The species Protium heptaphyllum
(Burseraceae) is popularly known in Amazon, Brazil as “breu-branco” and “almecegueira” [1
]. From the trunk of its trees is obtained a natural exuded oil-resin that is rich in volatile monoterpenes and triterpenes, especially α and β amirin (ABAM) (C30
]. Having a wide range of biological activities, triterpenes have aroused clinical interest [4
]. ABAM is a well-known natural mixture of isomeric triterpenes whose pharmacological properties include anti-inflammatory [5
], gastroprotective [6
], antitumor [7
], anxiolytic [8
] and hepatoprotective [6
]. The anti-inflammatory activity of ABAM has been demonstrated in different experimental models through the inhibition of the release of pro-inflammatory cytokines (IL-6, TNF-α and IL-1β) and the enzyme myelopereroxidase [6
Considering that ABAM has a pronounced lipophilicity and that in vivo studies using rats have shown low bioavailability for both its isolated form (0.86%) and its extract (3.83%) [10
], it becomes important to develop a system that is capable of enhancing the physicochemical properties of ABAM while making this new chemical entity feasible to be used as a new drug.
Cyclodextrins (CDs) are cyclic oligosaccharides of a variable number of glucose units, linked by α-(1,4) glycosidic bonds. They are characterized by a hydrophobic cavity and a hydrophilic exterior, and for this reason, CDs are able to host hydrophobic molecules in their interior, therefore, forming the so-called inclusion complexes (ICs) [11
]. Some drugs are already marketed in Europe, Japan, United States and Brazil as complexes with cyclodextrins, which includes dexamethasone, nimesulide, omeprazole, piroxicam, indomethacin and itraconazole [12
CDs have the ability to bind to the host molecule by non-covalent interactions in both aqueous and solid states, where such interactions have been capable of modifying the physicochemical properties of the bound molecule, which results in greater safety, solubility, stability and bioavailability [14
]. Several studies have demonstrated the ability of CDs in enhancing the safety, bioavailability and pharmacological activity of substances with anti-inflammatory properties [15
The aim of this work was to develop inclusion complexes (ICs) of ABAM with β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin (HPβCD) with the purpose of improving the physicochemical properties of ABAM. In addition, the in vitro anti-inflammatory activity of ABAM-CDs complexes was investigated using lipopolysaccharide-stimulated mouse macrophage cell line J774.
Complexation with cyclodextrins is one of the most commonly used strategy to increase the solubility, stability and safety of poorly soluble molecules [11
]. Thus, the physicochemical characterization of such complexes is of utmost importance in order to prove that complexation has taken place. In this current study, physicochemical characterization in addition to in vitro assays were carried out with the purpose of evaluating the feasibility of the formation of inclusion complexes between ABAM and cyclodextrins as well as the resulting improvement of its anti-inflammatory activity.
FTIR spectroscopy allows the identification of vibrational patterns of CDs and host molecules as well as changes in the characteristic bands of the ligand’s vibrational pattern such as disappearance, widening, changes in peak intensity or deviations in their wavenumbers. These changes may be a strong indicative of interaction between the host molecule and the CDs [37
]. When an inclusion complex (IC) is formed between a molecule and CD, this can mask or hinder the characteristic peaks of the host molecule, especially when the complexation occurs within the internal cavity of the CD [38
]. In this study, the characteristic bands of ABAM were masked as a result of ABAM-CDs complexation through PM and KND methods. The masking of the bands attributed to the host molecule, even when the drug-CD complex is prepared by simple PM, usually means that the drug is complexed inside the CD cavity [39
]. A similar result was reported by Quintans [40
], where a sapogenin was complexed with βCD and the bands attributed to the sapogenin were masked after the IC was obtained by PM.
SEM is widely used to morphologically identify the formation of inclusion complexes due to the fact that such complexation usually involves changes in the surface, particle size and appearance of the parent drug [41
]. When a drug is complexed within the CD cavity, the morphology of the obtained IC tends to be distinct from that of each individual component [42
]. As shown in Figure 2
, the morphological aspects of both ABAM- βCD and ABAM- HPβCD were markedly different from those of ABAM, βCD and HPβCD.
XRD determines the crystalline nature of solids and therefore, has been used for the characterization of ICs [44
]. By comparing the diffractograms of each individual substance with those of the corresponding ICs one can infer that complexation has taken place if changes between the diffractograms are observed [45
]. In this current work, the characteristic crystalline profile of ABAM was changed after complexation with CDs by both methods of preparation, whose decrease in the crystalline profile might result in an enhancement of the ABAM’s aqueous solubility.
Thermal analyses aim to investigate the thermal behavior of compounds submitted to variations of heating and cooling in a controlled manner as defined in 2006 by the International Confederation for Thermal Analysis and Calorimetry [47
]. TG evaluates mass losses due to heating as a function of time and therefore, has been used to identify the formation of ICs as evident changes in the mass loss profile are observed when a CD complexes with a host molecule [37
]. On the other hand, DSC is used to identify melting, degradation and recrystallization processes. In most cases, molecules in the crystalline state, such as ABAM, exhibit a melting event that can be evidenced as an endothermic peak. When ICs are formed, this endothermic peak disappears due to the loss of the crystalline structure as a result of the complexation [37
]. Even though the ICs still present a small event related to the ABAM’s melting, the observed event is of lower intensity, as confirmed by the low enthalpy, therefore, it can be suggested that such complexation favored a greater thermal protection to ABAM.
The results imply that the formation of inclusion complexes between ABAM and the two cyclodextrins improved the physicochemical properties of ABAM. Therefore, the in vitro anti-inflammatory model and the MTT assay were performed with the purpose of evaluating the viability of these inclusion complexes.
Inflammation is a natural and pathophysiological response of the body against lesions and infectious agents [49
]. However, inflammation becomes harmful when the process progresses and becomes a chronic condition, which usually requires pharmacological intervention. Nitric oxide (NO●
) is one of the main mediators of inflammation and its overproduction is associated with chronic inflammatory diseases. Therefore, NO●
has been used as the main marker of inflammation.
The MTT assay is commonly used to assess cell viability and proliferation by quantifying a blue-violet product called formazan crystal that is formed by enzymatic reduction in the cytoplasm of viable cells, which means that the higher its quantification, the greater the cell viability [50
]. In this study, it was demonstrated that ABAM at the concentration of 20 μg/mL was able to inhibit the production of nitric oxide in LPS-stimulated macrophages J774 cells. However, considering that the inclusion complexes were obtained at a molar ratio of 1: 1 (ABAM:CD), we can infer that ABAM-CD has a higher NO●
inhibition activity in comparison with ABAM alone due to the fact that its concentration is twice of that in the ABAM-CD system, which means that although the concentration of ABAM in each sample was 20 µg·mL−1
, and its concentration in the ICs was around 10 µg·mL−1
. In addition, the MTT assay showed that all the analyzed samples did not interfere significantly in the cell viability, which seems to indicate lack or very low toxicity.
Thus, the formation of inclusion complexes with cyclodextrins seems to be an effective and promising alternative to enhance the safety of ABAM.