG regarding the formation of inclusion complexes.

X-ray diffraction

Further information on the structure of the inclusion complexes was obtained with the use of XRD (Fig. 4). It was observed that the pure B4CMET presented a more crystalline structure when compared to the inclusion complexes, since in the complexes occurred the disappearance of the peaks of diffraction of B4CMET. This difference represents a loss of crystallinity with the formation of a less organized system, indicating that the formation of inclusion complexes occurred with the interaction between B4CMET and βCD.
Fig. 4

Diffractogram of the biphenyl-4-methyl carboxylate, β-cyclodextrin and inclusion complexes

Microbiological aspects

Antifungal activity

In evaluating antifungal activity, it was possible to observe a considerable improvement of antimicrobial activity in the inclusion complexes compared to that presented by the isolated B4CMET, as evidenced by the decreased minimum inhibitory concentration (Table 2).
Table 2

Minimum inhibitory concentration of the B4CMET and IC tested against Candida spp

 

Microorganisms tested

Candida albicans/µg mL−1

Candida tropicalis/µg mL−1

Candida parapsilosis/µg mL−1

B4CMET

 Batch 1

250.00

125.00

125.00

 Batch 2

250.00

125.00

125.00

 Batch 3

250.00

125.00

125.00

IC

 Batch 1

62.50

31.25

31.25

 Batch 2

62.50

31.25

31.25

 Batch 3

62.50

31.25

31.25

The increased activity of IC may be due to the higher solubility in culture medium (agar) of B4CMET from IC, since a compound’s increased solubility contributes to raising the substance’s area of contact with the microorganism, thereby favoring its action [25].

The increased action observed with the inclusion complexes of B4CMET corroborates the results of Kfoury et al. [26], who, when assessing the antifungal activity of several free substances and in complexes with cyclodextrin, observed improved inhibition when the inclusion complexes were tested. Similarly, Zang et al. [27] observed the antioxidant activity of inclusion complexes of extracts of Chimonanthus praecox, which marked an improvement in pharmacological activity.

Conclusions

The results showed the formation of inclusion complexes of B4CMET with βCD, thereby stressing its greater stability and potential use as an antifungal agent against Candida spp. Thus, with thermoanalytical techniques it was possible to observe the increased thermal stability, with FTIR changes of characteristic bands were verified, with XRD the disappearance of diffraction peaks of the B4CMET was verified, and with the microbiological assay it was possible to visualize increased antifungal activity. Such results suggest that inclusion complexes of B4CMET with βCD can be promising aids for developing future stable and effective pharmaceutical forms, representative of an alternative treatment for fungal disease.

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Copyright information

© Akadémiai Kiadó, Budapest, Hungary 2018

Authors and Affiliations

  • Fernando José de Lima Ramos Júnior
    • 1
    • 2
  • Karla Monik Alves da Silva
    • 1
    • 2
  • Deysiane Oliveira Brandão
    • 1
    • 2
  • José Venâncio Chaves Junior
    • 2
  • Jonh Anderson Borges dos Santos
    • 2
  • Fabrício Havy Dantas de Andrade
    • 1
    • 2
  • Rayanne Sales de Araújo Batista
    • 2
  • Taynara Batista Lins
    • 2
  • Damião Pergentino de Sousa
    • 3
  • Ana Cláudia Dantas Medeiros
    • 4
  • Marta Maria Conceição
    • 5
  • Rui Oliveira Macêdo
    • 1
    • 2
  • Fábio Santos de Souza
    • 1
    • 2
  1. 1.Programa de Pós-graduação em Ciências Farmacêuticas, Departamento de Ciências FarmacêuticasUniversidade Federal de PernambucoRecifeBrasil
  2. 2.Laboratórios Unificados de Desenvolvimento e Ensaios de Medicamentos, Departamento de Ciências FarmacêuticasUniversidade Federal da ParaíbaJoão PessoaBrasil
  3. 3.Laboratório de Química Medicinal, Departamento de Ciências FarmacêuticasUniversidade Federal da ParaíbaJoão PessoaBrasil
  4. 4.Centro de Ciências Biológicas e da Saúde, Laboratório de Desenvolvimento e Ensaio de MedicamentosUniversidade Estadual da ParaíbaCampina GrandeBrasil
  5. 5.Centro de Tecnologia e Desenvolvimento RegionalUniversidade Federal da ParaíbaJoão PessoaBrasil

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