Solubility Enhancement and Preparation of Antifungal Gel of Lawsone
Atul S. Sayare1*, Pallavi P. Kamble1, Prashant D. Ghode1, Shweta P. Ghode2,
Vrushali V. Pawar1, Shivani R. Yeole1, Pranjali A. Mashakhetri1
1Department of Pharmaceutical Quality Assurance, JSPM’s Rajarshi Shahu College of Pharmacy and Research, Pune, (M.S.) India.
2Department of Pharmacognosy, Rasiklal M. Dhariwal Institute of Pharmaceutical Education and Research,
Pune (MS), India.
*Corresponding Author E-mail: atulsayare@gmail.com
ABSTRACT:
Lawsone is the principle colouring compound of Henna, Lawsonia inermis Linn. (Fam. Lythraceae). Lawsone shows low bioavailability because it is insoluble in water and less soluble in other solvents. The objectives of the study were to increase the solubility and dissolution rate of lawsone using by forming β-cyclodextrin (β-CD) inclusion complex and formulating this into a gel formulation for topical use. Method: The inclusion complex were prepared by taking lawsone to β-CD weight ratios of 1:1, 1:2, 1:4 and 1:8. By this technique solubility and dissolution rate of lawsone wassignificantly increased. The inclusion complex was characterized by FTIR and DSC. Results: Antifungal activity of lawsone gel was evaluated on Candida albicans fungi. The in-vitro drug release study was performed on goat skin. Antifungal activity of lawsone and β-CD complex (1:2) showed the biggest zone of inhibition as compared to other inclusion complexes. Conclusion: The antifungal activity of gel of inclusion complex of lawsone and β-CD showed significant antifungal activity.
KEYWORDS: Lawsone, β-cyclodextrin, Inclusion complex, Carbopol 940, Gel, Antifungal activity.
INTRODUCTION:
Henna, Lawsonia inermis Linn. (Fam. Lythraceae) contains a red-orange coloured compound, known as Lawsone (2-hydroxynaphthalene-1,4-dione) (Figure 1)1. It has limited solubility in water at 0.2%, soluble in ethanol, methanol, ethyl glycol and dimethyl formamide2. Henna is well known to be useful in treating skin infections like tinea and also possess antibacterial property which is mainly due to the lawsone content3. But lawsone has very low bioavailability because of its limited water solubility and rapid rate of elimination from the body4.
Figure 1: Chemical Structure of Lawsone
Cylodextrin (CD) inclusion complexation is one of the approaches used to enhance the solubility and bioavailability of poorly water soluble drugs5. There are numerous examples in the literature of β-CD complexes of drugs used to improve solubility and bioavailability6,7.
Therefore, the key objective of present study was to prepare and evaluate the inclusion complex of lawsone using β-CD to increase the solubility and bioavailability of the drug. Another objective of this study was to prepare a topical gel by using lawsone-β-CD inclusion complex and to evaluate its antifungal activity.
MATERIALS AND METHODS:
Chemicals and reagents:
Standardized lawsone (99%) was obtained from Sigma Aldrich, India. β-CD, carbopol-940, polyethylene glycol, triethanolamine, methyl paraben, propyl paraben, ethanol were purchased from Thermosil Fine Chem Industries, Pune, India. Distilled water was used throughout this work.
Preparation of Inclusion Complex:
Inclusion complexes of lawsone and β-CD (1:1, 1:2, 1:4, and 1:8) were prepared by solvent evaporation. Briefly, solutions of lawsone in methanol (25ml) and β-CD in water (50ml) were mixed and stirred for 1 hour using a magnetic stirrer. Methanol was removed by heating (50°C, reduced pressure) and water was evaporated under reduced pressure using rotary evaporator. The mixture was further dried in an oven at 50 °C, ground to fine powder and stored in a closed container8. The different complexes of lawsone F1, F2, F3 and F4 are mentioned in Table1.
Table 1: Formulation of gel
|
Ingredients |
Formulation |
|||||
|
Lawsone |
Lawsone-β-CD complex (1:2) |
Lawsone-β-CD complex (1:4) |
||||
|
GL1 |
GL2 |
GL3 |
GL4 |
GL5 |
GL6 |
|
|
Lawsone /Inclusion Complex |
1% |
2% |
1% |
2% |
1% |
2% |
|
Carbopol 940 |
1% |
1% |
1% |
1% |
1% |
1% |
|
PEG 400 |
2.5% |
2.5% |
2.5% |
2.5% |
2.5% |
2.5% |
|
Methyl paraben |
0.1% |
0.1% |
0.1% |
0.1% |
0.1% |
0.1% |
|
Propyl paraben |
0.01% |
0.01% |
0.01% |
0.01% |
0.01% |
0.01% |
|
Ethanol |
1% |
1% |
1% |
1% |
1% |
1% |
|
Triethanolamine |
q.s. |
q.s. |
q.s. |
q.s. |
q.s. |
q.s. |
|
Distilled water |
q.s. |
q.s. |
q.s. |
q.s. |
q.s. |
q.s. |
Characterization of Inclusion Complex of Lawsone:
Phase solubility stud:
Different concentrations of β-CD (2-20mM) in water (50ml) was prepared and spiked with an excess amount lawsone (0.1g). Equilibrium was established by shaking the flask continuously at 30°C for 3 days. Samples (3ml) were withdrawn, filtered through 0.45 µm nylon membrane filter and analysed in triplicate at 452nm after appropriate dilution9. Following equation was used to calculate the stability constant (Kst).
