INTRODUCTION:

Usnic acid has the chemical name 2,6-diacetyl-7,9-dihydroxy-8,9bdimethyl-1,3(2H,9bH)-dibenzo-flurandione with the molecular formula C₁₈H₁₆O₇, with a molecular weight of 344g/mol¹^,². Known for its antimicrobial properties against both human and plant pathogens³^,⁴^,⁵, usnic acid has demonstrated antiviral, antiprotozoal, antiproliferative, anti-inflammatory, analgesic², and antitumor activities⁶.

The bioavailability and gastrointestinal absorption of usnic acid are affected by its poor solubility and high membrane permeability, since it is a class II medication in the Biopharmaceutical Classification System (BCS). The rate of dissolution is the determining factor in the oral absorption of usnic acid.

Therefore efforts are needed to increase the solubility and dissolution rate to increase the oral bioavailability of usnic acid⁷. To increase the solubility of insoluble substances, several methods can be used, namely the preparation of solid dispersions⁸^,⁹, inclusion complexation¹⁰, spherical agglomerates¹¹, nanoparticles¹², spray drying¹³^,¹⁴, crystal modification¹⁵, and polymorphism¹⁶.

The inclusion combination of usnic acid with β-cyclodextrin is one of the experiments that have been carried out to enhance the bioavailability and stability of usnic acid. When b-cyclodextrin was present, the solubility of usnic acid (7.3µg/mL) rose by more than five times, and its bioavailability was enhanced by the usnic acid inclusion complex with b-cyclodextrin¹⁷. Another study found that by using the solvent evaporation method to create cocrystals of usnic acid N-Methyl-D-Glucamine, the solubility of usnic acid may be increased by a factor of 24 and the dissolution rate by a factor of 23, respectively¹⁸ The most effective complexing agent for dissolving moss chemicals was shown to be cyclodextrin derivatives. The solubility of fumarprotocetraric acid increased the highest, almost tripling from 0.03mg/ml in water to 8.98mg/ml, with the help of 2-hydroxypropyl β-cyclodextrin¹⁹.

The drug compound's hydrophobicity, which interacts with the cyclodextrin cavity, and the drug compound's shape and size are other factors that impact the formation of inclusion complexes. The drug's stability, bioavailability, dissolving rate, and solubility are all improved by these complexes²⁰. The co-grinding method, involving milling active pharmaceutical ingredients and the carrier matrix together using ball milling or vibration milling, stands out as a commonly employed technique for reducing drug particle size²¹.

A chemical modification of cyclodextrin called hydroxypropyl-β-cyclodextrin is available for use in parenteral formulations. With a solubility of 50g/100 mL, hydroxypropyl-β-cyclodextrin is less hazardous than β-cyclodextrin²⁰. Substitution of hydroxyl groups (forming hydrogen bonds) can give a marked increase in water solubility, including random substitution with lipophilic methoxy groups. This random substitution converts the natural cyclodextrin from the crystalline phase into an amorphous mixture of physically stable isomers. Improved solubility of cyclodextrin derivatives certainly has an impact on the level of solubility of the inclusion complex formed to be better²¹.

According to previous studies, Using the co-precipitation approach, inclusion complexes containing cyclodextrins and usnic acid were synthesized. Using the co-precipitation approach, solid-state molecular inclusion complexes containing usnic acid, b-cyclodextrin, and 2-hydroxypropyl b-cyclodextrin were produced in a 1:1 mol ratio. Molecular encapsulation is the process that makes usnic acid more soluble in water. Uncomplexedusnic acid had a solubility of 0.06mg/cm³, but the inclusion complex with b-cyclodextrin had a solubility of 0.3mg/cm³ and hydroxyprofil b-cyclodextrin of 4.2mg/cm³. The solubility of usnic acid is increased by 70 times when hydroxyprofil-b-cyclodextrin is added, as previously explained²².

According to the previous explanation, the researcher is keen on investigating the co-grinding approach using a planetary ball mill to determine the development of inclusion complexes between usnic acid and hydroxypropyl-β-cyclodextrin. A variety of analytical tools were used to characterize the inclusion complexes that were produced. These tools included FT-IR, XRD, DSC, solubility tests, and dissolution profile determination.

