A Novel Mucilage from Ficus glomerata Fruits for Transdermal Patches: Taking Indomethacin as a Model Drug

1. Introduction

      Transdermal delivery has many advantages over conventional modes of drug administration as it avoids hepatic first pass metabolism and improves patient compliance. Intensive research has shown that transdermal route is a potential rout of delivery of lipophilic drugs to the systemic circulation [1].

      Indomethacin is an arylacetic acid derivate, which has excellent anti-inflammatory, analgesic and antipyretic efficacy. Indomethacin has been widely used for treating conditions such as chronic rheumatoid arthritis, osteoarthritis, spondylosis deformans, acute gout, periarthritis and humeroscapularis [2]. The drug has a biological half- life of about 5 to 10 h and a plasma clearance of 1 to 2.5 ml/kg/min which make it a suitable candidate for administration by transdermal route [3].

       Various experimental reports indicated that indomethacin is a suitable drug in controlled release formulations. In this study, Ficus glomerata fruit mucilage was used as a matrix forming polymer for controlling the release of indomethacin.

2. Materials and methods

2.1. Materials

       Indomethacin was obtained as a gift sample from Waksman Selman Pvt. Ltd, Anantapur, India. F. glomerata fruits were obtained from the main market of Anantapur and were authenticated by the Botany department of Sri Krishnadevaraya University, Anantapur. Glycerin, propylene glycol, span-80, methyl paraben and propyl paraben were procured from S.D Fine chemicals, Mumbai. All of the reagents used were of AR grade. Indomethacin sample was characterized by its solubility, pH and UV spectrophotometric method for its authentication.

2.2. Extraction of the mucilage

       The fruits of F. glomerata were thoroughly washed with water to remove dirt and debris then cut it into two pieces. The seeds which were present inside the fruit were removed. The pulps of the fruits were crushed and soaked in water for 5-6 h, boiled for 30 min and left to stand for 1 h to allow complete release of the mucilage into the water. The mucilage was extracted using a multi layer muslin cloth bag to remove the marc from the solution. Acetone (three times the volume of filtrate) was added to precipitate the mucilage. Then the mucilage was separated, dried in an oven at 40 °C, collected, ground, passed through a #80 sieve and stored in desiccator at 30 °C and 45% relative humidity before use [4].

Figure 1. DSC thermo gram of Indomethacin pure drug.

 Cfu= Colony forming units; NMT= Not more than

2.3. Purification of the mucilage

       The crude mucilage (1%) was homogenized (Potter homogenizer) with cold dilute trichloro acetic acid solution (5%). The solution was centrifuged (3500 rpm for 20 min), neutralized with sodium hydroxide by drop wise addition and then dialyzed for 30 h against distilled water. The mucilage was precipitated with ethanol (in the quantities of three times the volumes) and washed successively with ethanol, acetone and diethyl ether [4].

2.4. Characterization of the mucilage

       The collected mucilage was evaluated for physicochemical characteristics viz., morphological characteristics, identification by chemical tests, solubility, melting range, pH, swelling index, ash values, presence of foreign organic matter, test for lead and arsenic, loss on drying, destiny, compressibility index and angle of repose etc. [5]. The values are shown inTable 1.

2.5. Preparation of transdermal patches

       Various propositions of F. glomerata mucilage was taken in beaker add propylene glycol as plasticizer, span-80 as penetration enhancer, propyl paraben and methyl paraben as preservatives, water as solvent and finally Indomethacin (100 mg) was added with continuous stirring in magnetic stirrer for 30 min at 500 rpm. The above mixture was poured within the glass bangles (6.1 cm diameter) placed on mercury surface in a petri dish. The rate of evaporation was controlled by inverting a funnel over the petri dish. The patches were dried at 40±2 °C for 24 h after the dried patches were taken out and stored in desiccator till use  [6]. The formulae of various transdermal patches are shown in Table 2.

Figure 2. DSC thermo gram of Ficus glomerata fruit mucilage.

2.6. Differential scanning calorimetry studies

        Differential Scanning Calorimetry (DSC) curves were obtained by a differential scanning calorimeter (Schimadzu DSC-50, Tokyo, Japan) at a heating rate of 10 °C/min from 25-250 °C in nitrogen atmosphere (20 ml/min) with a sample weight of 3 mg. The DSC thermo grams were shown in Figures 1 to 3.

2.7. Fourier transform infra-red (FT-IR) spectral analysis

         Fourier Transformed Infrared (FT–IR) spectrums of indomethacin with F. glomerata fruit mucilage were obtained individually and in combinations on a Fourier-Transform Infrared (FT-IR) spectrophotometer (Perkin Elmer, spectrum-100, Japan) using the KBr disk method (5.2510 mg sample in 300.2502 mg KBr). The scanning range was 400 to 4000 cm^-1 and the resolution was 1 cm^-1. This spectral analysis was employed to check the compatibility of drugs with the polymers used. The FT- IR spectrums are shown in Figures 4 to 6.