m
Kst = ---------------------
(1-m) SO
Where,
m = the slope obtained from the plot of concentration of lawsone against β-CD concentration.
SO = the equilibrium solubility of lawsone in water.
Solubility stud:
Excess amount of lawsone-β-CD inclusion complex was mixed with 50ml of distilled water. The flasks were vortex-mixed (3 min) and swirled for 72hours in a water bath (120rpm, 30°C). Samples (3ml) were withdrawn, filtered through 0.45µm membrane filter and analysed in triplicate at 452nm after appropriate dilution using UV-visble spectrophotometer (Shimadzu 1800, Japan)8.
Determination of lawsone contents in inclusion complexes:
The lawsone-β-CD inclusion complex (20mg) was mixed with 20ml NaOH (0.05M), sonicated for 10 min and the volume was topped up with 0.05M of NaOH solution. The solution was analysed in triplicate at 452 nm after appropriate dilution with same solvent10.
Fourier Transform Infrared Spectroscopy (FTIR):
The IR spectra (Brucker Alpha 1) of lawsone, β-CD and inclusion complex were recorded to study the interaction between lawsone and β-CD.
Differential Scanning Calorimetry (DSC):
The DSC spectra (5mg of the sample hermetically sealed in an aluminium pan, mettler, Star SW 12.10) using empty aluminium pan as a reference. The DSC temperature was micro calibrated using indium as standard and sample was heated from 25°C to 300°C at a rate of 1°C/min11.
Preparation of gel formulation:
Carbopol 940 was dispersed in distilled water with constant stirring using magnetic stirrer and mixed with quantity of lwasone/ lawsone-β-CD complex, polyethylene glycol 400 (PEG 400), methyl paraben and propyl paraben (Table 1) with constant stirring. Finally gel was formed using Triethanolamine12.
Evaluation of Gel:
pH of formulation:
The pH of the gel was measured by dissolving accurately weighed quantity (0.5g) distilled water (50 ml) help of calibrated digital pH meter12.
Lawsone content:
Accurately weighed quantity of gel (100mg) was dissolved 10ml methanol, filtered and lawsone content was determined by analysing spectrophotometrically at 452nm10.
Viscosity study:
Brookfield viscometer (spindle number 64 at 10rpm ) was used to measure the viscosity of prepared gel13.
Spreadabilit:
The required quantity of gel (0.5 g) was placed on a glass slide within a circle (1 cm) and another slide was placed on it. Change in diameter after application of 500 g of weight on upper side was observed14.
In vitro permeation study:
Franz diffusion cell was used for the in-vitro permeation study of the gel formulation. Briefly, the gel was applied on the dermal surface of goat skin and the diffusion media (phosphate buffer saline pH 5.0, temperature 32± 0.5°C) was continuously stirred with magnetic stirrer for 6 hours. Sample (0.5ml) were withdrawn at predetermined time intervals (0.5, 1, 2, 4, and 6 hours) and replaced with the same diffusion media. Lawsone content of the solutions was calculated by UV spectrophotometer at 452nm and the cumulative percentage release of lawsone from the inclusion complex gel and lawsone gel were calculated15,16.
Antifungal study:
The antifungal activity of prepared gel formulations were evaluated using agar cup method against Candida albican strain. Fungal strain suspensions was inoculated in cup (10 mm) made aseptically in Subouraud dextrose agar. Gel was added in the holes and put it in incubator. The zone of inhibition of the lawsone-β-CD gel was compared with those of pure lawsone and lawsone gel17.
RESULTS AND DISCUSSION:
Phase Solubility Study:
Solubility of lawsone in β-CD was determined using phase solubility study. Figure 2 shows that as the concentration of β-CD increases the solubility of lawsone in β-CD also increases proportionally. The formation of stable the inclusion complex is apparent from the value of the stability constant (Ks=100.43 M-1).
Figure 2: Phase solubility diagram
Solubility Studies:
The solubility values of lawsone-β-CD inclusion complexes are presented in Table1. Lawsone solubility was significantly increased with the addition of β-CD. Lawsone solubility was significantly increased from 0.19mg/ml to 2.29mg/ml (lawsone/β-CD ratio at 1:1) and 2.88mg/ml (lawsone/β-CD ratio at 1:2).