MATERIALS AND METHODS:

Materials:

Usnic acid (STIFARM Central Laboratory Isolation Padang, Indonesia), hydroxypropyl-β-cyclodextrin (NandR industries, China), Potassium dehydrogen phosphate (KH₂PO₄) (Merck, Germany), and aquadest (PT Novalindo, Indonesia).

Formulation design:

Table 1: Design formulation of inclusion complexation 1:1, inclusion complexation 1:2, and physical mixture.

Composition	Inclusion Complexation 1:1 (mol)	Inclusion Complexation 1:2 (mol)	Physical Mixture 1:1(mol)
Usnic acid	0.344	0.344	0.344
Hydroxypropyl-β-cyclodextrin	1.4	2.8	1.4
Total	1.744	3.144	1.744

The co-grinding method was employed for the preparation of the inclusion complex involving usnic acid and hydroxypropyl-β-cyclodextrin:

The two components were blended in ratios of 1:1 and 1:2mol, and the resulting mixture underwent milling using a planetary ball milling apparatus (Retsch Type PM 100, Germany). The milling process, lasting 2 hours at 120rpm and utilizing 30 balls, facilitated the formation of the inclusion complex²³.

Preparation of a physical mixture of usnic acid and hydroxypropyl-β-cyclodextrin:

The physical combination was made using a 1:1 mol ratio of usnic acid and hydroxypropyl-β-cyclodextrin. The mortar contains usnic acid and hydroxypropyl-β-cyclodextrin. Once combined, use a spatula to whisk the ingredients together until they are completely smooth. The final physical combination is weighed after passing through a 40 mesh filter, and it is then dried in a desiccator until needed²²^,²⁴.

Fourier Transform Infra-Red (FT-IR) spectroscopic analysis:

An American-made Perkin Elmer L1600300 Spectrum Two FT-IR spectrometer was used for the study. Analysis was performed on samples of usnic acid, hydroxypropyl-β-cyclodextrin, the 1:1 inclusion complex, the 1:2 inclusion complex, and the physical combination. To ensure consistency, 10milligrams of KBr was mixed with 1 to 2milligrams of sample powder in a mortar. The subsequent step included moving the mixture to a die and applying a vacuum pressure of 800 kPa to compress it into a disc. The infrared absorption spectra of all the samples were captured by recording their spectra within the 400–4000 cm^-1wavenumber range. The purpose of this research was to uncover spectra that described the functional groups found in the compounds that were formed²⁵^,²⁶.

X-ray Diffraction (XRD) analysis:

An X-ray diffraction (XRD) apparatus from Philips X'Pert Pro-PANalytical in the Netherlands was used to do the study at room temperature. The following parameters were used for the measurements: using the following parameters: target metal Cu, filter Kα, voltage 40kV, current 30 mA, and analysis conducted within the 2 theta 5 - 35° range. During preparation, the sample was leveled in a glass sample holder to avoid particle orientation. Our objective in doing this investigation was to uncover the diffraction patterns of usnic acid, hydroxypropyl-β-cyclodextrin, the 1:1 inclusion complex, the 1:2 inclusion complex, and the physical mixture that resulted from these combinations²⁵^,²⁶.

Differential Scanning Calorimetry (DSC) analysis:

Individual substances, including usnic acid, hydroxypropyl-β-cyclodextrin, the 1:1 inclusion complex, the 1:2 inclusion complex, and physical mixtures, underwent analysis through Differential Scanning Calorimetry (DSC) using the Setaram DSC 131 Evo instrument from France. A small quantity of each sample was positioned in an aluminum housing, and the instrument's temperature was programmed within the range of 50°C to 300°C, with a heating rate set at 10°C per minute²⁵^,²⁶.

Solubility test:

The solubility examination was conducted on pure usnic acid, the 1:1 inclusion complex, the 1:2 inclusion complex, and the physical mixture. In this test, each sample was transformed into a saturated solution using CO₂-free distilled water. Specifically, 10mg of usnic acid equivalent samples were dissolved in 100mL Erlenmeyer flasks and agitated on an orbital shaker for 24hours at room temperature. Subsequently, the samples underwent filtration through a 0.45μm filter (Whatman filter paper), and the absorbance was measured utilizing a UV-Vis spectrophotometer at the maximum absorption wavelength. The obtained absorbance values were then input into the regression equation derived from the calibration curve, facilitating the determination of the dissolved content²⁶^,²⁷^,²⁸.