 Figure 3. DSC thermogram of formulation blend F5.

2.8. Evaluation of physicochemical parameters

2.8.1. Thickness

        The thickness of the formulated patches was determined using a digital caliper (BAKER-EC 10, Hyderabad, India). The mean thickness was measured at five different locations of the patch [7].

2.8.2. Tensile strength

        Tensile strength of formulated patches was determined by using computerized balance (Chiksan balance) with modifications. A 1×1cm patch was taken and subjected to studies [7].

2.8.3. Flatness and elongation breake

Longitudinal patches were cut out from the prepared transdermal patches. The flatness was determined at different by using vernier calipers [8]. The length before and after the breake point was considered in calculating the elongation breake [7]. Mathematically, it can

be expressed in the eq.1.

Elongation (%)=L₁–L₂×100/ L₂         Eq1

Where

L1=final length of each patch

L2=initial length of each patch

Figure 4. FTIR spectrum of indomethacin pure drug.

Figure 5. FTIR spectrum of Ficus glomerata fruit mucilage.

2.8.4. Folding endurance

       It can be determined by repeatedly folding a small piece of the patch (2×2 cm) at the same place till it breaks. The number of times the formulated patch can be folded without breaking was the folding endurance of the patch [7].

2.8.5. Moisture content

       The formulated patches were individually pre-weighed and kept in a desiccator containing activated silica at 30 °C for 12 h. The patches were individually reweighed until a constant weight was obtained. The change in the weight from the initial gives the percent moisture content of the patch. The formulated patches were cut into 20×50 mm strips. The patch was weighed and kept in a desiccator (contained saturated solution of calcium chloride) at 30 °C and dried for at least 12 h till the patch shows constant weight. The moisture content value was the difference between the constant and the initial weight [9].

2.8.6. Moisture uptake

      The physicochemical studies like moisture content and moisture uptake provide the information regarding the stability of the formulation. The moisture content was determined by keeping the drug matrix patches in a desiccator containing activated silica until they showed constant weight. The weight difference between relative weights to the final gives the percentage moisture content. The water absorption capacities of various patches were determined at 75% and 93% relative humidity (RH). Patches were cut into 25×60 mm strips. A patch was weighed and kept in a desiccator at 40 °C for 24 h. Then the patch was kept in a desiccator with RH of 75% (containing saturated solution of sodium chloride) and 93% (containing saturated solution of ammonium hydrogen phosphate) at room temperature. Then the patches were measured periodically to constant weights and the water absorption capacity was calculated [9].

 Figure 6. FTIR spectrum of formulation blend (F5).

Figure 7. Zero order plots of prepared transdermal patches.

2.8.7. Drug content of the patch

       Four pieces each of 1×1 cm of prepared patches were cut and took in a stoppered conical flasks containing 100 ml of dissolution medium (0.1 NHCl:CH3OH) and stirred for 6 h, filtered and suitable dilutions were made.

      Absorbance was observed using UV-Visible double beam spectrophotometer (Elico SL 210, Mumbai, India) at 238 nm against a blank [9].

Table3. Result of mechanical properties of formulated transdermal patches.

Number of trials (n)=3

 Figure 8. First order plots of prepared transdermal patches.

2.8.8. In vitro skin permeation studies

      The epidermal hair of rabbit was removed and skin was cleaned from tissues and blood vessels. The skin was mounted overnight (12 h) on the receptor phase to remove any material (UV absorbing). The in-vitro skin permeation of indomethacin from various transdermal patches was studied using KC diffusion cell [10]. The KC diffusion cell consists of upper donor compartment which contains the formulated patch and the bottom receptor compartment which contains dissolution medium, the water jacket (for maintaining the temperature conditions) and a sampling port. The effective permeation area of the diffusion cell was 1.0 cm² and receptor cell volume was 17.5 ml. The receptor compartment contained a phosphate buffer saline of pH 7.4 which was stirred by a magnetic stirrer. The temperature was maintained at 37±2 °C. One ml samples were withdrawn through the sampling port of the KC diffusion cell for the period of 48 h. The withdrawn samples were checked for absorbance at 238 nm. Three trials were made and blanks were also run and the average values were noted [9]. The in-vitro skin permeation data was treated with kinetic modeling and the plots were shown in Figures 7 to 11.

2.8.9. Skin irritation studies

      The skin irritation studies were carried out using modified Draize test [11]. Totally six rabbits were selected. The hairs at the dorsal area of rabbits were removed by shaving 24 h before test. One side of the rabbit’s back (untreated skin area) serves as the control and other as experimental side. The formulated patch was applied on experimental side and the non-medicated patch on the control side of the rabbits. The medicated patches (F5) were changed after 48 h and the fresh patches were adhered at the same site without changing the control side. The patches were secured on the back of the rabbit for seven days. After seven days, the patches were removed and examined for any sign of erythema or edema.