Determination of lawsone Content in Inclusion Complexes:
The content of lawsone was greater than 91% for all inclusion complexes, showing that the drug was distributed uniformly (Table 2).
Table 2: Solubility data of lawsone β-CD complex
|
Batch No. |
Molar ratio of lawsone: β-CD |
Solubility (mg/ml)
|
Phase solubility (Ks value) |
Drug content |
|
F1 |
1:1 |
2.294±0.02 |
116M-1 |
91% |
|
F2 |
1:2 |
2.888±0.04 |
128M-1 |
94% |
|
F3 |
1:4 |
3.352±0.04 |
137M-1 |
91% |
|
F4 |
1:8 |
4.169±0.04 |
148M-1 |
97% |
Fourier transform infrared spectroscopy (FTIR):
IR spectra of β-CD, lawsone and the β-CD-lawsone inclusion complexes are shown in Figure 3. For β-CD, peak at 3320 cm-1 (vibrations of the O-H bonds in the primary hydroxyl groups) was observed. However, the peak at 1660 cm−1 corresponded to C=O group in lawsone disappeared in inclusion complexes, indicating a chemical interaction between lawsone and the polar region of β-CD.
(a)
(b)
(c)
Figure 3. FTIR spectrum of (a) β-cyclodextrin, (b) lawsone and (c) lawsone β-CD complex
Differential scanning calorimetry (DSC):
A broad endothermic peak corresponding to melting point of lawsone (198°C) was observed in the DSC spectra of lawsone. The inclusion complex of lawsone and β-CD showed two major peaks at 121°C and at 220 °C. The slight increase in temp may be due to interaction between lawsone and β-CD. So the lawsone and β-CD have some interaction. The DSC thermogram of lawsone and lawsone-β-CD complex are showed in Figure 4.
(a)
(b)
Figure 4: DSC thermogram of (a) lawsone and (b) lawsone β-cyclodextrin complex
Evaluation of gel:
The prepared gel formulations were orange transparent coloured, homogenous, and without air bubbles. All the formulations showed a pH in between 5-6 which is appropriate prevent skin irritation. Viscosity of all the formulation was measured by using Brookfield viscometer (Table 3). The formulation batch G4 showed maximum viscosity and satisfactory texture and hence it was selected for further study.
Table No. 3 Evaluation of gel formulation
|
Formulation batch |
pH
|
Viscosity (cps) |
Appearance
|
|
GL1 |
6.2 |
55671±1.52 |
Transparent |
|
GL2 |
6.5 |
51929±2.51 |
Transparent |
|
GL3 |
6.8 |
52904±3.01 |
Transparent |
|
GL4 |
6.2 |
58493±1.24 |
Transparent |
|
GL5 |
6.5 |
56695±2.081 |
Transparent |
|
GL6 |
6.4 |
54736±2.01 |
Transparent |
Texture profile analysis (TPA) of gel:
Good hardness, adhesiveness and adhesive force was observed with G4 formulation. The gel formulation G4 was found to be adhesive and has good texture as indicated by adhesiveness value (2.9 mJ). The hardness value of G4 formulation was found to be 249g i.e, it has good hardness value (Figure 5).
Figure 5: Texture profile analysis (TPA) of gel
In-vitro drug release study:
For the formulation GL4 (2% of complex, 1:2 molar ratio) 94.20% cumulative percentage drug release up to 6 hours was observed. The in-vitro drug release graph is shown in Figure 6.
Figure 6. The In-vitro drug release
Antifungal study:
The antifungal activity was measured by zone of inhibition. The results of all formulae were satisfactory as the greatest activity was observed with G4 (22 mm) which contains the 2% lawsone-β-CD inclusion complex in the 1:2 ratios, while the lowest activity was found with G1 (13 mm) which contains 1% of pure lawsone (Figure 7).
Figure 7: The antifungal activity of % lawsone-β-CD inclusion complex
CONCLUSION:
The inclusion complex of lawsone and β-CD was prepared by solvent evaporation method. The solubility and dissolution rate of lawsone increased by this method. Batch F2 containing 1:2 molar ratio of Lawsone and β-CD formed good inclusion complex. Based on above study the inclusion complex of F2 shows good antifungal activity as compared to other complexes. Of all the gel formulations, better drug content and texture was observed in the GL4. Hence the gel formulation GL4 (2% of 1:2 ratio of lawsone: β-CD inclusion complex) can be considered as the most suceesful formulation based on its drug release.
ACKNOWLEDGEMENT:
The authors would like to thank Dr. K. R. Khandelwal, Principal, Rajarshi Shahu College of Pharmacy and Research for providing necessary facilities to carry out the research work. Authors are also thankful to Sigma Aldrich, India for providing drug sample
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Received on 31.12.2020 Modified on 22.02.2022
Accepted on 27.10.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(4):1776-1780.
DOI: 10.52711/0974-360X.2023.00292