Dissolution rate profile study:

RESULT AND DISCUSSION:

The initial step of the research was the preparation of the inclusion complexation and the physical mixture of usnic acid-hydroxypropyl-β-cyclodextrin. The inclusion complexation was prepared using the co-grinding method, each material was weighed according to the composition (table 1), grinding was carried out for 2hours, grinding using 30 zirconium balls was carried out at a speed of 120rpm. In milling, one ball variant is used with the aim that there are no cavities between the balls so that the powder can be perfectly ground by the balls and the more balls, the finer the powder we get so that the particle size will be smaller where the smaller the particle size, the surface area will be the larger the solubility increases.

The inclusion complexation and physical mixture were characterized to see the nature or character of the inclusion complexation and the resulting physical mixture and also compared with pure usnic acid. The characterization used is Fourier Transform Infra Red (FT-IR), X-Ray Diffraction (XRD), and Differential Scanning Calorimeter (DSC). Furthermore, the solubility test and determination of the usnic acid dissolution profile in the inclusion complexation were carried out.

To gain insight into the compounds' structures, qualitative investigation using FT-IR spectroscopy was conducted. Interactions between complexes and medicines are often investigated using infrared spectroscopy³⁰. One way to tell whether hydrogen bonds are present is to use infrared spectroscopy. An organic compound's functional groups and its structure were revealed by infrared spectroscopy analysis by comparing fingerprint areas. The results of the investigation are shown graphically, showing the percentage of differences in transmittance at various infrared radiation frequencies³¹. Based on the analysis of the FT-IR spectrum of usnic acid (table 2), there are functional groups at wave number 1632.22cm^-1 (C=C); 3435.88cm^-1 (O-H); and 1692.58cm^-1 (C=O).

Table 2: FT-IR spectrum analysis of usnic acid, hydroxypropyl-β-cyclodextrin, inclusion complexation 1:1, inclusion complexation 1:2, and physical mixture.

Sample	Functional Groups and Wavenumber (cm^-1)
Sample	C=C (1675-1500)	O-H (3650- 3200)	C=O (1708-1613)
Usnic acid	1632.22	3435.88	1692.58
HPβCD	1650.91	3400.68	1650.91
Inclusion complexation 1:1	1633.28	3771.08	1692.85
Inclusion complexation 1:2	1632.06	3400.40	1691.02
Physical mixture	1633.03	3400.30	1692.56

Figure 1: Fourier transform infrared spectroscopic analysis of (a) usnic acid, (b) hydroxypropyl-β-cyclodextrin, (c) inclusion complexation 1:1, (d) inclusion complexation 1:2, (e) physical mixture.

Hydroxyprofil-β-cyclodextrin has a wave number of 1650.91cm^-1 (C=C); 3400.68cm^-1 (O-H); and 1650.91cm^-¹ (C=O). The results of FT-IR analysis on inclusion complexation 1:1 show that there is 1633.28cm^-¹; 3771.08cm^-¹ (O-H); 1692.85cm^-¹ (C=O). The results of the FT-IR analysis of inclusion complexation 1:2 are the functional groups of the wave number 1632.06cm^-¹ (C=C); 3400.40cm^-¹ (O-H); 1691.02cm^-¹ (C=O). FT-IR analysis results on the physical mixture successively from wave numbers 1633.03cm^-¹ (C=C); 3400.30cm^-¹ (O-H); and 1692.56cm^-¹ (C=O). The results of the Fourier Transformation Infra Red (FT-IR) spectroscopy can be concluded that there is a shift in wave number (Table 2, Figure 1).

X-ray diffraction is a reliable method for characterizing the interaction between two solid components and determining whether or not a new crystalline phase is formed³²^,³³. If a new crystalline phase is formed as a result of the interaction of the two components, it will be observed in a real way from the X-ray diffractogram. In the X-ray diffraction test, the diffractogram of pure usnic acid (Figure 2a) shows sharp and clear peaks at an angle of 2 theta 9.9791° with an intensity of 1791.973 units, 10.3431° with an intensity of 4855.684 units, 21.3671° with an intensity 1199.079 units, 22.7191° with an intensity of 1039.663 units.