Figure 9. Higuchi’s plots of prepared transdermal patches. 

Table 4.Result of mean weights, moisture content, moisture uptake and dug content of formulated transdermal patches.

Number of trials (n)³

2.8.10.  Scanning electron microscopy (SEM) studies

       The selected formulation’s (F5) surface morphology was studied by using Scanning Electron Microscope (FE-SEM, Carl Zeiss, Germany). The SEM photographs are shown in Figure12.

2.8.11.  Accelerated stability studies

       The optimized formulation (F5) was subjected to accelerated stability studies were conducted according to the International Conference on Harmonization (ICH) guidelines by storing the transdermal patches at 40±0.5 °C and 75±5% RH for 3 months [12].

3. Results and discussion

3.1. Pre formulation studies

      The DSC scan of indomethacin showed a short endothermic peak at 215.50 °C. The thermogram of formulated transdermal patches showed an endothermic peak of drug at 215.07 °C and 215.68 °C, respectively, indicating a slight change in terms of shifting towards the lower temperature. This minor change in the melting endotherm in the drug could be due to the mixing of the drug and excipients which lower the purity of each component in the mixture and may not necessarily indicate potential incompatibili-ty. The characteristic peaks in FTIR spectrum of indomethacin were also observed even in formulation blend, indicating that there is no incompatibility between the indomethacin and the excipients used.

Figure 10. KorsmeyerPeppa’s plots prepared transdermal patches.

Table 5. Results of skin irritation test of formulated transdermal patches.

Visual observation values are expressed as Mean ±SEM, n=6;* Significant compared to formalin (p < 0.05); F-5=Indomethacin Ficus glomerata fruit mucilage patch;

Blank=Patch without drug

3.2. Post formulation studies

      The thicknesses of formulated matrix transdermal patches (630±35.6 to 690±25.6 µm) showed uniformity in thickness. The tensile strength of formulated patches (0.285±0.25 to 0.326±0.10 kg/cm²) and the elongation of the patches (15.33±0.89 to 26.23±0.84 N/mm²) were within the limits. The prepared patches did not show any signs of cracking, which might be attributed to the addition of the plasticizer, propylene glycol. The folding endurance of the patches (98±1.8 to 124±0.9) indicates that the formulated patches maintain their integrity without breaks up on the general usage of patch. All these values were shown in Table 3. The formulated patches showed uniformity in weight (1.561±0.51 to 1.597±0.01 g). The moisture content (2.645±0.35 to 2.854±0.56%) in the formulated transdermal patches was low, which helps the patches to remain stable and from being a completely dried and reduce brittleness during storage. The patches showed uniformity in drug content (97.4±0.02 to 100.7±0.45%). All these values are shown in Table 4. The patches did not show any visible erythema or edema with the formulation or the base used. The results of skin irritation studies are shown in Table 5. The drug permeation from prepared patches was sustained within the therapeutic range. The drug release mechanism from the formulated patches was non-Fickian transport (Figures 7 to 11). The SEM photograph (Figure12 A, 12 B and 12 C) indicates the uniform dispersion of polymeric solution with drug molecule and the Ficus glomerata based patch shown porous surface, which may be suitable for the matrix system. In the present work accelerated stability studies were carried out for selected formulation (F5) at 40±0.5 °C and 75±5% RH for 3 months. The values of physicochemical properties before and after accelerated stability studies are shown in Table 5. The accelerated stability studies indicate that the formulation is quite stable at accelerated environmental conditions. Physical parameters before and after accelerated stability study of optimized F5 patches are shown in Table 6.

Figure 11. Hixson Crowell’s plots of prepared transdermal patches.

4. Conclusion

       This investigation revealed that F. glomerata fruit mucilage appears to be suitable for use as a matrix former in the manufacturing of transdermal patches because of its satisfactory physical and mechanical properties. The formulated patches did not show any visible erythema or edema with the F. glomerata fruit mucilage, which indicates that the formulated patches are compatible with the skin and devoid of potential skin irritation and hence it can be used for transdermal application. The drug permeation from prepared patches was sustained within the therapeutic range. The accelerated stability studies indicate that the formulation is quite stable at accelerated storage conditions. The in-vitro permeation data revealed that dried F. glomerata fruit mucilage can be used as a matrix former in transdermal patches.

Figure 12. SEM of formulated transdermal patches (A, B and C) at various magnifications.

Acknowledgments

       The authors are thankful to Waksman Selman Pvt. Ltd, Anantapur, India, for providing a gift sample of indomethacin.