(a)

(b)

(c)

(d)

(e)

Figure 2: X-ray diffraction analysis of (a) usnic acid, (b) hydroxypropyl-β-cyclodextrin, (c) inclusion complexation 1:1, (d) inclusion complexation 1:2, (e) physical mixture.

Figure 3: Overlay X-ray diffraction analysis (a) usnic acid, (b) hydroxypropyl-β-cyclodextrin, (c) inclusion complexation 1:1, (d) inclusion complexation 1:2, (e) physical mixture.

The 1:1 inclusion complexation diffractogram (Figure 2c) shows sharp and clear peaks at an angle of 2 theta 9.9791° with an intensity of 514.9717 units, 10.3431° with an intensity of 544.8093 units, 21.3671° with an intensity of 262.8316 units, 22.7191° with an intensity of 265.2165 units. The 1:2 inclusion complexation diffractogram (Figure 2d) shows sharp and clear peaks at an angle of 2 theta 9.9791° with an intensity of 654.3281 units, 10.3431° with an intensity of 664.6202 units, 21.3671° with an intensity of 209.2712 units; 22.7191° with an intensity of 221.2445. The physical mixture diffractogram (Figure 2e) shows sharp and clear peaks at an angle of 9.9791° with an intensity of 427.3012 units, 10.3431° with an intensity of 446.6943 units, 21.3671° with an intensity of 199.8883 units, 22.7191° with an intensity 228,2162 units.

From the results of the diffractogram of usnic acid, it shows peaks with high intensity which shows the crystal shape, in the diffractogram of inclusion complexation 1:1, inclusion complexation 1:2, and the physical mixture of usnic acid and hydroxypropyl-β-cyclodextrin shows an amorphous shape and looks increasingly close to the diffraction pattern of hydroxypropyl-β-cyclodextrin with increasing peaks similar to hydroxypropyl-β-cyclodextrin can be seen in Figures 2 and 3, and there was a very sharp decrease in the intensity of the usnic acid peaks. The fact that the usnic acid molecule becomes more prominent in the hydroxypropyl-β-cyclodextrin diffractogram34 indicates that it has penetrated the cavity structure of the molecule³⁴.

(a)

(b)

Figure 4: DSC thermogram (a) inclusion complexation 1:1 (b) inclusion complexation 1:2.

Figure 5: Differential scanning calorimetry analysis of (a) usnic acid, (b) hydroxypropyl-β-cyclodextrin, (c) inclusion complexation 1:1, (d) inclusion complexation 1:2, (e) physical mixture.

DSC thermal analysis is a thermal analyzer that can be used to determine the heat capacity and enthalpy of a material. The energetic observations between usnic acid and hydroxyprofil-β-cyclodextrin were thermally analyzed using DSC. Lattice energy is known to have a correlation with the solubility and dissolution properties of a compound. The lattice energy can be qualitatively observed through the melting point of the preparation at³⁵.

Based on the research, usnic acid showed a sharp endothermic peak at 207.369°C with an enbutment of 86.132 J/g, according to the literature the melting point of usnic acid was 204.8°C³⁶. The hydroxypropyl-β-cyclodextrin thermogram shows a broad endothermic peak at 87.228^oC with an entropy of 76.708 J/g. In the inclusion complexation thermogram 1:1 (Figure 4a), two endothermic peaks were also seen, namely at a temperature of 78.896^oC with an enthalpy of 79.874 J/g and the second at a temperature of 201.12^oC with an enthalpy of 4.311 J/g. At inclusion complexation 1:2 (Figure 4b) two endothermic peaks were seen, namely at 79.032^oC with an enthalpy of 92.557 J/g and the second at 206.114^oC with an enthalpy of 11.328 J/g. In the physical mixture, there are two endothermic peaks, namely at a temperature of 90.353^oC with an enthalpy of 257.871 J/g and the second at a temperature of 202.776^oC with an enthalpy of 8.536 J/g. In the DSC analysis the results of inclusion complexation 1:1, inclusion complexation 1:2, and physical mixtures show that the melting point shifts are not too different, and there is also a shift in enthalpy values, when compared to the enthalpy values of pure usnic acid and hydroxypropyl-β- cyclodextrin (Fig. 5). The characterization results showed inclusion complexation 1:1 and inclusion complexation 1:2 had a widened endothermic peak unlike pure usnic acid (Figure 5). It can be concluded that the inclusion complexation showed an amorphous shape. This indicates that the guest molecule enters the cavity of the host³⁷.

The solubility test was carried out on pure usnic acid, inclusion complexation, and physical mixtures. The solubility test was conducted to determine how the development of inclusion complexes affected the solubility of usnic acid. To start, in distilled water that is devoid of CO₂, find out what the maximum wavelength is at which usnic acid absorbs light. Pure usnic acid has a water solubility of 0.113 µg/mL. With a 1:1 inclusion complexation, the solubility result was 1.008 µg/mL, whereas with a 1:2 inclusion complexation, it was 1.138 µg/mL. A concentration of 0.479 µg/mL is obtained from the physical mixture. Solubility test results for 1:1 inclusion complexation were nine times quicker than those for pure usnic acid, while findings for 1:2 inclusion complexation were ten times faster. As seen in Table 3, inclusion complexation has the ability to enhance solubility.

Table 3: Results of solubility test of pure usnic acid, inclusioncomplexation, and physical mixture in CO2-free aquadest solvent.

Compound	Solubility (mg/mL)	Enhancement (times)
Usnic acid	0.113 ± 0.02	-
Inclusion complexation 1:1	1.008 ± 0.14	9
Inclusion complexation 1:2	1.138 ± 0.29	10
Physical mixture	0.479 ± 0.02	4

[mean±SD, n= 3]

In determining the dissolution profile of pure usnic acid, inclusion complexation, and physical mixture, it was carried out with phosphate buffer medium pH 7.4 using a type 2 device, namely the paddle method with a rotation speed of 50rpm at a temperature of 37±0.5°C for 60minutes. This test was carried out by taking 5mL of solution at 5, 10, 15, 30, 45, and 60 minutes and measured at a wavelength of 289.20 nm.

Table 4: Dissolution efficiency of pure usnic acid, inclusion complexation, and physical mixture in phosphate buffer medium pH 7.4.

Compound	Dissolution Efficiency (%)	Enhancement (times)
Usnic acid	2.154 ± 0.09	-
Inclusion complexation 1:1	11.546 ± 1.20	5
Inclusion complexation 1:2	11.929 ± 0.46	6
Physical mixture	5.750 ± 0.17	3

[mean±SD, n= 3]

Figure 6: Dissolution profile curves of pure usnic acid, inclusion complexation, and physical mixtures

The dissolution results showed that pure usnic acid had the slowest dissolution rate than the inclusion complexation and physical mixture. Inclusion complexation 1:1 has an increase in dissolution efficiency of 5 times and inclusion complexation 1:2 has an increase of 6 times. The findings demonstrated that the dissolution rate of usnic acid was higher when the inclusion complexation of hydroxypropyl-β-cyclodextrin usnic acid was formed, in comparison to pure usnic acid. The active ingredient complexes with a matrix to produce cavities, the inside of which is hydrophobic and the outside of which is hydrophilic. This causes the dissolution rate to rise. Substances having issues with solubility and dissolution rate may enhance medication bioavailability and absorption rate by including the hydroxypropyl-β-cyclodextrin complexer³⁸.

CONCLUSION:

Based on the FT-IR data showing functional group changes, a drop in XRD intensity, and a decrease in DSC melting point, it can be concluded that the physicochemical characteristics of pure usnic acid may be improved by the characterisation of the usnic acid-hydroxypropyl-β-cyclodextrin inclusion complexation. The solubility of usnic acid may be increased by a factor of 9 in a 1:1 inclusion complexation and by a factor of 10 in a 1:2 inclusion complex by forming an inclusion complexation between usnic acid and hydroxypropyl-β-cyclodextrin. Usnic acid-hydroxypropyl-β-cyclodextrin also showed an increase in its dissolving rate, where the dissolution efficiency of 1:1 inclusion complexation increased 5 times and 1:2 inclusion complexation increased 6 times.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank STIFARM Padang for support in the use of the laboratory.

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Received on 16.08.2023 Modified on 14.11.2023

Research J. Pharm. and Tech 2024; 17(5):2206-2212.

DOI: 10.52711/0974-360X.2024.00347